th
8 Cyanobacterial
Molecular Biology
Workshop
August 25-29, 2004
HÔTEL LE CHANTECLER
Ste. Adele, Quebec
Canada
August 10, 2004
Dear Colleagues,
The organizers would like to welcome you to the 8th Cyanobacterial Molecular Biology
Workshop. It has been our honor and pleasure to organize this latest gathering of
cyanobacteriologists and we thank all participants for their contributions to what ought to be
a highly stimulating meeting in a new and beautiful location. The assistance and advice of
previous organizers, along with the help of Mike Schroder at Virgina Tech has been invaluable
to us. As in previous meetings, the major objective of the workshop is encourage productive
interactions among reserarchers of cyanobacteria and give students, postdocs, and junior faculty
a chance to voice their latest findings alongside more seasoned scientists.
This event is also the 20th anniversary of the first Workshop, and we are happy that
several of the original participants, including the founding organizer Bob Haselkorn, are
presenting their work. The original 1984 program for the first workshop is appended to this
book to offer perspective on what continues to be a vibrant and progressive portion of the
scientific community. Thank you for joining us and we look forward to a great meeting!
Rob Burnap
George Bullerjahn
1
Table of Contents
Brief History of the Workshop
3
Workshop Schedule
4
Poster Presentations
10
Session I Oral Presentation Abstracts
15
Session II Oral Presentation Abstracts
24
Session III Oral Presentation Abstracts
31
Session IV Oral Presentation Abstracts
40
Session V Oral Presentation Abstracts
48
Session VI Oral Presentation Abstracts
57
Session I Poster Presentation Abstracts
64
Session II Poster Presentation Abstracts
72
Session III Poster Presentation Abstracts
81
Session IV Poster Presentation Abstracts
90
Session V Poster Presentation Abstracts
99
Session VI Poster Presentation Abstracts
105
Participants
111
Program of the 1st Cyanobacterial
Molecular Biology Workshop (September 1984)
114
2
Brief History of the Workshop
The first Cyanobacterial Molecular Biology Workshop was convened in
September 1984 by Robert Haselkorn at the University of Chicago. At that time,
molecular genetic analyses were being developed for many unicellular and
filamentous cyanobacteria, thus it was appropriate to bring together the major
research groups to discuss and share current molecular approaches and future
research directions. The 1st Workshop set the tone for subsequent meetings by
encouraging graduate students and postdoctoral fellows to present their work.
Following the success of the 1st Workshop, subsequent meetings were arranged so
as not to conflict with other meetings such as the Photosynthesis Gordon
Conference and the ISPP (International Symposium on Phototrophic
Prokaryotes). As a result, the 2nd and 3rd Workshops were held as satellite
meetings to the ASPP Annual meetings in St. Louis and Toronto (1986 and 1989).
Following the Toronto Workshop, the conference moved to Asilomar Conference
Center in Pacific Grove, CA in 1992. At the conclusion of the 2001 meeting, the
attendees agreed that the Workshop be moved to a new location, and that a
Canadian site (perhaps aligned with the Photosynthesis Congress) would be an
appropriate venue. The site and timing of the 8th Workshop thus reflects the
decision to stage it as a satellite meeting immediately prior to the 13th
International Congress on Photosynthesis in Montreal (August 29 – September 3,
2004).
3
Section One:
Workshop Schedule
4
Schedule of Events
Program Schedule: 8th Cyanobacterial Molecular Biology Workshop
Wednesday, August 25, 2004
3:00 - 6:00 p.m.
Registration/Poster set-up
6:00 - 7:30 p.m.
Dinner
7:45 - 8:00
Opening remarks and welcome: Rob Burnap and George Bullerjahn
8:00 - 9:00
Keynote Address: Robert Haselkorn, University of Chicago, “Heterocyst
differentiation in Anabaena: old wine, new bottles.”
9:00 p.m.
Poster Viewing
Thursday, August 26, 2004
7:30-9:00 a.m.
Breakfast
Session I: Physiology, Metabolism and Global Responses (I):
Session Chair: Günter Peschek, University of Vienna
9:00 - 9:20
Rakefet Schwarz, “NblC, a novel modulator of pigment level during
nutrient limitation in Synechococcus elongates PCC 7942”
9:20 - 9:40
Yukako Hihara, “A small transcriptional regulator, Ssl0564, is involved in
a novel mechanism of redox regulation in a cyanobacterium Synechocystis
sp. PCC 6803”
9:40 – 10:00
Galyna Kufryk, “Functional complementation of the cytochrome c oxidase
deletion mutant by transposon mutagenesis in Synechocystis sp.
PCC6803”
10:00 – 10:20
Miriam Martin, “The myriad lifestyles of Nostoc punctiforme: an array of
possibilities”
10:20 - 10:40 a.m.
Coffee break
10:40 – 11:00
Aaron Kaplan, “Photoreduction of O2, mediated by two A-type
flavoproteins, may consume large fraction of the electrons produced by
water cleavage in cyanobacteria but does not produce H2O2”
11:00 – 11:20
Jean-Charles Cadoret, “Cyclic nucleotides and response to a UV-B stress
in Synechocystis PCC 6803”
5
Schedule of Events
11:20 – 11:40
”
11:40 – 12:00
12:00 – 1:00 p.m.
Jessica Brown, “A cool twist on RNA helicase expression”
Günter Peschek, “Dissection and reassembly of a cyanobacterial
respiratory chain using recombinant electron transport proteins from
Synechocystis sp. PCC6803 throughout”
Lunch
Session II: Heterocysts and nitrogen metabolism:
Session Chair – Karl Forchammer, Justus-Liebig Universität Giessen
1:00 – 1:20 p.m.
Alicia Muro-Pastor, “The role of NtcA in heterocyst development and function.”
1:20 – 1:40
Mani Maheswaran, “Arginine biosynthesis in Cyanobacteria is subjected to global
nitrogen control via PII signal transduction”
1:40 – 2:00
Teresa Thiel, “Developmental and metal regulation of the modA gene of
Anabaena variabilis ATCC 29413”
2:00 – 2:20
Qing Fan, “Identification of Anabaena sp. PCC 7120 genes required
specifically for heterocyst formation and function”
2:20 – 2:40
Francisca Fernandez-Piñas, “Calcium is required for heterocyst
differentiation in Anabaena sp. PCC 7120”
2:40 - 4:20
Poster viewing (authors stand by odd-numbered posters)
4:20 - 6:00
Jeff Elhai, “Workshop: BioLingua, a knowledge-base and programming
environment for the analysis of cyanobacterial genes and genomes
6:00 - 7:30
Barbeque; Le Chantecler Terrace
8:00 - 9:00
Seminar: James Golden, Texas A&M University, “Regulation of heterocyst
development and pattern formation”
9:00 p.m.
Poster viewing
Friday, August 27, 2004
7:30-9:00 a.m.
Breakfast
Session III: Physiology, Metabolism and Global Responses (II):
Session Chair - Francis X. Cunningham, Jr., University of Maryland
9:00 – 9:20 a.m.
Robert J. Jeanjean, “Study on the transcription of genes encoding for
putative peptide synthetases in Anabaena PCC7120”
6
Schedule of Events
9:20 – 9:40
Karen E. Chapman, “Characterisation of three adenylate cyclase Nostoc
punctiforme ATCC 29133 mutants shows phenotypic disparity”
9:40 – 10:00
Mathew Carberry, “Pervasive cyanophage in a Laurentian Great Lakes:
applications of molecular techniques to gain insight on their distribution and
ecology”
10:00 – 10:20
Christophe Six, “Evidences for two novel phycobilisome linkers in the
marine cyanobacterium Synechococcus sp. WH8102, impact of high light
and ultraviolet radiations on the phycobilisome structure and composition
10:20 - 10:40 a.m.
Coffee break
10:40 - 11:20
Martin Hagemann, “Salt stress response in cyanobacteria: mechanisms
involved in the salt-regulation in Synechocystis sp. strain PCC 6803”
11:20 – 11:40
Mary Allen, “Acid stress response in two strains of Synechocystis species”
11:40 – 12:00
Francisco Laganés, “Characterization of mrpA, a gene with roles in pH
adaptation and resistance to Na+ in the cyanobacterium Anabaena sp.
PCC7120”
12:00 - 1:00
Lunch
1:00 - 3:00 p.m.
Poster Viewing (authors stand by even-numbered posters)
Session IV: Photosynthesis and responses to light:
Session Chair - John Cobley, University of San Francisco
3:00 – 3:20 p.m.
Masayuki Muramatsu, “Promoter analysis of genes encoding subunits of
photosystem I in Synechocystis sp. PCC 6803”
3:20 – 3:40
Anthony Kappell, “Identification of a cis-acting element involved in
negative control of the hliA gene of cyanobacteria in response to light”
3:40 – 4:00
John Cobley, “PsoR is a regulator of phycobilisome abundance in the
cyanobacterium, Fremyella diplosiphon”
4:00 - 4:20 p.m.
Coffee break
4:20 – 4:40
Natalia Ivleva, “LdpA: a component of the circadian clock senses redox
state of the cell”
4:40 – 5:00
You Chen, “Global functional analysis of circadian clock genes in
Synechococcus elongatus PCC 7942: strategy and progress”
7
Schedule of Events
5:20 – 5:40
Masato Nakajima, “The mechanism of circadian regulation of gene
expression in the cyanobacterium Synechococcus elongatus PCC 7942”
5:40 – 6:00
Yao Xu, “Circadian mechanisms in cyanobacteria”
6:00 - 7:30
Dinner
8:00 - 9:00
Seminar: Petra Fromme, Arizona State University; “Structure and
Function of Photosystem I and II"
9:00 p.m.
Poster Viewing
Saturday, August 28, 2004
7:30 - 9:00 a.m.
Breakfast
Session V: General Structural Aspects:
Session Chair - Cheryl Kerfeld, UCLA
9:00 – 9:20
Nataliya Yeremenko, “Supramolecular organization and dual function of
the IsiA chlorophyll-binding proteins in cyanobacteria”
9:20 – 9:40
Wendy Schluchter, “Identification of a new family of phycobiliprotein
lyases in cyanobacteria: characterization of a phycocyanin lyase”
9:40 – 10:00
Darryl Horn, “Synechococcus sp. PCC 7002 PetB arginine 214: a key
residue for quinone-reductase function and possible oxygen radical
production in the cytochrome b6/f complex
10:00 – 10:20
Cheryl Kerfeld, “Structure and function of protein complexes in
photoprotection and carbon fixation: the orange carotenoid protein and the
carboxysome”
10:20 - 10:40 a.m.
Coffee break
10:40-11:00
Gouzhong Shen, “Functional genomics of genes for biogenesis of Fe/S proteins in
cyanobacteria”
11:00 – 11:20
Marc Nowaczyk, “Preliminary structural characterization of the 33-kDa protein
(PsbO) in solution studied by site-directed mutagenesis and NMR spectroscopy”
11:20 – 11:40
Archana Mukopadhyay- “Identification of a low molecular weight protein
tyrosine phosphatase and its potential substrates in Synechocystis sp. PCC
6803”
11:40 - 1:00 p.m.
Lunch
8
Schedule of Events
Session VI: Carbon metabolism:
Session Chair – Dean Price, Australian National University
1:00 – 1:20 p.m.
Martin Cann, “Signal transduction molecules directly regulated by
bicarbonate and sodium ions”
1:20 – 1:40
Suzanne Burey, “Response to low carbon dioxide in the glaucocystophyte
alga, Cyanophora paradoxa”
1:40 – 2:00
Michael Summers, “Identification and analysis of akinete specific genes in
Nostoc punctiforme”
2:00 – 2:20
Fiona Woodger, “Regulation of the cyanobacterial CO2 concentrating
mechanism
2:20 – 2:40
Graciaela Solerno, “Regulation of sucrose metabolism in salt-treated cells of
Nostoc sp. PCC 7120: towards the understanding of the role of sucrose in
cyanobacteria”
2:40 - 4:40
Poster viewing – then posters to be taken down
4:40 - 6:00
Free time (and time for break-out groups to discuss specific areas)
6:00 - 7:30
Dinner
8:00 - 9:00
Seminar: Murray Badger, Australian National University; “Cyanobacterial
photosynthetic CO2 concentrating mechanisms: solutions employing many
Ci transporters, two carboxysomes types and diverse carbonic anhydrases”
Sunday, August 29, 2004
7:30-9:00 a.m.
Breakfast
Depart Ste. Adele.
In Montréal, the 13th International Congress of Photosynthesis registration starts at 1:00 p.m. in
Hall Bleury (1001 Place Jean-Paul-Riopelle) and will be followed by a mixer buffet at 6:00 p.m
in panoramic room 710. The opening ceremony is Monday, August 30th, at 8:30 in room 210-A.
9
Poster Listing Section
Poster Presentation Listing
Session I: Physiology, Metabolism and Global Responses (I):
Session Chair: Günter Peschek, University of Vienna
1. Akiko Tomitami- Isolation and characterization of Nostoc punctiforme ATCC 29133
mutants unable to differentiate into hormogonia
2. Alicia M. Muro-Pastor- Identification of a cis-acting antisense RNA potentially
regulating furA expression in Anabaena sp. PCC 7120.
3. Shannon R. Cannales-Differential circadian regulation of psbA gene expression in
Synechococcus elongatus PCC 7942
4. Laura Patterson-Fortin- Novel expression regulation and biochemical activities of a
redox-regulated RNA helicase
5. George W. Owtrim- RNA Structural Rearrangements by the Synechocystis RNA
Helicase, CrhR
6. Kirsten Gutekunst- Transcriptional regulation of the bidirectional NiFe-hydrogenase in
Synechocystis sp. PCC 6803
7. Ninja Backasch, The role of tocopherol in preventing oxidative damage at the
cyanobacterial photosystem II from Synechocystis sp.PCC 6803
8. Young Mok Park- Novel Interaction between Two CheA-like Molecules Involved in
Gliding Motility of Cyanobacterium Synechocystis sp. PCC 6803
Session II: Heterocysts and nitrogen metabolism:
Session Chair –Karl Forchhammer, Justus-Liebig Universität Giessen
9. Rebecca Thayer- Characterization of Anabaena sp. strain PCC 7120 genes alr4311 and
all4312.
10. Ignacio Luque- Nitrogen control of the glutamyl-tRNA synthetase in Tolypothrix sp. PCC
7601
11. Karl Forchhammer- Nitrogen regulation in cyanobacteria: new insights in responses
mediated by the PII signal transduction protein
12. Jeff Elhai- Possible role of a non-coding RNA in the initiation of heterocyst
differentiation
13. Natalia Ivanikova- Construction of a nitrate responsive Synechocystis sp. strain PCC
6803 bioreporter for estimating nitrate bioavailability in freshwater
10
Poster Listing Section
14. Martha Ramirez- Characterization of the DNA-binding activity of Anabaena 7120 devH
protein
15. Sigal Lechno-Yossef- alr1086 is an ORF required for regulation of heterocyst
polysaccharide synthesis in Anabaena sp. strain PCC 7120
16. Vladimir Krasikov- The response of Synechocystis sp. PCC 6803 to nitrogen starvation:
transcriptomics versus proteomics
Session III: Physiology, Metabolism and Global Responses (II):
Session Chair: Francis X. Cunningham, Jr., University of Maryland
17. Akito Nishizawa-Genetic analysis of nonribosomal peptide synthetase genes in
cyanobacteria
18. Florence Gleason- Ribonucleotide reduction in the cyanobacteria
19. Jens Appel- Protein trans-splicing of the -subunit of the DNA-polymerase III of
Synechocystis sp. PCC 6803: does it exert a regulatory role?
20. Sousuke Imamura- Characterization of group 2 sigma factors of RNA polymerase and
their roles in the cyanobacterium Synechocystis sp. strain PCC 6803
21. Aaron Kaplan- The composition and dynamics of the phytoplankton assemblage in Lake
Kinneret is strongly affected by cyanobacterium - dinoflagellate communication
22. Douglas Graham- Investigation of carbon and light on cyanobacterial toxin production
23. Yuichi Fujita- “Chlorophyll Pasteur point”, a critical atmospheric oxygen level for
ancestral chlorophyll biosynthesis
24. Ramakrishna Boyanpalli- Construction of cyanobacterial bioreporters for detecting
nutrient deficiency in marine waters.
25. George S. Bullerjahn- Expression and mutagenesis of mapA, a Synechococcus sp. PCC
7942 iron responsive gene: evidence for oxidative stress protection
26. Katie M. Shea, Acid stress response in two strains of Synechocystis species
Session IV: Photosynthesis and responses to light:
Session Chair - John Cobley, University of San Francisco
27. Georg Schmetterer- Cyanobacterial respiratory terminal oxidases
28. Kazuki Terauchi- Circadian rhythm of gene expression and cloning of clock genes in the
facultative filamentous cyanobacterium Plectonema boryanum
29. Eugenia Clerico- Unveiling the presence of more than one oscillator in S. elongatus
PCC7942
11
Poster Listing Section
30. Jayna Ditty- Role of kaiBC transcriptional timing in the circadian clock mechanism of
Synechococcus elongatus PCC 7942
31. Gogang Dong- Homotypic interactions of central oscillator components in the
cyanobacterial circadian clock
32. Mitsunori Katayama- Alterations in the gene expressions by the disruption of genes
encoding phytochrome-related protein in Synechocystis sp. PCC 6803
33. Aaron Kaplan- Activation of photosynthesis and resistance to photoinhibition in
cyanobacteria within biological desert crust.
Session V: Structural aspects:
Session Chair: Cheryl Kerfeld, UCLA
34. Hans C. P. Matthijis- Photosystem I cyclic electron transfer pathways and function
35. Robert L. Burnap- In situ effects of mutations of the extrinsic cytochrome c550 of
Photosystem II in Synechocystis sp. PCC6803
36. Gaozhong Shen- Progress in sequencing, assembly and annotation of the genome of the
marine unicellular cyanobacterium Synechococcus sp. PCC 7002
37. C. Kay Holtman- First fruits of the Synechococcus elongatus PCC 7942 functional
genomics project
38. Jingdong Zhao- Deletion of large chromosomal fragments of Anabaena sp. PCC 7120
with a Cre/loxP system
39. Marc Nowaczyk,- Preliminary structural characterization of the 33-kDa protein (PsbO) in
solution studied by site-directed mutagenesis and NMR spectroscopy
Session VI: Carbon metabolism:
Session Chair – Dean Price, University of Durham
40. Dean G. Price- Identification of a new class of bicarbonate transporter from the marine
cyanobacterium, Synechococcus PCC7002
41. Louis A. Sherman- Pleiotropic regulation of carbohydrate metabolism by Hik8 (a SasA
orthologue) in Synechocystis 6803
42. Aaron Kaplan- Towards resolving the Glucose sensing in Synechocystis PCC 6803
43. Ben M. Long-A proteomic study of carboxysomes from -cyanobacteria
44. Robert L. Burnap - Global patterns of gene expression in Synechocystis sp. PCC 6803 in
response to inorganic carbon limitation and the inactivation of ccmR, a LysR family
regulator
12
Oral Presentation Abstracts
Abstracts for oral presentations
13
Oral Presentation Abstracts
Keynote Talk
Heterocyst differentiation in Anabaena: old wine, new bottles
ROBERT HASELKORN
Department of Molecular Genetics & Cell Biology, University of Chicago, 920 East 58 Street,
Chicago IL 60637 USA
Our first foray into biology, as opposed to the biochemistry of nucleic acids, was a study of
the plant virus Turnip Yellow Mosaic Virus. After moving to Chicago, we took up bacterial
viruses, whose great virtue was the ability of single particles to infect. When cyanophages were
discovered in 1963, they were added to the mix because their hosts, the cyanobacteria, did plantlike photosynthesis and looked gorgeous on plates. Numerous students (Ron Luftig, Lou
Sherman, Jim Mackenzie and Ken Adolph) did their theses on cyanophages. When Honoree
Fleming came along, she was encouraged to work on cyanophage development too. Instead, she
studied cellular development, namely the differentiation of heterocysts in Anabaena in response
to starvation for a fixed source of nitrogen. She used the newly introduced method of
polyacrylamide gel electrophoresis of proteins to show that nitrogenase was made chiefly in the
differentiating heterocysts and that many other proteins appeared in the differentiating cells
according to a program not unlike that of a viral infection of bacteria. We subsequently
determined to study that program from the point of view of the regulated transcription of sets of
genes. Gene cloning came into the lab in the late 1970s, introduced by Doug Rice and Barbara
Mazur, and exploited first by Moshe Mevarech, Pete Lammers, Rich Fisher, Steve Robinson, Jim
Golden, Nilgun Tumer, Stephanie Curtis, Sandy Nierzwicki-Bauer, Bill Belknap, Jean Lang,
George Schneider, R. Nagaraja and later by Bill Buikema, Martin Mulligan, Dulal Borthakur,
Herbert Boehme, Chris Bauer, Kristin Bergsland, Jihong Liang, Bianca Brahamsha, Brian
Palenik and Kristin Black. The most recent people to join the program were Kathryn Jones and
Sean Callahan. Most of the individually cloned and sequenced genes were used for two
purposes: to see when and where they were transcribed during heterocyst differentiation and to
try to identify promoter elements governing the program. Specific efforts were made to
characterize modification of the transcription machinery during differentiation but nothing
striking was found beyond the unusual structure of the RNA polymerase core. Subsequently,
around 1990, it became possible to isolate mutants that were defective in heterocyst
differentiation and to isolate the genes that complement these mutations, using a plasmid
complementation system introduced by Wolk and Elhai and then optimized by Bill Buikema.
The latter program revealed the master regulator HetR, whose amazing properties were
elucidated later by Zhao’s group in Beijing. Buikema also introduced a method for controlling
gene expression with a Cu++-regulated promoter, making it possible to turn essential or
potentially lethal genes on or off at will. These genetic tools, together with the complete genome
sequence from the group at Kazusa and the global transcription profiles of gene expression
during heterocyst differentiation by Ehira and Sato, make it reasonable to expect a complete
molecular description of cellular differentiation in Anabaena in the near future.
14
Session II Oral Presentation Abstracts
Session I: Physiology, Metabolism and Global Responses (I)
Session Chair: Günter Peschek, University of Vienna
15
Session II Oral Presentation Abstracts
NblC, a novel modulator of pigment level during nutrient limitation in
Synechococcus elongates PCC 7942
ELEONORA SENDERSKY, ROXANE LAHMI, JUDITH SHALTIEL, ALEXANDER
PERELMAN AND RAKEFET SCHWARZ*.
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Modulation of pigment level in response to environmental cues is an essential process that allows
photosynthetic organisms to adjust light harvesting to the metabolic needs of the cell and
minimizes the photo-oxidative damage resulting from surplus excitation. Mutants that, unlike
wild type cells, do not degrade their light harvesting pigments under sulfur and nitrogen
starvation have been used to identify components of the degradation pathway. Starved mutant
cultures appear blue green rather than yellowish or bleached as starved wild type cultures, and
therefore the phenotype was termed non-bleaching (nbl). Four components of the nbl-pathway
have been previously identified: NblS and NblR were assigned a regulatory function whereas
NblA and NblB appear to be involved in the degradation process. The specific role of the latter
components is not clear, neither the mechanism of pigment degradation or its regulation by
nutrient availability. It was therefore desirable to isolate novel non-bleaching mutants to further
elucidate the mechanism underlying modulation of pigment level. We have developed an
efficient screening method, which employs Fluorescence Activated Cell Sorter (FACS) and
isolated novel non-bleaching mutants. Characterization of one of these mutants uncovered a new
component of the degradation pathway designated NblC. Inactivation of nblC resulted in a nonbleaching phenotype during nitrogen, sulfur or phosphorus starvation. Therefore, NblC, similarly
to NblR, belongs to a cascade of events resulting in a general acclimation response. Over
expression of NblC by a foreign promoter resulted in pigment degradation under the inducing
conditions. Interestingly, a strain of nblR-mutant in which NblC was over expressed retained its
pigmentation, suggesting dependence of NblC on NblR or on a gene product modulated by the
latter. Transcription of nblC is induced during sulfur, nitrogen and phosphorous starvation.
Furthermore, the NblC-mutant exhibited reduced viability under nutrient limitation as compared
to wild type cells. The role of NblC during nutrient starvation will be discussed in light of
transcription analysis of nblA and cpc operon (encoding for the subunits of phycocyanin) in the
wild type, in nblC-mutant and in wild type and mutant strains over expressing NblC or NblR.
* Corresponding author: [email protected]
16
Session II Oral Presentation Abstracts
A small transcriptional regulator, Ssl0564, is involved in a novel mechanism of
redox regulation in a cyanobacterium Synechocystis sp. PCC 6803.
KINU NAKAMURA and YUKAKO HIHARA*
Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-okubo,
Saitama 338-8570, Japan
ssl0564, a small ORF of a cyanobacterium Synechocystis sp. PCC 6803, encodes one of the
smallest protein belonging to the LuxR family of transcriptional regulators. Its 89-amino-acid
sequence is similar over its entire length to the DNA binding domain of this protein family,
including a putative helix-turn-helix motif. We found that purified Ssl0564 protein formed a
dimer structure that could be disrupted to yield monomers by the addition of DTT or mercaptoethanol. Three cysteine residues at the amino-terminal domain are well-conserved
among cyanobacterial species and therefore seemed to be involved in dimerization through the
formation of intermolecular disulfide bonds. In order to identify the target genes for Ssl0564,
DNA microarray analysis of ssl0564-disrupted mutant was performed. It was revealed that chlL,
chlN and chlB genes encoding subunits of light-independent protochlorophyllide reductase, katG
encoding catalase-peroxidase and slr1957 were up-regulated and ssl0564-sll0296 operon, ndhD2
encoding a subunit of NADPH dehydrogenase and rpe encoding pentose-5-phosphate-3epimerase were down-regulated by Ssl0564 under normal growth conditions. Furthermore, by
northern blot analysis, we verified that the transcript levels of ndhD2 and rpe were regulated by
Ssl0564 under different growth conditions. In the wild type cells, transcripts of these genes
accumulated within 15 min after the shift to high light conditions and this up-regulation was
suppressed by addition of DCMU, methyl viologen or hydrogen peroxide. On the other hand, in
ssl0564-disrupted mutant, these transcript levels were less affected by the environmental changes
and always higher than those in the wild type. This suggests that Ssl0564 is a transcriptional
regulator that can perceive the redox state of the acceptor side of photosystem I. When
generation of the reducing power by photosynthetic electron transport chain was slowed down
and redox components located downstream of photosystem I became oxidized, Ssl0564 may
form the intermolecular disulfide bonds to become “active” dimeric form.
* Corresponding author
17
Session II Oral Presentation Abstracts
Functional complementation of the cytochrome c oxidase deletion mutant by
transposon mutagenesis in Synechocystis sp. PCC6803
GALYNA KUFRYK* and WIM VERMAAS
School of Life Sciences, and Center for the Study of Early Events in Photosynthesis, Arizona State University, P.O.
Box 874501, Tempe, AZ 85287-4501
Cyanobacteria represent an interesting system for studies of photosynthesis and respiration.
Components of these two electron transport systems are located in cytoplasmic membrane, and
some of them, such as plastoquinone (PQ) and the cytochrome b6/f complex, are used in both
respiration and photosynthesis.
Cytochrome c oxidase catalyses reduction of the final electron
acceptor (molecular oxygen) in Synechocystis sp. PCC6803. Deletion of the gene coding for
subunit I of this enzyme (slr1137) impacts the ability of the organism to grow at low light
intensity (less than 5 µmol photons m-2 s-1). This probably is a result of overreduction of the
plastoquinone pool that cannot be alleviated by the photosynthetic activity of photosysem I.
Therefore, the cytochrome c oxidase deletion mutant (Cta- strain) is a good model to study
regulation of redox state of the PQ pool in this cyanobacterium. Transformation of this strain
with the interruption library of Synechocystis sp. PCC6803 obtained by transposon mutagenesis
resulted in several mutants that retained the original deletion of cytochrome c oxidase but were
able to grow at low light intensity. Interruption mutation in one of them was mapped to 71 bp
upstream of the ssl3342 coding region. This open-reading frame is the first gene of a possible
operon that contains three other genes, sll1749, sll1750, and sll1751. The ssl3342 gene codes for
a 84-residue protein of unknown function that has two predicted transmembrane domains. A
homologue of this gene can be found in Arabidopsis thaliana. The next gene of the possible
operon, sll1749, also codes for an unknown protein, whereas the product of the third gene,
sll1750, has been annotated to be the urease alpha subunit. The function of Sll1751 remains
unknown. Absorption spectra and low temperature fluorescence spectra showed that the
photosystem II complexes in this mutant were fully assembled with normal antenna size. The
phenotype of the mutant strains is being investigated.
* Corresponding author.
18
Session II Oral Presentation Abstracts
The myriad lifestyles of Nostoc punctiforme: an array of possibilities
MIRIAM MARTIN* and JACK MEEKS
Section of Microbiology, University of California-Davis, One Shields Ave Davis, CA, 95616
The filamentous cyanobacterium Nostoc punctiforme inhabits diverse ecological niches and its
vegetative cells can differentiate into an unusually wide range of cell types: nitrogen-fixing
heterocysts, motile hormogonia, and perrenating akinetes. The phenotypic plasticity of N.
punctiforme is reflected in its relatively large ~9 MB genome with more than 7300 genes. While
a number of heterocyst determinants and biosynthetic components have been defined through
traditional genetics, the bulk of the regulatory proteins remain elusive. Similarly, the proteins
that signal and implement the differentiation of vegetative cells into hormogonia and akinetes are
largely unknown. To accelerate our studies of N. punctiforme differentiation, we are
constructing a DNA array with which we will compare the transcriptional complement of all four
cell types over the course of development. The array will consist of ~7000 PCR products,
representing all ORFs with similarity in the database and all ORFs over 240 bp in length
encoding hypothetical proteins. Only one member of families of nearly identical ORFs will be
amplified for printing and 311 highly similar transposase genes will not be arrayed. Each PCR
product contains terminal adaptamers that allow further amplification of the entire genome with
minimal effort and expense. Following the printing of the array, dual hybridizations with
fluorescently-labeled cDNA will be employed to quantitatively compare the transcript level of
each gene in ammonium grown vegetative cells with mRNA from cultures possessing
heterocysts, akinetes or hormogonia. Genes that are preferentially expressed in non-vegetative
cells will be targeted for disruption by insertional mutagenesis to explore a possible role for that
protein in the differentiation or function of that particular cell type(s). Genomic approaches are
also being complemented by mutational and reporter gene analysis of several known regulatory
elements of heterocyst development, patS, patN and hetR, to determine the epistatic relationships
between these elements in coordinating the initation of patterned heterocyst differentiation.
*Corresponding author
19
Session II Oral Presentation Abstracts
Photoreduction of O2, mediated by two A-type flavoproteins, may consume
large fraction of the electrons produced by water cleavage in cyanobacteria
but does not produce H2O2.
YAEL HELMAN1, DAN TCHERNOV4, LEONORA REINHOLD1, MARI SHIBATA2,
TERUO OGAWA2, RAKEFET SCHWARZ3, ITZHAK OHAD1, BOAZ LUZ1, AARON
KAPLAN1*
1
Minerva Centre for Photosynthesis under Stress, The Hebrew University of Jerusalem, Israel
Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan
3
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
4
Interuniversity Institute for Marine Science, Eilat, Israel
2
Photoreduction of O2 by photosynthetic electron transfer, the Mehler reaction, is observed in
all groups of oxygenic photosynthetic organisms. Although reported over 50 years ago the
electron transport chain that mediates this reaction has not yet been identified. Our study
provides the first evidence for the involvement of A-type flavoproteins. Mutants of
Synechocystis sp. strain PCC 6803 defective in genes encoding A-type flavoproteins, flv1 and
flv3, failed to exhibit O2 photoreduction but performed normal photosynthesis and respiration.
We show that the light-enhanced O2 uptake was not due to respiration or photorespiration. After
dark acclimation, photooxidation of P700 was severely depressed in mutants flv1 and flv3
(confirmed by steady state fluorescence measurements) but recovered following light activation
of CO2 fixation, which provides P700 with an additional electron acceptor. Inhibition of CO2
fixation prevented recovery but scarcely affected P700 oxidation in the wild type where the
Mehler reaction serves as an alternative route for electrons. We conclude that the source of
electrons for photoreduction of O2 is PSI; and that A-type flavoproteins Flv1 and Flv3 are
essential for this process in vivo (1).
Isolated Flv3 protein, expressed in E. coli, showed typical absorbance of an A-type flavoprotein
and consumed O2 when supplied with NADPH, in vitro. We propose that, in contrast to the case
in eukaryotes, the Mehler reaction in cyanobacteria, reduces O2 directly to water in vitro, does
not produce reactive oxygen species and that it may be evolutionarily related to the response of
anaerobic bacteria to O2.
Application of the three stable O2 isotope methodology (2) showed that photoreduction of O2
can be distinguished from other O2 consuming reactions and that, depending on the
environmental conditions, this reaction may consume as much as 40 % of the electrons leaving
PSII.
1. Helman, Y., Tchernov, D., Reinhold, L., Shibata, M., Ogawa, T., Schwarz, R., Ohad, I. and A. Kaplan
(2003) Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria
Current Biology 13: 230-235.
2. Luz, B and B. Barkan (2000) Assessment of oceanic productivity with the triple-isotope composition of
dissolved O2. Science 288: 2028-2031.
*
Corresponding author
20
Session II Oral Presentation Abstracts
Cyclic nucleotides and response to a UV-B stress in Synechocystis PCC 6803
CADORET J.-C.1, PEREWOSKA I.1, ROUSSEAU B.1, ETIENNE, A.-L.1, VASS I.2 and
HOUMARD J.1*
1
Organismes Photosynthétiques et Environnement, Ecole Normale Supérieure, FRE2433, 46 rue d'Ulm, 75230 Paris
cedex 05, France. 2 Institute of Plant Biology, Biological Research Center, P.O. Box 521, 6701 Szeged, Hungary.
Cyclic nucleotides (cAMP and cGMP) are ubiquitous signalling molecules from prokaryotes
to higher eukaryotes that mediate adaptative responses of cells. They act as second messengers,
and regulate gene expression, enzyme or channel activity, for example. Among prokaryotic
organisms, cyanobacteria present the peculiarity of having the two types of cyclic nucleotides,
and their levels vary with environmental changes (1). A strict control of cAMP and cGMP
homeostasis is achieved by cyclases (for the synthesis) and phosphodiesterases (PDEs, for their
degradation). In Synechocystis PCC 6803, cya1 codes for an adenylyl cyclase (1), cya2 for a
guanylyl cyclase (2), and two ORFs (sll1624 and slr2100) were found by in silico analysis to
exhibit similarities with eukaryotic PDEs (3).
His-tagged Slr2100 was constructed and purified. In vitro it possesses a cGMP
phosphodiesterase activity. In parallel, fully segregated slr2100 and sll1624 mutants were
obtained by interposition. Under standard conditions, both have wild type growth rates. By
measuring O2 evolution and performing fluorescence relaxation experiments on cells subjected to
a UV-B stress, we found that slr2100 but not sll1624 was more sensitive to UV-B than the wild
type. UV-B radiations are known to cause important damages to the photosynthetic apparatus, in
particular to PSII, leading to a decreased O2 evolution. More specifically we found that slr2100
is impaired in PSII repair when illuminated with UV-B, and that the wild type behaves similarly
to slr2100 upon addition of dipyridamol, a specific inhibitor of cGMP phosphodiesterases.
Comparative macroarray analyses and quantitative PCRs were performed to monitor changes in
gene expression. Altogether, the data show that, during a UV-B stress, cGMP play an important
role in signal transduction, and that in Synechocystis PCC 6803 photoacclimation mechanisms
are linked to the intracellular levels of cyclic nucleotides.
References
1. Cann, M. (2003) New Phytologist 161: 23–34.
2. Terauchi, K. & Ohmori, M. (1999) Plant Cell Physiol. 40: 248-251.
3. Ochoa de Alda, J.A.G., Ajlani, G. & Houmard, J. (2000) J. Bacteriol. 182: 3839-3842.
4.Ochoa de Alda, J.A.G.. & Houmard, J. (2000) Microbiology 146: 3183-3194.
* Author for correspondence; email: [email protected]
21
Session II Oral Presentation Abstracts
A cool twist on RNA helicase expression
J.M. BROWN, D. CHAMOT, and G.W. OWTTRIM*
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9
Cyanobacterial survival in a wide array of ecosystems depends on their ability to rapidly
and specifically sense and respond to prevailing conditions. Our lab studies a cyanobacterial
DEAD-box RNA helicase, CrhC, whose expression is specifically regulated by temperature.
RNA helicases are key players in many diverse cellular processes involving all aspects of RNA
metabolism. Previously, we have proposed that CrhC performs a role in adaptation to reduced
temperature in Anabaena sp. strain PCC 7120, as both transcript and protein accumulate at
growth temperatures below 30oC. The regulatory mechanism(s) providing temperature dependent
expression of CrhC is not known. At the transcriptional level, characterization of protein binding
to the crhC promoter revealed a binding site for a putative 60 kDa repressor, phosphorylation of
which represses crhC promoter activity above 30oC. An AT-rich region within the binding site is
essential for temperature-dependent regulation of crhC expression at both the transcript and
protein levels. At the post-transcriptional level, crhC expression from a constitutive promoter
indicates that the 5’ UTR confers temperature-dependent transcript stability below 30oC. Thus,
CrhC expression is regulated through a combination of transcriptional and post-transcriptional
temperature-dependent processes. The data are discussed with respect to potential functional
role(s) performed by CrhC in cyanobacterial adaptation to environmental change.
22
Session II Oral Presentation Abstracts
Dissection and reassembly of a cyanobacterial respiratory chain using recombinant
electron transport proteins from Synechocystis sp. PCC6803 throughout.
M. Paumann1, R. Duran2, C. Obinger3 and G. A. Peschek1
1
Molecular Bioenergetics Group, Institute of Physical Chemistry, University of Vienna,
Althanstrasse 14, A-1090 Wien, Austria.
2
Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla, Americo Vespucio s/n,
SP-41092 Sevilla, Spain.
3
Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Wien,
Austria.
E-mail: [email protected]
The genes encoding cytochrome- c6 and plastocyanin (PC) in Synechocystis 6803 ( petJ and
petE) as well as the part of the ctaC gene that encodes the soluble, bimetallic CuA.CuA-binding
domain of subunit II of the cytochrome-c oxidase, were overexpressed (either with or without his
tag) in E. coli. After purification, the recombinant proteins were thoroughly characterized
physicochemically and spectroscopically. All their properties were identical to published values
obtained from corresponding native proteins. Of special interest were the thermodynamic and
kinetic parameters of electron transfer reactions from reduced cytochrome-c6 and PC to the
bimetallic CuA-centre which mimick the native situation of the terminal respiratory electron
transport in the membranes (both cytoplasmic and thylakoid membranes, CM and ICM, both of
which are known as potential sites of RET). Conforming to previous observations, both
cytochrome-c6 and PC proved to be efficient and alternatively indispensable electron donors not
only to P700 (in photosynthesis) but also to the cytochrome -c oxidase in respiration, and the
dependence of the reactions on the ambient ionic strength as reflected by Brönsted plots (using
different cyt-c and PC species of different IEPs) were in perfect agreement with the Marcus
theory. The results will be discussed in detail and in view of the close relationship and partial
identity of RET and PET in cyanobacteria.
(Literature: G. A. Peschek, C. Obinger and M. Paumann: Physiol. Plant. 120:358-369 (2004).)
23
Session II Oral Presentation Abstracts
Session II: Heterocysts and nitrogen metabolism
Session Chair: Karl Forchammer, Justus-Liebig
Universität Giessen
Keynote speaker: James Golden, Texas A&M University,
“Regulation of heterocyst development and pattern
formation”
24
Session II Oral Presentation Abstracts
Keynote Talk
Regulation of heterocyst development and pattern formation
JAMES W. GOLDEN
Department of Biology, Texas A&M University, College Station, Texas 77843-3258 USA
Anabaena (Nostoc) sp. strain PCC 7120 is a filamentous cyanobacterium that reduces atmospheric
dinitrogen to ammonia in specialized differentiated cells called heterocysts. The differentiation of a
photosynthetic vegetative cell into a nitrogen-fixing heterocyst requires extensive changes in gene
expression that result in substantial morphological and physiological changes. As a postdoc in Robert
Haselkorn's lab, I found that two DNA rearrangements affecting nitrogen fixation operons were
programmed to occur during heterocyst differentiation. These rearrangements were found to result from
the site-specific excision of 11,289-bp and 59,428-bp DNA elements from the open reading frames
(ORFs) of the nifD and fdxN genes, respectively. The xisA gene on the nifD element encodes a phage
integrase-family site-specific recombinase that is required for excision of the element. Excision of the
fdxN element requires three genes present on the element: xisF, which encodes a resolvase-family sitespecific recombinase, and the nearby xisH and xisI genes. Later, a third programmed rearrangement was
found to involve the excision of a 9,435-bp element from within the heterocyst-specific hupL gene. We
have recently shown that the xisC gene on the hupL element is required for programmed excision of the
element and that XisC and XisA are members of a distinct subclass of the phage integrase-family. During
diazotrophic growth, filaments of Anabaena PCC 7120 produce a developmental pattern of single
heterocysts separated by 10 to 15 vegetative cells. The patS gene encodes a 13 (or 17) amino acid peptide,
which is thought to serve as a diffusible cell-to-cell signal that contributes to the regulation of heterocyst
pattern by lateral inhibition. Overexpression of patS inhibits heterocysts, and a patS deletion mutant
forms multiple contiguous heterocysts and abnormally short vegetative-cell intervals between heterocysts.
Addition of sub-micromolar concentrations of a synthetic peptide representing the C-terminal 5 amino
acids of PatS (PatS-5, RGSGR) to growth medium inhibits heterocyst development. The current data
support a model in which PatS signaling is important for resolution of pairs and clusters of differentiating
cells. However, a variety of data indicate that factors other than PatS, such as the supply of nitrogen from
heterocysts and signals requiring the hetN gene, also must be involved in heterocyst pattern formation.
We are using several approaches to characterize the PatS signaling pathway. A set of patS minigenes
encoding only the last 4, 5, 6, 7, and 8 codons were constructed, and all except the smallest suppressed
heterocyst development. A GFP-PatS-5 fusion protein, a PatS-6His fusion, and two ORFs (other than
patS and hetN) that encode an internal RGSGR sequence all suppress heterocysts when overexpressed in
vegetative cells. These data are consistent with a PatS receptor located in the cytoplasm rather than on the
plasma membrane. Genetic screens for bypass suppressors of the patS overexpression phenotype have
identified several genes potentially involved in PatS signaling or the control of heterocyst development.
For example, strains containing a hetR R223W allele fail to respond to PatS or other pattern formation
signals, and overexpression of the R223W allele results in a lethal phenotype because all cells
differentiate within a few days after nitrogen step-down. Recent work from Zhou's lab indicates that HetR
binds DNA and that this activity is inhibited by PatS-5 pentapeptide, indicating that HetR is the PatS
receptor, which is consistent with our studies. The continued analysis of heterocyst development will
provide a better understanding of how a prokaryotic multicellular organism can perform both
photosynthetic carbon fixation and nitrogen fixation simultaneously.
25
Session II Oral Presentation Abstracts
The role of NtcA in heterocyst development and function.
ALICIA M. MURO-PASTOR*, ELVIRA OLMEDO-VERD, ANA VALLADARES,
ANTONIA HERRERO, and ENRIQUE FLORES
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Avda. Américo Vespucio 49, E-41092,
Seville, Spain.
Global nitrogen regulation is operated in cyanobacteria by the CRP family transcriptional
regulator NtcA, which activates or represses expression of genes whose products are involved in
nitrogen assimilation (1). In heterocyst-forming cyanobacteria, such as Anabaena sp. PCC 7120,
NtcA is also required for heterocyst development and diazotrophic growth (2, 3), thus linking the
process of heterocyst differentiation to nitrogen deprivation. NtcA is required for induction of
hetR, wich encodes one of the earliest-acting proteins that are required during the sequence of
events that leads to heterocyst differentiation (4, 5). In addition, because HetR is also required
for induction of ntcA during heterocyst development, expression of hetR and NtcA exhibits a
mutual dependency (6). Several genes whose expression is activated by NtcA during heterocyst
differentiation are also dependent on HetR. In order to investigate whether this double
dependence is actually operated at the transcriptional level via NtcA, with HetR being required
to increase NtcA levels, we constructed strains that over-express NtcA both in the wild-type and
hetR backgrounds. Increased levels of NtcA did not promote expression of heterocyst specific
genes in the presence of combined nitrogen, corroborating that the activity of the NtcA protein is
subjected to regulation in response to N availability (7). However, when the cells were subjected
to nitrogen deficiency, although mature heterocysts were not developed in the hetR background,
expression of the NtcA- and HetR-dependent devBCA operon (8) took place in both NtcA
overexpressing strains. Additionally, NtcA-overexpressing strains did not exhibit a requirement
for HetR for the excision of the nifD-intervening11-kb element. Thus, for the expression of some
genes, the requirement for HetR can be bypassed by increased levels of NtcA. The promoters of
some NtcA-activated genes involved in heterocyst differentiation or function, e.g., those of hetC,
devB or glnA, perfectly match the Class II canonical promoter defined for NtcA-activated genes
(9). However, as the NtcA regulon becomes larger, different promoter architectures are being
identified. Thus, the promoters of the coxBAC2 and coxBAC3 gene clusters, which encode two
different cytochrome oxidases required for diazotrophic growth and are expressed in an NtcAdependent manner (10), contain NtcA binding sites that are located further upstream than in
canonical NtcA-activated promoters. We are currently investigating whether the different
features found in NtcA-dependent promoters, together with the cellular level of active NtcA
protein, have a role in the determination of the hierarchy of gene activation during the process of
heterocyst differentiation.
(1) Herrero, A., A. M. Muro-Pastor, and E. Flores. 2001. J. Bacteriol. 183:411-425.
(2) Frías, J. E., E. Flores, and A. Herrero. 1994. Mol. Microbiol. 14:823-832.
(3) Wei, T.-F., T. S. Ramasubramanian, and J. W. Golden. 1994. J. Bacteriol. 176:4473-4482.
(4) Buikema, W. J., and R. Haselkorn. 1991. Genes Dev. 5:321-330.
(5) Black, T. A., Y. Cai, and C. P. Wolk. 1993. Mol. Microbiol. 9:77-84.
(6) Muro-Pastor, A. M., A. Valladares, E. Flores, and A. Herrero. 2002. Mol. Microbiol. 44:1377-1385.
(7) Luque, I., M. F. Vázquez-Bermúdez, J. Paz-Yepes, E. Flores, and A. Herrero. 2004. FEMS Microbiol. Lett.
in press.
(8) Fiedler, G., A. M. Muro-Pastor, E. Flores, and I. Maldener. 2001. J. Bacteriol. 183:3795-3799.
(9) Herrero, A., A. M. Muro-Pastor, A. Valladares, and E. Flores. 2004. FEMS Microbiol. Rev. in press.
(10) Valladares, A., A. Herrero, D. Pils, G. Schmetterer, and E. Flores. 2003. Mol. Microbiol. 47:1239-1249.
26
Session II Oral Presentation Abstracts
Arginine biosynthesis in Cyanobacteria is subjected to global nitrogen control
via PII signal transduction.
MANI MAHESWARAN, ANNETTE HEINRICH, ULRIKE RUPPERT and KARL
FORCHHAMMER*
Institut für Mikrobiologie und Molekularbiologie; Justus-Liebig Universität Giessen; IFZ
Heinrich-Buff-Ring 26-32; D 35392 Giessen
The first receptor protein for signal perception from the PII signal transduction protein in the
cyanobacterium Synechococcus elongatus PCC 7942 is described. To identify proteins that
interact with the PII signalling protein, a yeast two hybrid screening was conducted with glnB
encoding PII as bait against a S .elongatus PCC 7942 genomic library. Among the positive
clones, we found the argB product, encoding N-acetyl L-glutamate kinase (NAGK), the key
enzyme of the arginine biosynthetic pathway. The interaction between PII and NAGK was
confirmed by pull-down assays. To investigate complex formation between PII and NAGK in
more detail, gel-filtration experiments were conducted. PII and NAGK form a tight complex in
the absence of effector molecules and the unmodified seryl-residue 49 (the site of PII
phosphorylation) is critical for complex formation. Binding of PII to NAGK strongly affects the
catalytic activity of NAGK: The KM for N-acetylglutamate decreases and Vmax increases
considerably. Furthermore, feedback inhibition of NAGK activity by arginine was reduced
Titration of NAGK activity with PII and analytical ultracentrifugation experiments revealed that
one PII trimer binds to one hexameric NAGK. The role of metabolite effector on PII and NAGK
complex formation was studied using BIAcore and pull down experiments. The substantial
effects that could be observed by ATP, ADP, 2-oxoglutarate and divalent cations will be
reported.
In accord with the data that complex formation was impaired in PII mutants of Ser49, NAG
kinase activity in S. elongatus extracts correlated with the phosphorylation state of PII, with high
NAG kinase activities corresponding to non-phosphorylated PII (nitrogen-excess condition) and
low activities to increased levels of PII phosphorylation (nitrogen –poor condition), thus
subjecting the key enzyme of arginine biosynthesis to global nitrogen control.
1) Heinrich, A., Maheswaran, M. Ruppert,U and Forchhammer, K (2004)
Mol.Microbiology.52:1303-1314
*corresponding author
27
Session II Oral Presentation Abstracts
Developmental and metal regulation of the modA gene of Anabaena variabilis
ATCC 29413
BRENDA PRATTE and TERESA THIEL*
Department of Biology, University of Missouri-St. Louis, One University Drive, St. Louis MO
63121
Molybdenum (Mo) is a trace element required for the function of many important enzymes
including nitrogenase and nitrate reductase. Hence, many organisms have high-affinity Mo
transport systems to scavenge Mo when it becomes scarce. The high-affinity molybdate transport
system is an ABC-type transport system comprising four known genes: modA, modBC (fused
into one ORF in A. variabilis), and modE. ModA binds molybdate in the periplasm, ModBC
channels the molybdate into the cytoplasm of the cell using ATP, and ModE represses
transcription of modA and modBC in the presence of molybdate. Although the structural
components of the Mo-uptake system in A. variabilis closely resemble those found in E. coli, the
regulation of this system is more complex. In a modE mutant, the rate of 99Mo-uptake is 10-fold
higher in the presence of Mo than it is in the wild-type strain. However, in the modE mutant, the
rate of molybdate transport is only about 20% of the rate measured for mutant cells starved for
molybdate; therefore, other unknown factors also contribute to Mo-dependent repression of
molybdate transport. In order to better understand the transcriptional control of this transport
system, we constructed various size modA promoter::lacZ fusions to determine the specific
regions that are necessary for transcription and for repression by molybdate. ß-galactosidase
assays demonstrated a fragment spanning from –40 from the transcription start site to the start of
modA was required for modA expression. The smallest fragment capable of transcription showed
incomplete repression of ß-galactosidase activity in cells grown with either molybdate or
tungstate, indicting that the region most critical for repression by molybdate is downstream of
the –40 region. Complete repression of the modA transcription, however, required the region
–125 to –225 from the transcription start site. In situ fluorescence localization of ß-galactosidase
activity in filaments of A. variabilis has shown that the transcription of m o d A is also
developmentally controlled. The modA gene appears to be controlled by both a vegetative cell
promoter and heterocyst-specific promoter. Molybdate represses transcription of both
promoters.
28
Session II Oral Presentation Abstracts
Identification of Anabaena sp. strain PCC 7120 genes required specifically for
heterocyst formation and function
QING FAN1, GUOCUN HUANG1, SIGAL LECHNO-YOSSEF1, ELIZABETH WOJCIUCH1,
YI LI1, C. PETER WOLK*1, SATOSHI TABATA2, AND TAKAKAZU KANEKO2, 1MSU-DOE
Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, U.S.A. and
2
Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba, 292-0818, Japan
Anabaena sp. strain PCC 7120 (hereinafter, Anabaena) has been used extensively for genetic
studies of cell differentiation to form heterocysts, cells specialized for the fixation of N2 under
aerobic conditions. To facilitate the elucidation of mechanisms underlying Anabaena
differentiation, we seek to identify the complement of genes required specifically for heterocyst
formation and N2 fixation. We mutated Anabaena with transposon Tn5-1063 [1], which confers
resistance to neomycin and other antibiotics. Mutant colonies presumptively unable to fix
dinitrogen in the presence of oxygen (denoted the Fox- phenotype) were identified by their
persistent change of color from blue-green to yellow, and lack of protracted growth, in response
to nitrogen-deprivation. Such colonies were grown with nitrate supplemented with neomycin for
selection, and their DNA was extracted. DNA contiguous with the transposons was amplified by
inverse PCR, the PCR products were sequenced, and the sequences were localized within the
genome of Anabaena. We isolated 1652 such mutants and identified the chromosomal positions
of 1079 of them. In addition to known Fox genes, we identified many candidates for such genes.
We are using complementation and insertional mutagenesis to test for which of these mutants the
Fox-phenotype is really due to the transposon insertion. With few exceptions, complementation
experiments were carried out initially with BAC (bacterial artificial chromosome) clones
(conferring resistance to chloramphenicol and erythromycin) whose position was mapped as part
of the Anabaena sequencing project [2]. Conjugative plasmid pRL443 [3] and a mobilizable
methylating plasmid derived from pRL1124 [3] were introduced into Escherichia coli strain
DH10B bearing a BAC. The resulting strain was mated di-parentally with a corresponding
mutant, with selection for resistance to neomycin and erythromycin. To date, 76 mutated ORFs
not previously shown to be Fox genes have been complemented by the introduced clones. The 76
include 10 ORFs, each, in the Hep and Hgl islands [4], and 56 ORFs elsewhere in the genome.
Few ORFs that showed only a single transposon insertion were found to be Fox genes. Further
experiments are being carried out with potentially complementing, mobilizable plasmids, based
on RSF1010 or pDU1, that each carries only a single complete ORF. To date, we have finished
complementation tests of 62 ORFs with such single-gene constructs. Of these ORFs, 47 were
complemented, perhaps in some instances as a result of recombination. The other 15 are being
tested for complementation by downstream ORFs, and by a combination of the mutated ORFs
and their downstream ORFs. Presumptively regulatory genes identified as Fox genes include
alr0117 (recently reported [5]), all0187, alr1086, alll1711 and all2760, although not all possible
polar effects have been excluded.
1. Wolk, C.P., et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5355-5359 (1991). 2. Kaneko, T., et al., DNA Res. 8: 205-213
(2001). 3. Elhai, J., et al., J. Bacteriol. 179: 1998-2005 (1997). 4. Ehira, S., et al., DNA Res. 10: 97-113 (2003). 5.
Ning, D., and X. Xu, Microbiology. 150: 447-453 (2004).
29
Session II Oral Presentation Abstracts
Calcium is required for heterocyst differentiation in Anabaena sp. PCC7120
I. TORRECILLA, F. LEGANÉS, I. BONILLA, J.C. FERNÁNDEZ-MORALES & *F.
FERNÁNDEZ-PIÑAS
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid
28049, Spain
The impact of calcium signals in virtually all cells has lead to the study of its implication in
prokaryotic organisms as a stress response modulator. Cell differentiation in adverse conditions
is a common Ca2+- requiring response. Nitrogen starvation induces the differentiation of N2fixing heterocysts in the filamentous cyanobacterium Anabaena sp. PCC7120. Here we used a
recombinant strain of this organism, expressing the photoprotein aequorin (1), to monitor the
intracellular free calcium concentration during the course of heterocyst differentiation. A specific
calcium signature that is triggered exclusively when cells are deprived of combined nitrogen and
generated by intracellular calcium stores was identified. We manipulated the intracellular
calcium signal by treatment with specific calcium drugs, and subsequently assessed the effect of
such manipulation on the process of heterocyst differentiation. Suppression, magnification or
poor regulation of this signal prevented the process of heterocyst differentiation, thereby
suggesting that a calcium signal with a defined set of kinetic parameters may be required for
differentiation. Calcium transients in response to nitrogen stepdowns in the non differentiating
hetR and hetP mutant strains expressing apoaequorin are also under analysis.
1.
Torrecilla, I., Leganés, F., Bonilla, I. and Fernández-Piñas, F. 2000. Use of recombinant aequorin to study
calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria. Plant
Physiology 123:161-175.
*
Author for correspondence; email: [email protected]
30
Session III Oral Presentation Abstracts
Session III: Physiology, Metabolism and Global
Responses (II)
Session Chair: Francis X. Cunningham, Jr., University
of Maryland
31
Session III Oral Presentation Abstracts
Study on the transcription of genes encoding for putative peptide synthetases
in Anabaena PCC7120.
R. J
JEANJEAN, C.-C. ZHANG
LCB-CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
The genome of Anabaena PCC 7120 has been sequenced, showing the presence of several
clusters of genes encoding microcytin synthetases, peptide synthetases and polyketide synthases
(for example: all2643, all1649 etc). Nethertheless, Anabaena is known as a non-toxic strain
which raises some question about the function of these genes and their expression. We have
undertaken a study on the environmental conditions that activates the transcription of these
genes. Semi-quantitative RT-PCR allowed us to estimate the level of transcription of the genes
of the different clusters in total RNA extracted from cells having undergone several stress
(starvation for iron, growth with molecular nitrogen, high light, salt stress, heat shock etc..). The
trancripts were generally at a very low basal level under usual growth conditions and they were
significantly increased when cells were grown in a medium depleted of iron.
32
Session III Oral Presentation Abstracts
Characterisation of three adenylate cyclase Nostoc punctiforme ATCC 29133
mutants shows phenotypic disparity
K.E.CHAPMAN1*, P.S.DUGGAN1, N.A.JOSEPH2 & D.G.ADAMS1
1) School of Biochemistry & Microbiology, University of Leeds, Mount Preston Street,
Leeds, LS2 9JT, UK.
2) Tecan UK, Theale Court, 11-13 High Street, Theale, RG7 5AH, UK.
Adenylate cyclase (AC), encoded by the cya gene, is an enzyme that catalyses the formation
of the cyclic nucleotide, adenosine 3’,5,-cyclic monophosphate (cAMP) from ATP (1). This
cyclic nucleotide is an important molecular messenger in both prokaryotic and eukaryotic
organisms, mediating signals from outside the cell to target proteins that regulate gene
expression, cell division and / or enzyme activity (2). cAMP is widely distributed in bacteria and
although large amounts of the nucleotide have been detected in cyanobacteria, in particular those
with filamentous morphology, physiological roles have yet to be confirmed (2). Six potential
ACs have been identified in Anabaena PCC 7120, five (cyaA, cyaB1, cyaB2, cyaC & cyaD)
through complementation analysis (3) & most recently, cyaE by searching the Anabaena genome
project (4 & 5). All have catalytic domains near the C-terminal region with significant structural
similarity to the catalytic domains of eukaryotic ACs but have different regulatory components
from each other upstream of the catalytic domain (3). It is apparent that, like Anabaena PCC
7120, Nostoc punctiforme has several putative adenylate cyclase genes, including cyaA, cyaB,
cyaC & cyaD. cyaC encodes somewhat of a novel protein, consisting of a number of distinct Nterminal domains including two response regulator domains & one histidine kinase domain (3).
Work conducted at the University of Leeds (6), whereby a N. punctiforme cyaC mutant (H1) was
generated through transposon mutagenesis, suggests a possible role for CyaC in the control of
cellular differentiation, as well as in the formation of plant-cyanobacterial symbiotic
associations. Subsequently, a site-directed mutant (KC1) was created by inserting a kanamycin
resistance gene into cyaC close to the N-terminus. Unlike H1, which infected the liverwort
Blasia pusilla at a significantly higher rate than WT 29133, KC1 infected the plant auricles at
low frequency. The domain disrupted by the transposon Tn5 in H1 was the proposed catalytic
domain of cyaC and so a second mutant (KC2) was created, whereby the kanamycin resistance
gene was inserted towards the C-terminus within the putative catalytic domain. Physiological
analysis unveiled similarities between KC2 and H1, both infecting
Blasia at a significantly higher frequency than wild-type or KC1. cAMP assays,
complementation analysis and cyaC expression studies are being carried out in order to
investigate these phenotypes further, with the ultimate aim of elucidating possible explanations
behind this phenotypic disparity.
1)
2)
3)
4)
5)
6)
Botsford, J.L. & Harman, J.G. (1992). Cyclic AMP in prokaryotes. Microbiol Revs; 56: 100-22
Ohmori, K. & Ohmori, M. (1993). J Gen Appl Microbiol; 39: 247-50
Katayama, M. & Ohmori, M. (1997). J Bacteriol; 179: 3588-93
Kasahara et al. (2001). J Biol Chem; 276: 10564-10569
Ohmori et al (2001). DNA Res; 8: 271-284
Joseph, N.A. (2001). PhD Thesis, University of Leeds
33
Session III Oral Presentation Abstracts
Pervasive cyanophage in a Laurentian Great Lakes: applications of molecular
techniques to gain insight on their distribution and ecology
MATHEW J. CARBERRY and STEVEN W. WILHELM*
Department of Microbiology, The University of Tennessee, Knoxville, TN 37996
Viruses play important roles in aquatic microbial communities. Although marine viruses have
received significant attention in recent years, freshwater viruses have received comparatively
little attention, despite the importance of our freshwater resources. Invasive species have also
received significant attention from the scientific community, but the focus has been on
macrofaunal invasions, with virtually no attention paid to microbial invaders. During recent
studies of the viral community in Lake Erie, we have discerned the presence of viruses capable
of infecting marine cyanobacteria of the genus Synechococcus. These viruses appear to be well
distributed throughout the lake. Preliminary sequence analysis suggests a relationship between
this new virus group and previously characterized marine cyanomyoviridae. Applications of
quantitative polymerase chain reaction (qPCR) to this system allow researchers to quantify the
copies of specific genes in a sample and infer the abundance of these viruses in the absence of
cumbersome infection assays. Data obtained from these experiments will be presented in
comparison with classical enumeration techniques to assess the effectiveness of qPCR for
enumeration of active cyanobacterial viruses.
34
Session III Oral Presentation Abstracts
Evidences for two novel phycobilisome linkers in the marine cyanobacterium
Synechococcus sp. WH8102, impact of high light and ultraviolet radiations on
the phycobilisome structure and composition.
1*
Christophe SIX, 2Jean-Claude THOMAS, 3Brian PALENIK, 4Yves LEMOINE, 1Frederic
PARTENSKY
1
Station Biologique, UMR 7127 CNRS and University Paris 6, 29682 Roscoff, France.
Ecole Normale Superieure, UMR 8543, Laboratoire Organismes Photosynthetiques et
Environnement. 75230 Paris, France.
3
Marine Biology Research Division, Scripps Institution of Oceanography, University of
California, La Jolla, CA 92093-0202, USA.
4
Equipe de Phycologie et Production Primaire, UMR CNRS-USTLille 8013 ELICO Villeneuve
d'Ascq France.
2
The recent release by the Joint Genome Institute of the whole genome sequence of
Synechococcus sp. WH8102 provides unprecedented insights into the structure of a marine
Synechococcus phycobilisome (PBS). Genome analyses showed that PBS rods of WH8102 are
constituted by one type of phycocyanin (R-PCII) and two types of phycoerythrins (C-PEI and CPEII) which is a unique feature of marine Synechococcus. Absence of cpcC and cpcD genes
further suggests that there is only one disc of R-PCII per rod. Genome analyses alsoreveal the
presence of 9 genes encoding putative linker polypeptides. Five of these linkers (including two
novel ones) are likely phycoerythrin (PE)-associated. Biochemical analyses showed that at least
8 predicted linkers were present and that four of them were chromophorylated, including the two
new ones. The first one (SYNW2000) is unusually long (548 residues) and apparently results
from the fusion of homologues of MpeC, a phycoerythrin-II linker, and of CpeD, a
phycoerythrin-I linker, therefore suggesting that SYNW2000 may coordinate the junction
between PEI and PEII hexamers. The second one (SYNW1989) has a more classical size (300
residues) and is also a MpeC homologue. The effect of growth irradiance on the structure and
pigmentation of these PBSs was also investigated. Besides evidences of a decrease of PE in the
PBS with increasing culture irradiances, we also observed a weak but significative decrease of
the phycoerythrobilin (PEB) to phycourobilin (PUB) ratio at high light which can be interpreted
as a decrease in the PEII to PEI ratio. This indicates that PBS acclimation to high light involves a
partial degradation of PEII, the most distal part of PBS, as confirmed by observation of a
decrease in the relative proportion of MpeC with regard to all other linkers. Effects of UV
radiations on the PBS structure and composition have also been investigated.
Corresponding author : Christophe SIX.
35
Session III Oral Presentation Abstracts
Evidences for two novel phycobilisome linkers in the marine cyanobacterium
Synechococcus sp. WH8102, impact of high light and ultraviolet radiations on
the phycobilisome structure and composition.
1*
Christophe SIX, 2Jean-Claude THOMAS, 3Brian PALENIK, 4Yves LEMOINE, 1Frederic
PARTENSKY
1
Station Biologique, UMR 7127 CNRS and University Paris 6, 29682 Roscoff, France.
Ecole Normale Superieure, UMR 8543, Laboratoire Organismes Photosynthetiques et
Environnement. 75230 Paris, France.
3
Marine Biology Research Division, Scripps Institution of Oceanography, University of
California, La Jolla, CA 92093-0202, USA.
4
Equipe de Phycologie et Production Primaire, UMR CNRS-USTLille 8013 ELICO Villeneuve
d'Ascq France.
2
The recent release by the Joint Genome Institute of the whole genome sequence of
Synechococcus sp. WH8102 provides unprecedented insights into the structure of a marine
Synechococcus phycobilisome (PBS). Genome analyses showed that PBS rods of WH8102 are
constituted by one type of phycocyanin (R-PCII) and two types of phycoerythrins (C-PEI and CPEII) which is a unique feature of marine Synechococcus. Absence of cpcC and cpcD genes
further suggests that there is only one disc of R-PCII per rod. Genome analyses alsoreveal the
presence of 9 genes encoding putative linker polypeptides. Five of these linkers (including two
novel ones) are likely phycoerythrin (PE)-associated. Biochemical analyses showed that at least
8 predicted linkers were present and that four of them were chromophorylated, including the two
new ones. The first one (SYNW2000) is unusually long (548 residues) and apparently results
from the fusion of homologues of MpeC, a phycoerythrin-II linker, and of CpeD, a
phycoerythrin-I linker, therefore suggesting that SYNW2000 may coordinate the junction
between PEI and PEII hexamers. The second one (SYNW1989) has a more classical size (300
residues) and is also a MpeC homologue. The effect of growth irradiance on the structure and
pigmentation of these PBSs was also investigated. Besides evidences of a decrease of PE in the
PBS with increasing culture irradiances, we also observed a weak but significative decrease of
the phycoerythrobilin (PEB) to phycourobilin (PUB) ratio at high light which can be interpreted
as a decrease in the PEII to PEI ratio. This indicates that PBS acclimation to high light involves a
partial degradation of PEII, the most distal part of PBS, as confirmed by observation of a
decrease in the relative proportion of MpeC with regard to all other linkers. Effects of UV
radiations on the PBS structure and composition have also been investigated.
Corresponding author : Christophe SIX.
36
Session III Oral Presentation Abstracts
Acid stress response in two strains of Synechocystis species
K.M. SHEA, T.N. NGUYGEN, C.Z. YANONNI, J.J. HUANG and M.M. ALLEN
Department of Biological Sciences, Wellesley College, Wellesley, MA 02481, U. S. A.
The aim of this research is to characterize and compare the acid stress response in
Synechocystis sp. strains PCC 6803 and 6308. Exponentially growing cells were transferred to
either buffered or unbuffered BG-ll media ranging in pH, and growth and external pH were
monitored over time. Both strains are incapable of growing in media with a pH of less than six,
and they have a cell density-dependent ability to increase the external pH when placed at pH 4 or
above. Measurement of ammonium concentration in unbuffered medium after acid shock shows
that ammonia is excreted by cells, causing an increase in external pH. Neither strain appears to
have an acid tolerance response since growth at mildly acidic pH does not increase the ability of
cells to later grow in media with a pH lower than 4. Protein synthesis during acid stress, in
medium buffered at pH 5.5, was studied using one-dimensional and two-dimensional gel
electrophoresis, as well as autoradiography. One-dimensional autoradiography showed that
many proteins, including phycocyanin, were differentially expressed. Data analysis of 2D gels,
using Compugen Z3 software, identified approximately 100 proteins that exhibit differential
expression in Synechocystis sp. strain 6803. MALDI-TOF-MS analysis indicated that the
expression of the protein synthesis elongation factor Tu increases with acid stress while DnaK
shows variable expression. Additional differentially expressed proteins are being identified by
mass spectrometry.
*
M.M. Allen
37
Session III Oral Presentation Abstracts
Characterization of mrpA, a gene with roles in pH adaptation and resistance
to Na+ in the cyanobacterium Anabaena sp. PCC7120.
A. BLANCO-RIVERO, F. FERNÁNDEZ-PIÑAS, E. FERNÁNDEZ-VALIENTE & *F.
LEGANÉS
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid
28049, Spain
Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain,
PHB11, that grew poorly at external pH values above 10. The mutant strain was also sensitive to
increasing Na+ concentrations and this sensitivity was higher under basic conditions. Mutant
strain PHB11 also showed a significant inhibition of both photosynthesis and respiration,
independently of the external pH, although the inhibition became more pronounced under
alkaline pH. Interestingly; the mutant was also unable to fix N2 both under aerobic and anaerobic
conditions, differentiating immature heterocysts. Reconstruction of the transposon mutation of
PHB11 in the wild type strain reproduced the phenotype of the original mutant. The wild type
version of the mutated gene was cloned and the mutation complemented. In vivo expression
studies indicated that mrpA is induced with increasing external Na+ concentrations and alkaline
pH. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven
ORFs operon with significant homology to a family of bacterial operons that are believed to code
a novel multiprotein cation/proton antiporter involved in pH adaptation and resistance to salt
stress. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation) following
the nomenclature of the Bacillus subtilis operon; based on the highest homology with the
Bacillus mrp genes, the ORF mutated in PHB11 was denoted mrpA. The mrp operon is also
present in the genome of the unicellular Synechocystis sp. PCC6803, Thermosynechococcus
elongatus BP1 and Synechococcus sp. PCC7942 , the marine filamentous Trichodesmium
erythraeum and the heterocystous Anabaena variabilis but is absent from the marine unicellular
Prochlorococcus marinus strains MED4 and MIT91313, Synechococcus WH-8102, the
thylakoid-less Gloeobacter violaceus and the heterocystous Nostoc punctiforme. The
cyanobacterial operons differ from the bacterial ones in that gene arrangement within the operon
is mrpCDEFGAB instead of mrpABCDEFG; also mrpA is fourfold shorter than its bacterial
homologues. Computer analysis suggested that all seven predicted Anabaena Mrp proteins were
highly hydrophobic with several transmembrane domains; in fact, the predicted protein
sequences coded by mrpA, mrpB and mrpC show a clear homology to hydrophobic subunits of
the proton pumping NADH:ubiquinone oxidoreductase (Complex I). The Synechocystis sp
PCC6803 mrp operon consists of 9 ORFs instead of 7 probably due to duplication of ORFs C
and D. Interestingly; Wang and coworkers (1) found in Synechocystis that two ORFs of a large
transcriptional units were upregulated in response to a CO2 downshift. We have identified the
two ORFs as mrpD and its probable duplicate and have found that Anabaena mrpA is also
induced twofold under Ci limitation. We propose, as it has been postulated in heterotrophic
bacteria, that the cyanobacterial Mrp complex may be an unusual multicomponent cation/proton
antiporter energized (al least partially) by electron transport through the subunits that resemble
the hydrophobic core of complex I. Finally, the Na gradient created may drive nutrient uptake
i.e. HCO3-, as suggested in Synechocystis (1).
1.
*
Wang H-L., Postier B. L. and Burnap R.L. 2004. The Journal of Biological Chemistry 279:5739-5751.
Author for correspondence; email: [email protected]
38
Session III Oral Presentation Abstracts
Special Workshop
BioLingua, a knowledge-base and programming environment
for the analysis of cyanobacterial genes and genomes
JEFF ELHAI,1*, JP MASSAR,2 JEFF SHRAGER,3 MIKE TRAVERS,4
AUSTIN HESS,5 JAMES MASTROS,1 SARAH COUSINS,1 MATTHEW BERGINSKI6
1
Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond
VA 23284; [email protected]; 3Dept. of Plant Biology, Carnegie Institute of Washington,
Stanford CA 94305; 4MDL Information Systems, San Leandro CA 94577; 5Dept. of Biology,
Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; 6School of Biology,
Georgia Institute of Technnology, Atlanta GA 30332
BioLingua (1) puts in one place, behind one interface, all information we can gather
concerning cyanobacteria whose genomes have been at least partially sequenced. This includes
genomic information and annotation, of course, but also knowledge about metabolism and mass
experimental results. Just as important, it provides a simple language that enables you to access
and analyze the information in a way that (unless you're an expert programmer) would not have
been possible to you before. The workshop will illustrate the simplicity of the language and
power to answer important biological questions through several examples. Examples include:
(a) What are the characteristics of genes that are found in heterocystous cyanobacteria but
not other cyanobacteria?
(b) What cyanobacterial proteins are induced under conditions of high intensity light that are
within a given size class?
(c) Can upstream sequence motifs be identified that are unusually common amongst those
genes upregulated by nitrogen starvation?
Other examples will be taken from the interests of the participants. BioLingua is freely available online (2) to the
cyanobacteriological community, along with an extensive description of its capabilities and its use (3).
1.
Massar JP, Travers M, Elhai J, Shrager J (2004). BioLingua: A Biologist's Computational `Workbench.
Submitted to Bioinformatics.
BioLingua Server, Cyanobacterial Edition. http://nostoc.stanford.edu:8002/biologin
BioLingua Help. http://ramsites.net/~biolingua/help/
2.
3.
*
Jeff Elhai, Center for the Study of Biological Complexity, 1000 W. Cary St., Virginia Commonwealth University,
Richmond VA 23284. E-mail: [email protected]; Tel: 1-804-828-0794
39
Session IV Oral Presentation Abstracts
Session IV: Photosynthesis and responses to light
Session Chair: John Cobley, University of San
Francisco
40
Session IV Oral Presentation Abstracts
Promoter analysis of genes encoding subunits of photosystem I in
Synechocystis sp. PCC 6803
1
1
2
MURAMATSU, M., 1 SONOIKE, K. and 2Y. HIHARA*
Department of Integrated Biosciences, The University of Tokyo, Japan,
Department of Biochemistry and Molecular Biology, Saitama University, Japan,
In cyanobacteria, the amount of photosystem
(PS) I complex decreases under high-light
conditions. This decrease seemed to be mainly achieved by the coordinate down-regulation of
promoter activities of PS I genes in Synechocystis sp. PCC 6803. Namely, upon the shift of the
cells to high-light conditions, transcript levels and promoter activities of PSI genes were downregulated to 5-10% of the level of low-light conditions (1). On the other hand, when cells were
returned to low-light conditions, transcript levels and promoter activities of PSI genes rapidly
recovered. However, the mechanism by which PS I promoter activities are modulated is totally
unknown. In this study, we investigated promoter architectures of psaAB genes encoding
reaction center subunits and psaD gene encoding a small subunit of PS I. As a first step, the
transcriptional start points of psaAB and psaD genes were determined. There existed two major
transcriptional start points in psaA gene, -45 and –144 relative to the translational start point
(+1), and a single major transcriptional start point at –35 in psaD. Next, to identify cis-regulatory
elements, various fragments of upstream region of psaAB and psaD were fused to luxAB reporter
gene, and luminescence levels from each construct were compared. It was found that psaAB had
two promoters (P1, P2) corresponding to each of the two transcriptional start points, and each
promoter possessed positive and negative cis-elements. psaD gene also had two promoters (P1,
P2), although we could not identify transcriptional start point corresponding to P1. DNA
mobility shift assay showed that both promoters of psaAB bound protein factor(s) but we could
not detect any specific protein binding in psaD promoters so far. Furthermore, we found at least
three light-responsive elements in psaAB promoter region. The molecular mechanism of light
response of PSI genes will be discussed.
(1) Muramatsu and Hihara, Planta 216: 446-453, 2003
* Corresponding author
41
Session IV Oral Presentation Abstracts
Identification of a cis-acting element involved in negative control of the hliA
gene of cyanobacteria in response to high light
ANTHONY D. KAPPELL and *LORRAINE G. VAN WAASBERGEN.
Department of Biology, University of Texas at Arlington, Arlington, TX 76019
The high-light-inducible proteins (HLIPs) of cyanobacteria are involved in protecting the
cells from high-intensity light (HL). The hli genes encoding the HLIPs are expressed in response
to HL or low-intensity blue/UV-A light (a response that appears to be related to the HL
response). We undertook an analysis of the region upstream of hliA from Synechococcus
elongatus PCC 7942 for cis-acting elements involved in regulation of the gene. Expression of
hliA was monitored following various deletions and mutations made to the upstream region of
gene fused translationally to a GUS reporter gene on a plasmid that replicates autonomously in
the cyanobacterium. Constructs with deletions to -134 (relative to the transcriptional start site)
showed no significant difference in expression from the undeleted construct in low light (LL),
HL, or UV-A light. However, a deletion to –25 (on the plasmid pHG-del) or an 18-bp Pho box
inserted (originally for other purposes) between -30 and –28 resulted in 8-9 fold higher
expression of the gene in LL and 2-3 fold higher expression in HL and UV-A light, suggesting
that these changes interrupted the binding of a repressor protein that is most active in LL. A
substitution at -26 and -27 from TT to CG (on the plasmid pHG-stu) practically abolished
expression under all light conditions, suggesting that the change resulted in increased affinity for
binding of the putative repressor (or decreased ability for RNA polymerase to bind to the
promoter). Partial deletion by interposon mutagenesis of the apparently essential NblS sensor
kinase known to control hliA expression resulted in enhanced expression of the gene in LL, HL
and UV-A light, further suggesting that hliA is under negative control (and that NblS is involved
in that control). Gel mobility shift assays were done to identify the activity of DNA-binding
proteins using the sequence from -34 to -17 of the hliA gene (and equivalent 18-bp regions of the
altered sequences present in pHG-del and pHG-stu) and crude protein extracts from cells grown
in LL and HL. The assays showed a commonly shifted band in each case, with the shifted
complex being more abundant in LL than in HL. The abundance of the complex was much less
in both LL and HL using the pHG-del sequence than using the wild-type sequence and much
greater in LL using the pHG-stu sequence. These results support the hypotheses regarding the
binding of a repressor protein to the sequences based on the GUS assays. Similar mobility shift
assay results were obtained using Synechocystis sp. strain PCC 6803 extracts and a significantly
similar 18-bp sequence located in the regions upstream of several hli genes from Synechocystis,
indicating the conserved nature of this HL-responsive element. We are currently involved in
attempts to identify the putative repressor protein that binds to this site.
*Author for correspondence; email: [email protected]
42
Session IV Oral Presentation Abstracts
PsoR is a regulator of phycobilisome abundance in the cyanobacterium,
Fremyella diplosiphon.
JOHN COBLEY*, QIAN WANG, KATRINA SHIEH, JENNIFER SEKI & JEFFREY ODA.
Dept. of Chemistry, University of San Francisco, 2130, Fulton St., San Francisco, CA 94117, USA.
In Fremyella diplosiphon green light induces the synthesis of phycoerythrin (PE) and represses
the synthesis of phycocyanin (PC). This acclimation to the color of ambient light results in
maximum light absorption for photosynthesis, and has been shown to involve the green-lightregulated transcription of four operons. Members of a class of mutants of F. diplosiphon ("blue"
mutants) overproduce phycobilisomes (PBSs).
Strain F. diplosiphon SF2412-15 is a
characteristic "blue" mutant, and was generated by transposon mutagenesis (1). Recovery of
the transposon from F. diplosiphon SF2412-15, followed by sequencing of the DNA flanking the
transposon has led to the identification of an ORF which we have called psoR (phycobilisome
abundance regulator). Clones with the characteristic "blue" phenotype could be recreated when
a frameshifted allele of psoR was exchanged into the wild-type genome by homologous
recombination (2). PCR analysis of genomic DNA from the recreated "blue" clones has
demonstrated that, in these strains, allelic segregation is complete. Fluorescence emission
spectra at 77K show that the transposon mutant and the engineered psoR - strains all overproduce
PBSs to a similar extent. Conversely, overexpression of psoR+ from a shuttle plasmid
replicating in F. diplosiphon + resulted a dramatic 10-20 fold decrease in the PBS/chlorophyll a
ratio. Collectively these data suggest that PsoR is an important negative regulator of PBS
abundance. Considerable genetic information has already been obtained about the regulation of
PBS degradation (3). psoR is the first gene implicated the regulation of PBS abundance.
Genes highly similar to psoR are found in the genomes of all PBS-containing cyanobacteria for
which a genome sequence is available, with two exceptions; Gloeobacter violaceus PCC 7421
and Synechococcus sp. WH8102. No gene similar to psoR is present in any of the three
Prochlorococcus species for which a genome sequence is available. An NCBI Conserved
Domain Search predicts PsoR from F. diplosiphon (554 amino acids) to be "an exonuclease of
the beta-lactamase fold involved in RNA processing" (COG1236, with random expectation of 2
x 10-57). Our data strongly suggest that PsoR plays a crucial a role in a post-transcriptional step
in the regulation of phycobilisome synthesis.
Pleiotropy can be especially valuable in helping to understand relationships between different
regulatory processes. The psoR - mutants of F. diplosiphon discussed above show two changes
in phenotype; not only do these mutants overproduce phycobilisomes (PBSs), but they are also
strongly impaired in the synthesis of PE in response to green light. This pleiotropy of psoR mutants suggests that, in F. diplosiphon, the regulation of phycobilisome abundance and the
regulation of phycobilisome composition (by the color of available light) are integrated
responses, and that a step which is key for both responses occurs at the level of RNA
metabolism.
(1) Cobley, J.G., Seneviratne, L., Drong, L., Thounaojam, M., Oda ,J.F., and Carrol, J. (1999) Transposition of Tn5
derivatives in the chromatically adapting cyanobacterium, F. diplosiphon. In The Phototrophic Prokaryotes.
Peschek, G., Löffelhardt, W., and Schmetterer, G. (eds). New York: Kluwer Academic/Plenum, pp. 443-451.
(2) Cobley, J.G., Clark, A.C., Weerasurya, S., Queseda, F.A., Xiao, J.Y., Bandrapali, N., D'Silva, I., Thounaojam,
M., Oda, J.F., Sumiyoshi, T., and Chu, M.-H. (2002) CpeR is an activator required for expression of the
phycoerythrin operon (cpeBA) in the cyanobacterium, Fremyella diplosiphon, and is encoded in the phycoerythrin
linker-polypeptide operon (cpeCDESTR). Mol Microbiol 44: 1517-1532.
(3) Grossman, A.R., Bhaya, D., and He, Q. (2001) Tracking the light environment by cyanobacteria and the dynamic
nature of light harvesting. J Biol Chem 276: 11449-11452.
43
Session IV Oral Presentation Abstracts
LdpA: a component of the circadian clock senses redox state of the cell
NATALIA B. IVLEVA1, MATT R. BRAMLETT2, PAUL A. LINDAHL2 and SUSAN S.
GOLDEN1*
1
Department of Biology and 2Department of Biochemistry and Biophysics, Texas A&M
University, College Station, Texas 77843, USA
Cyanobacteria are the only bacteria that have been shown to have circadian rhythmicity, an
endogenously controlled oscillation of physiological activities with a period of roughly 24 hours.
The circadian rhythms in the cyanobacterium Synechococcus elongatus PCC 7942 and other
organisms are entrained by a variety of environmental factors. In cyanobacteria the mechanism
of transduction of environmental input signals to the central oscillator of the clock is not known.
Our earlier study identified ldpA as a gene involved in light-dependent modulation of the
circadian period. Here we show that the LdpA protein contains two 4Fe-4S clusters and is redox
sensitive. Affinity purification demonstrated that LdpA copurifies with proteins previously
shown to be a part of circadian control. The data suggest that LdpA is able to sense the redox
state of a cell and is an intrinsic component of the ‘clockosome’ complex. These findings reveal
a novel input pathway to the circadian oscillator.
* Corresponding author
44
Session IV Oral Presentation Abstracts
Global functional analysis of circadian clock genes in Synechococcus elongatus
PCC 7942: strategy and progress
C. KAY HOLTMAN, YOU CHEN, PAMELA SANDOVAL, ALEJANDRA GONZALES,
MARK S. NALTY, PHILIP A. YOUDERAIN, and SUSAN S. GOLDEN*
Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA
The cyanobacteria are the first prokaryotes shown to possess a circadian clock, an internal
timing system that anticipates daily environmental changes, thereby allowing the organism to
adjust physiological activities for better performance. Most studies of cyanobacterial circadian
rhythms have employed Synechococcus elongatus PCC 7942, a unicellular fresh water obligate
photoautotroph. Several genes necessary for clock function were initially identified in PCC 7942
and subsequently found to be widespread among cyanobacteria, including kaiABC, a locus that
encodes the components of the central oscillator. However, the molecular mechanism of the
clock is still not clear. We aim to identify all genes required for clock function in cyanobacteria
to ascertain how the clock ticks at the molecular level, and to understand the physiological
significance of circadian rhythms. We have undertaken a transposon-mediated mutagenesis and
sequencing strategy to isolate insertional mutants of essentially every PCC 7942 gene. By
identifying sequence surrounding transposon insertions in a cosmid library of PCC 7942 we have
identified positions of insertions in 28 cosmids, which cover roughly 920 kb, or 34%, of the
genome. Integration of these data with the complete genome sequence, recently closed and
finished by the Joint Genome Institute, has allowed us to annotate the locations of all insertions
and identify specific clones for mutagenesis to complete the project. Transposon insertions have
been crossed into the PCC 7942 genome for inactivation of approximately 500 genes and the
resulting mutants have been screened for circadian phenotypes. Insertions in genes clpP2 and
clpX resulted in merodiploid cells with long circadian period phenotypes. We also identified
several other loci that show changes in circadian rhythm properties. At the end of the project,
archived mutagenesis templates will be available for nearly every locus. These functional data,
paired with the genome sequence and opportunities for cyanobacterial comparative genomics,
provide a valuable resource for understanding cyanobacterial physiology.
*Author for correspondence; email: [email protected]
45
Session IV Oral Presentation Abstracts
The mechanism of circadian regulation of gene expression in cyanobacterium
Synechococcus elongates PCC 7942.
MASATO NAKAJIMA, ALI AZAM TALUKDER, TAEKO NISHIWAKI, HIDEO IWASAKI,
*TAKAO KONDO
Division of Biological Science, Graduate School of Science, Nagoya University, Core Research
for Evolutional Science and Technology (CREST),
Circadian rhythms, biological oscillations with a period length of about 24 h, are found
ubiquitously among eukaryotes. In prokaryotes, only Cyanobacteria are known to have the
circadian clock. A gene cluster composed of kaiA, kaiB and kaiC has been cloned as central
components of cyanobacterial clock in the unicellular cyanobacterium Synechococcus elongatus
strain PCC 7942 (1). KaiC and KaiA likely act as negative and positive elements in the
molecular feedback loop of kaiBC expression, respectively. In eukaryotic models, cis-acting
elements and trans-acting factors are thought to be clock specific components. However, the
mechanism by which Kai proteins generate circadian rhythm still remains unknown. It was
recently reported that almost all of gene expressions in cyanobacteria showed circadian rhythm,
suggesting that the cyanobacterial clock system was genome-wide (2). Indeed, re-examination of
locations of Kai proteins clarified the association of Kai proteins with nucleoid, which was a
complex of genomic DNA with proteins. These associations were weak to release by washing
with low salt. Moreover, we could not detect associations of Kai proteins with DNA. Hence, the
mechanism of cyanobacterial circadian clock may be different from that of eukaryotic clock. We
will discuss, based on genetic and biochemical results, the mechanism of genome-wide circadian
gene expression.
(1) Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johonson, C.H. and
Kondo, T. (1998) Science, 281, 1519-1523
(2) Nakahira, Y., Katayama, M., Miyashita, H., Kutsuna, S., Iwasaki, H., Oyama, T. and Kondo, T. (2004) Proc.
Natl. Acad. Sci. USA, 101, 881-885
*corresponding author. E-mail: [email protected]
46
Session IV Oral Presentation Abstracts
Circadian mechanisms in cyanobacteria
Yao Xu, Tetsuya Mori, Mark.Woelfle, Carl H. Johnson
Department. of Biological Sciences, Vanderbilt University, Nashville, TN 37235 USA
Circadian (daily) rhythms are endogenous oscillations of biochemical, cellular,
developmental, and behavioral activities found in a wide variety of organisms, including the
prokaryotic cyanobacteria. The molecular machinery that permits the measurement of time is
referred as the “biological clock” or “circadian clock”. The fundamental properties of circadian
clocks in eukaryotes and in cyanobacteria are the same: surprisingly precise self-sustained
oscillations with an approximately 24 h period that are temperature compensated and entrainable
by environmental cycles. The ultimate explanation for the mechanism of these unusual
oscillators will require characterizing the structures, functions, and interactions of the molecular
components of circadian clocks. We have used the simplest model system, the cyanobacterium
Synechococcus elongates, to elucidate common themes and mechanisms in circadian
timekeeping systems. The circadian clock in cyanobacteria globally regulates the expression of
essentially all promoters in the organism. A cluster of three circadian clock genes encodes the
essential circadian clock components KaiA, KaiB and KaiC that interact each other and comprise
of a feedback loop. The KaiB and KaiC protein levels display robust rhythms, whereas the KaiA
protein abundance undergoes little circadian oscillation. KaiC appears to be the central protein
and forms the core of the KaiABC clock protein complex. Induction of the kaiC gene at specific
phases elicits phase resetting. KaiC can exist in both phosphorylated and non-phosphorylated
forms in vivo, and its phosphorylation status and the degradation rate are correlated with clock
speed in vivo. KaiC can auto-phosphorylate and auto-dephosphorylate in vitro. KaiA and KaiB
modulate the phosphorylation status of KaiC in vitro and in vivo: KaiA enhances KaiC
phosphorylation (and/or inhibits its dephosphorylation), while KaiB antagonizes the effects of
KaiA. KaiC forms hexameric ring structures that are reminiscent of related proteins that act
directly upon DNA; it can bind forked DNA substrates. The structure of KaiC reveals the
functional insights toward ATP binding, Kai-protein complex formation, and autophosphorylation sites, etc. We also addressed the adaptive significance of circadian rhythmicity
by testing the relative fitness under competition between various strains of cyanobacteria
expressing different circadian periods. Strains that had a circadian period similar to that of the
light/dark cycle were favored under competition. Arhythmic strains could not compete well
against wild-type strains in light/dark cycles, but they could compete effectively in constant
light. Our studies on circadian programming in cyanobacteria will be interpreted in the context of
a new model for the cyanobacterial clock in which circadian gene expression is orchestrated by
rhythmic structural changes in the chromosome that are in turn mediated by rhythmic changes in
the activity of a KaiC-containing complex.
47
Session V Oral Presentation Abstracts
Session V: General Structural Aspects
Session Chair: Cheryl Kerfeld, University of
California at Los Angeles
Keynote Speaker: Petra Fromme, Arizona State
University:
“Structure and Function of Photosystem I and II"
48
Session V Oral Presentation Abstracts
Keynote Talk
Structure and Function of Photosystem I and II
PETRA FROMME
Department of Chemistry and Biochemistry Physical Science Building
PS-C155 Arizona State University Tempe, AZ 85287-1604 USA
Photosystem I is the most complex membrane protein, whose structure had been determined.It
catalyzes the electron transfer from plastocyanin/cytochrome c6 at the lumenal side of the
thylakoid membrane to ferredoxin/flavodoxin at the stromal side by a chain of electron carriers..
Photosystem I of (cyanobacteria) consists of 12 protein subunits, to which more than 100
cofactors are non-covalently bound: one functional unit of Photosystem I contains 96
Chlorophyll a molecules, 22 carotenoids, 3 Fe4S4-clusters and 2 phylloquinones that perform the
complex function of light harvesting and charge separation. Photosystem I exists as a trimer in
the cyanobacterial membrane, with a molecular mass of more than 1 000 000 Da. The X-ray
structure of photosystem I at a resolution of 2.5 Å [Jordan 2001] shows the location of the
individual subunits and cofactors and provides new information on the protein-cofactor
interactions. In the talk, biochemical data and results of biophysical investigations are discussed
with respect to the X-ray crystallographic structure in order to give an overview of the structure
and function of Photosystem I with special focus on the electron transfer and excitation energy
transfer in PS I. The discussion will include the interaction sites of PS I with its soluble elctron
carriers plastocyanain/cytochrome c6 and ferredoxin as well as possible interaction sites with the
peripheral antenna systems of PS I. The structure of the oxygen-evolving Photosystem II was
determined at 3.8 Å resolution based on crystals of Photosystem II isolated from the
thermophilic cyanobacterium Synechococcus elongatus [Zouni 2001]. The structure shows a
field of 36 transmembrane -helices of which 22 were assigned to the major subunits of
Photosystem II. Information is also obtained about location, size and shape of the electron
transport chain, including the manganese cluster that catalyses water oxidation. Our structural
information of PS II from Synecococcus elongatus based on a preliminary 3.6 Å model will be
compared to the data published for PS II from Synechococcus vulcanus [Kamiya and Shen 2003]
and the structure at 3.5 A resolution by [Barber and Iwata in press(2003)]. Similarities and
differences will be pointed out and address in respect to the structure-function relationship in PS
II.
Zouni,A ., Witt,H.T., Kern,J., Fromme,P.., Krauß,N., Saenger,W. and Orth,P. (2001) Nature, 409, 739-743 Crystal
Structure of oxygen evolving Photosystem II from Synechococcus elongatus at 3.8 Å Resolution.
Jordan,P., Fromme,P.., Witt,H.T., O. Klukas , Saenger,W. and Krauß,N. (2001) Nature 411, 909-917 Threedimensional structure of cyanobacterial Photosystem I at 2.5 Å resolution.\
Kamiya N, Shen JR (2003) Proc Natl Acad Sci U S A 100: 98-103 Crystal structure of oxygen-evolving
photosystem II from Thermosynechococcus vulcanus at 3.7-A resolution.
49
Session V Oral Presentation Abstracts
Supramolecular organization and dual function of the IsiA chlorophyllbinding protein in cyanobacteria
NATALIYA YEREMENKO*1, ROMAN KOURIL2, JANNE A. IHALAINEN3, SANDRINE
D’HAENE3, NIELS VAN OOSTERWIJK2, ELENA G. ANDRIZHIYEVSKAYA3, WILKO
KEEGSTRA2, HENK L. DEKKER4, MARTIN HAGEMANN5, EGBERT J. BOEKEMA2,
HANS C.P. MATTHIJS1 AND JAN P. DEKKER3
1
Aquatic Microbiology, Institute of Biodiversity and Ecosystem Dynamics, Faculty of Science,
Universiteit van Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands;
2
Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands;
3
Division of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081,
1081 HV Amsterdam, The Netherlands;
4
Swammerdam Institute for Life Sciences, Mass Spectrometry Group, University of Amsterdam,
Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands;
5
Universität Rostock, Fachbereich Biowissenschaften, Pflanzenphysiologie, AlbertEinstein-Strasse 3a, 18051, Rostock, Germany
A significant part of global primary productivity is provided by cyanobacteria, which are
abundant in most marine- and fresh-water habitats. In many oceanographic regions, however, the
concentration of iron may be so low that it will limit growth (1). Cyanobacteria respond to this
condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes
a chlorophyll-binding protein known as IsiA or CP43 (2). It was recently shown that 18 IsiA
proteins encircle trimeric photosystem I (PSI) under iron-deficient growth conditions (3, 4). We
report here that after prolonged growth of Synechocystis sp. PCC 6803 in an iron-deficient
medium, the number of bound IsiA proteins can be much higher than known thus far. The largest
complexes bind 12-14 units in an inner ring and 19-21 units in an outer ring around a PSI
monomer. Fluorescence excitation spectra indicate an efficient light-harvesting function for all
PSI-bound chlorophylls. We also find that cyanobacteria accumulate IsiA in excess of what is
needed for functional light harvesting by PSI, and that a significant part of IsiA builds
supercomplexes without PSI. Because the further decline of PSI makes photosystem II (PSII)
increasingly vulnerable to photooxidation, we postulate that the surplus synthesis of IsiA shields
PSII from excess light. We conclude that IsiA plays a surprisingly versatile role in cyanobacteria,
by significantly increasing the light harvesting ability of PSI and providing light shielding for
PSII.
1. Martin, J. H., Coale, K. H., Johnson, K. S., Fitzwater, S. E., Gordon, R. M., Tanner, S. J., Hunter, C. N., Elrod, V.
A., Nowicki, J. L., Coley, T. L. et al. (1994) Nature 371, 123-129.
2. Burnap, R. L., Troyan, T., & Sherman, L. A. (1993) Plant Physiol. 103, 893-902.
3. Bibby, T. S., Nield, J., & Barber, J. (2001) Nature 412, 743-745.
4. Boekema, E. J., Hifney, A., Yakushevska, A. E., Piotrowski, M., Keegstra, W., Berry, S., Michel, K. P., Pistorius,
E. K., & Kruip, J. (2001) Nature 412, 745-748.
*Corresponding author: Nataliya Yeremenko
50
Session V Oral Presentation Abstracts
Identification of a new family of phycobiliprotein lyases in cyanobacteria:
characterization of a -phycocyanin lyase.
1
G. SHEN, 2N. A. SAUNÉE, 2E. GALLO, 2Z. BEGOVIC, 2W. M. SCHLUCHTER*, AND 1D. A.
BRYANT.
1
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, PA 16802 USA. 2Department of Biological Sciences, University of New
Orleans, New Orleans, LA 70148-2960, USA. *[email protected]
We have identified a new family of proteins that are involved in phycobiliprotein
biosynthesis in Synechococcus sp. PCC 7002. This gene family, which was first identified in
Fremyella diplosiphon as cpeS and cpeT (1), is not related to the cpcE and cpcF genes that
encode the -phycocyanin lyase. There are three cpeS-like genes and one cpeT-like gene within
the genome of Synechococcus sp. PCC 7002, an organism that does not synthesize
phycoerythrin. We created a cpeS1 knockout mutant that accumulates very low amounts of
phycocyanin (PC). The majority of the PC produced in this mutant is not incorporated within
phycobilisomes and can be recovered at the top of sucrose density gradients. This fraction of PC
has a blue-shifted absorbance maximum (600 nm) compared to WT PC (623 nm). When either
the phycobilisomes (PBS) or proteins isolated from the top of the sucrose gradient are separated
by SDS-PAGE followed by immunoblot analysis using anti--PC antibodies, we find that the
mutant contains two species of -PC. One has the same electrophoretic mobility as WT phycocyanin, and the other has slightly higher mobility on SDS-PAGE. Mass spectrometric
analysis of purified PBS from the mutant also showed that there were two types of -PC present
and that one had the mass expected for the WT -PC subunit. The other species present within
mutant PBS was 586.8 Da smaller than WT -phycocyanin. Free phycocyanobilin with a mass of
587.5 Da was also observed, indicating that this second species of PC has a non-covalently
bound phycocyanobilin in one of the two bilin-binding pockets. SDS-PAGE of formic acid
cleavage products from the mutant phycocyanin, with detection of bilin fluorescence after Znstaining, showed that the aberrant -PC in the mutant lacks the phycocyanobilin at the -82 site
but that the -153 site is apparently unaffected. Therefore, we conclude that CpeS1 is a
component of a lyase that attaches phycocyanobilin to the -82 site on phycocyanin. We also
demonstrate that recombinant CpeS1 copurifies with His-tagged apo--PC (HT-CpcB) but is
unable to attach phycocyanobilin to the HT-CpcB in vitro. Therefore, it is likely that one or
more additional subunits are required for the activity of this putative lyase. We are currently
testing whether one of the other two CpeS proteins, the CpeT protein, or some combination of
these proteins is required to form an active -PC lyase. In addition we have generated knockout
mutants for the other cpeS and cpeT genes. The results from these studies will be presented.
1.
Cobley, J. G., A. C. Clark, S. Weerasurya, F. A. Queseda, J. Y. Xiao, N. Bandrapali, I. D'Silva, M.
Thounaojam, J. F. Oda, T. Sumiyoshi, and M. H. Chu. 2002. CpeR is an activator required for
expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is
encoded in the phycoerythrin linker- polypeptide operon (cpeCDESTR). Molecular Microbiology 44:15171531.
51
Session V Oral Presentation Abstracts
Synechococcus sp. PCC 7002 PetB arginine 214: a key residue for quinonereductase (Qi) function and possible oxygen radical production in the
cytochrome bf complex
1
DARRYL HORN, 1MATTHEW NELSON, 2GIOVANNI FINAZZI,
3
QINGJUN WANG, 4JOHN WHITMARSH AND 1TOIVO KALLAS.
1
Department of Biology & Microbiology, University of Wisconsin-Oshkosh, Oshkosh, WI 54901,
2
CNRS UPR 1261, Institut de Biologie Physico-Chimique, 75005 Paris, FRANCE, and
3
Center of Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, and
4
NIH NIGMS, Bethesda MD USA 20892.
The cytochrome bf complex catalyzes the rate-limiting plastoquinol-oxidation step of
photosynthesis. Quinol oxidation involves bifurcated transfer of one electron to a high potential
chain (> Riekse protein > cytochrome f) and another to a transmembrane, low potential chain (>
heme b L > heme b H > quinone-reductase (Qi) site). Qi domains of cytochrome bf and
mitochondrial-type bc complexes differ markedly in structure and inhibitor specificity. To
investigate the bf Qi domain, we constructed mutation R214H in the Synechococcus 7002 petB
(cytochrome b6) gene, which makes the cytochrome bf complex more like the bc complex. The
converse H217R mutation in the bc complex alters the redox properties and function of the Qi
site (1). At high light intensity, the mutant grew ~2.5 fold slower than the wild type. Slower
growth arose from slower turnover of the bf complex. Specifically, the R214H mutation partially
blocked electron transfer to the Qi site (mimicking the effect of the Qi-site inhibitor NQNO, 2-nnonyl-4-hydroquinoline N-oxide) as shown by an increased amplitude of b-heme reduction and a
lower rate of b-heme oxidation following single flash turnovers. The wild type showed a
significant isotope effect on b-heme reduction/oxidation kinetics but D2O had little effect in the
mutant. This indicates that proton transfer events limited b-heme oxidation in the wild type,
whereas electron flow became limiting in the mutant. Redox titrations of membranes revealed
midpoint potentials (Em 7) of ~–30 mV and –120 mV for the two b hemes in the mutant similar
to those in the wild type (2). Events that impede electron flow to the Qi site may prolong the
reactive semiquinone at the quinol-oxidation (Qo) site leading to production of reactive oxygen
species (ROS). Preliminary data support this. The R214H mutant showed increased ROS
production relative to the wild type as measured by formation of formazan from XTT (2,3bis[Methoxy-4-nitro-5sulfo-phenyl]-2H-tetrazolium-5-carboxanilide) which reacts specifically
with superoxide. Our analysis defines cytochrome b6 arginine 214 as a key residue for quinonereducatse (Qi) site function and turnover of the cytochrome bf complex. In the recent cytochrome
bf structures (3, 4), R207 (Synechococcus R214) lies near the Qi pocket and the newly discovered
c’ (or x) heme. Our data suggest that arginine 214 may bind plastoquinone at the Qi site and may
have roles in modulating the redox potential of heme c' (x) and in proton translocation. These
findings will be discussed in light of the bf structures.
(Supported by NSF MCB 0091415)
1. Gray, K. A., Dutton, P. L. and F. Daldal. 1994. Biochemistry 33, 723-733.
2. Baymann, F., Rappaport, F., Joliot, P. and T. Kallas. 2001. Biochemistry 40, 10570-10577.
3. Kurisu, G., Zhang, H., Smith, J. L. and W. A. Cramer. 2003. Science 302, 1009-1014.
4. Stroebel, D., Choquet, Y., Popot, J.-L. and D. Picot. 3002. Nature 426, 413-418.
52
Session V Oral Presentation Abstracts
Structure and Functional Studies of Protein Complexes in Photoprotection
and Carbon Fixation: The Orange Carotenoid Protein and the Carboxysome
CHERYL A. KERFELD1, MICHAEL SAWAYA1, DAVID KROGMANN2 & TODD O. YEATES1
1
Molecular Biology Institute, UCLA, Los Angeles, CA and
2
Department of Biochemistry, Purdue University, West Lafayette, IN
Our structural studies focus on the structural basis of photosynthetic function. The structure of
the uniquely water-soluble orange carotenoid-binding protein (OCP) isolated from the
cyanobacterium Arthrospira maxima has been determined at a resolution of 2.1Å. The OCP is
presumably involved in photoprotection. The structure reveals the protein-pigment interactions
that influence the spectral properties of the carotenoid. A proteolytic product of OCP has been
isolated that appears red instead of orange. The OCP can also be converted into a red protein by
exposure to low pH. Circular dichroism data suggest that low pH changes the structure of the
protein. Results of experiments directed toward understanding the functional role of the OCP
will be presented.
We have also initiated structural studies of components of the carboxysome, a cage-like protein
complex involved in optimizing carbon fixation. Progress in these structural studies will be
described.
53
Session V Oral Presentation Abstracts
Functional genomics of genes for biogenesis of Fe/S proteins in cyanobacteria
Gaozhong Shen1, Ramakrishnan Balasubramanian1, Tao Wang1, Bhramara Tirupati1, J. Martin
Bollinger1,2, John H. Golbeck1,2 and Donald A. Bryant1
1
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, PA 16802, USA, 2Department of Chemistry, The Pennsylvania State University,
University Park, PA 16802, USA
Genomic sequencing of Synechococcus sp. PCC 7002 and other cyanobacteria has provided
useful tools in identifying genes with possible functions in Fe/S cluster assembly. Indeed, genes
for two different systems of Fe/S cluster biogenesis (named SUF and ISC) have been identified
in the genomes of several sequenced cyanobacteria. SufS and SufE are thought to be responsible
for sulfur mobilization. SufC functions as a versatile orphan ATPase and may work together with
the SufB and SufD proteins in iron assimilation and Fe/S cluster insertion. In cyanobacteria,
SufR functions in the regulation of expression of suf genes. We studied the functions of selected
genes in the SUF and ISC systems in cyanobacteria by reverse genetics. Such as for iscS gene,
no phenotype can be observed in the iscS1 mutant of Synechococcus sp. PCC 7002 and iscS
mutants or fdx mutant Synechocystis sp. 6803. Even the double mutant of iscS1 and iscS2 genes
can grow photoautotrophically. In contrast, we have not been able to fully segregate interruption
mutants of the sufD and sufS genes in Synechococcus sp. PCC 7002 or the sufS gene in
Synechocystis sp. PCC 6803. This demonstrates that these suf genes, unlike the isc genes, are
essential in cyanobactera. Surprisingly, no phenotype could be observed in the sufA and iscA
single or double interruption mutants of Synechococcus sp. PCC 7002. This result casts doubt on
the function of the IscA and SufA proteins to function as essential scaffolds in Fe/S cluster
biogenesis in photosynthetic organisms, as is the case in Azotobacter vinelandii. Components of
the SUF system found in cyanobacteria can be localized in the chloroplasts of higher plants.
These results show that oxygenic photosynthetic organisms rely primarily on the SUF rather than
the ISC system for biogenesis of Fe/S clusters for Photosystem I and/or other Fe/S proteins.
54
Session V Oral Presentation Abstracts
Preliminary structural characterization of the 33-kDa protein (PsbO) in
solution studied by site-directed mutagenesis and NMR spectroscopy
MARC NOWACZYKa,CARSTEN BERGHAUSb, RAPHAEL STOLLb AND
MATTHIAS RÖGNER*a
a
Plant Biochemistry, Faculty of Biology, and
Biomolecular NMR, Faculty of Chemistry,
Ruhr-University Bochum, D-44780 Bochum, Germany
b
Photosystem 2 (PS2) is located in the thylakoid membrane of chloroplasts and cyanobacteria
and performs one of the key reactions on our planet - the light-driven oxidation of water to
molecular oxygen. This process occurs in four one-electron oxidation steps (S-states) by the
manganese containing water-oxidising complex (WOC). Recent advances in X-ray structure
analysis of PS2 crystals (1,2) provide a detailed view of the structure and organisation of the
WOC. At the lumen side of PS2, the WOC is shielded by several extrinsic proteins. One of them,
the 33-kDa protein (PsbO), is of special interest, because it is ubiquitous in all photosynthetic
organisms performing water-splitting and its removal results in a strong decrease of the oxygen
evolving activity. Therefore, it is often referred to as manganese stabilizing protein (MSP).
Site-directed mutagenesis experiments combined with 1D and 2D NMR spectra provide a
preliminary insight into the structure and dynamics of the 33-kDa protein (PsbO) from T.
elongatus free in solution. The NMR spectra suggest that PsbO rather than forming a ‘molten
globule’ state or being ‘natively unfolded’, contains both a well folded core and highly flexible
regions. The PsbO protein shows a remarkable temperature-, pH-, and long-term-stability being
stable for at least four weeks under the conditions tested in this study. Due to this extraordinary
stability of PsbO, we characterized four cysteine mutants serving as local probes for structural
and dynamic properties of PsbO. The results indicate sites of increased accessibility/flexibility
which may be important for the docking to the PS2 core complex.
1. N. Kamiya and J.R. Shen, Proc Natl Acad Sci U S A, 2003, 100, 98.
2. K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, and S. Iwata, Science, 2004, 303, 1831.
55
Session V Oral Presentation Abstracts
Identification of a low molecular weight protein tyrosine phosphatase and its
potential substrates in Synechocystis sp. PCC 6803
ARCHANA MUKHOPADHYAY1 and PETER J. KENNELLY1*
Department of Biochemistry, Virginia Polytechnic Institute and State University, 105 Engel Hall,
Blacksburg, Virginia 240611
Abstract
The predicted protein product of open reading frame
slr0328 from Synechocystis sp. PCC
6803, SynPTP, possesses significant amino acid sequence identity with known low molecular
weight protein tyrosine phosphatases (PTPs). To determine gross functional properties of this
hypothetical protein, gene slr0328 was cloned, and its predicted protein product was expressed in
E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography.
The catalytic activity of SynPTP was tested toward several exogenous protein substrates that had
been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited
phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward
phosphotyrosyl 32P-casein was 1.5 nmole/min/mg). However, no phosphatase activity of SynPTP
was detected toward serine phosphorylated protein substrates. SynPTP displayed
phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl
phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl
phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that Km (0.6 mM) and
Vmax (3.2 µmole/min/mg) values for SynPTP toward pNPP are similar to those of other known
bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited
by sodium orthovanadate, a potent inhibitor for tyrosine phosphatases, but not by okadaic acid,
an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted
catalytic cysteine, Cys7, of SynPTP to serine abolished enzyme activity. Several phosphotyrosine
containing proteins were detected from the cell extracts of Synechocystis sp. PCC 6803 through
immunoreactions using anti-pTyr antibody. SynPTP was observed to dephosphorylate three of
these proteins in vitro. Further studies will be performed to identify these potential substrates of
SynPTP by peptide-mass fingerprinting analysis.
*Corresponding author. E-mail: [email protected]
Phone: (540) 231-4317
56
Session VI Oral Presentation Abstracts
Session VI: Carbon metabolism
Session Chair: Dean Price, Australian National
University
Keynote Speaker: Murray Badger, Australian
National University; “Cyanobacterial photosynthetic
CO2 concentrating mechanisms:
57
Session VI Oral Presentation Abstracts
Signal transduction molecules directly regulated by bicarbonate and sodium
ions.
ARNE HAMMER, MARTIN CANN*.
Department of Biological and Biomedical Sciences, University of Durham, South Road, Durham,
DH1 3LE, United Kingdom.
The ability to respond and adapt to abiotic stress is fundamental to the biology of all
organisms. Two key abiotic stressors that impact upon cyanobacteria are inorganic carbon
availability and environmental sodium concentration. Despite the importance of mechanisms that
enable an organism to act in response to salt and carbon stress, no directly sodium or carbon
responsive signalling molecules have been described. The identification of such molecules is of
paramount importance in delineating the mechanisms by which cyanobacteria respond to abiotic
stress. Here we describe signal transduction molecules directly regulated by bicarbonate and
sodium.
1) We present data for a novel class of bicarbonate stimulated adenylyl cyclase present in many
eukaryotic and prokaryotic species (including the cyanobacteria) and propose that the cAMP
signal transduction pathway mediates aspects of inorganic carbon detection among diverse
organisms.
2) We present recent work detailing the identification of an evolutionarily widespread sodium
responsive signalling domain and detail the role of this domain in the control of the salt stress
response of cyanobacteria.
* corresponding author
58
Session VI Oral Presentation Abstracts
Response to low carbon dioxide in the glaucocystophyte alga,
Cyanophora paradoxa
A
S. C. BUREY, BV. POROYKO, BN. OZTURK, AS. FATHI-NEJAD, AG. HAMMERSCHMIED,
A
C. SCHUELLER, AJ. M. STEINER, BH. J. BOHNERT AND A*W. LOEFFELHARDT
1A
Max F. Perutz Laboratories
University Departments at the Vienna Biocenter
Department of Biochemistry and Molecular Cell Biology and Ludwig Boltzmann Research Unit
of Biochemistry, 1030 Vienna, Austria
and
1B
Departments of Plant Biology and Crop Sciences
University of Illinois,
Urbana, IL 61801, USA
Cyanophora paradoxa is the best investigated member of the glaucocystophyte algae, the
most primitive phototrophic eukaryotes whose plastids (cyanelles) are surrounded by a
peptidoglycan layer, a clear indication of their descent from endosymbiotic cyanobacteria. As
many aquatic microorganisms which as a group contribute about 50% to global CO2 fixation, C.
paradoxa possesses an inorganic carbon concentration mechanism (CCM). Increase of the CO2
level in CCM microcompartments harboring Rubisco increases the efficiency of photosynthetic
carbon fixation. Operation of the CCM is triggered by growth at ambient (0.04%) CO2
concentrations with concomitant induction of CO2 and bicarbonate transporters and components
of these microcompartments. These electron-dense structures are carboxysomes in prokaryotes
and pyrenoids in eukaryotic algae. We postulate that the cyanelles of C. paradoxa did retain
another prokaryotic feature in addition to the peptidoglycan wall: the unique case of an
eukaryotic carboxysome. An isolation procedure for carboxysomes was developed enabling a
proteomics approach and the identification of carboxysome proteins other than Rubisco. Rubisco
activase was imported into isolated cyanelles and was shown to integrate into carboxysomes.
Two cDNA libraries of cells grown under ambient and high CO2 grown cells were constructed.
Around 450 genes were differentially expressed under high and ambient CO2. The potential
involvement of the corresponding gene products in the CCM of C. paradoxa will be discussed.
*for correspondence: e-mail [email protected]
59
Session VI Oral Presentation Abstracts
Identification and analysis of akinete specific genes in Nostoc punctiforme
M.L. Summers, C. Argueta, and K. Yuksek
California State University Northridge, Northridge, CA 91330-8303
The filamentous cyanobacteria Nostoc punctiforme is capable of dark heterotrophy and cellular
differentiation into nitrogen-fixing heterocysts, motile hormogonia, or spore-like akinetes. The
study of akinete differentiation at the molecular level has been limited by the asynchronous
development and limited number of akinetes formed within a filament. A system in which to
study the development and gene regulation of akinetes was investigated using a zwf mutant
lacking the initial enzyme of the oxidative pentose phosphate pathway. Following dark
incubation in the presence of fructose, the zwf- strain ceased growth and differentiated into
akinete-like cells where as the wild-type strain was capable of heterotrophic growth. Darkinduced zwf akinetes had increased resistant to the environmental stresses of desiccation, cold, or
treatment with lysozyme relative to vegetative cells of both strains. Dark-induced zwf akinetes
also exhibited PAS staining characteristics identical to that observed for wild-type akinetes, and
indicated that synchronous induction akinetes occurred in treated cultures. Transcription of the
avaK akinete marker gene was found to be fructose-induced in both wild-type and zwf strains,
but was increased strongly in zwf dark-induced akinetes two days after induction. This model
system was used in conjunction with the differential display technique to identify akinetespecific genes. Following screening of identified candidate genes by reverse transcriptase realtime Q-PCR, the promoter regions for several putative akinete-expressed genes were cloned into
GFP reporter vectors developed for this purpose. Four genes were confirmed to exhibit akinetespecific GFP expression, and one of these was strongly expressed in developing hormogonia.
Phenotypic analysis of insertional mutants will be presented. The phenotypic and genetic
evidence showing synchronous induction of dark-induced zwf akinetes coupled with the
identification of akinete-specific genes indicates this system will provide a valuable tool for the
continued study of akinete development in N. punctiforme.
60
Session VI Oral Presentation Abstracts
Regulation of the cyanobacterial CO2 concentrating mechanism
WOODGER, F.J., 1BADGER, M.R. AND 1*PRICE, G.D.
1. Molecular Plant Physiology, Research School of Biological Sciences, Australian
1
National University, ACT, 0200.
Approximately 50% of global CO 2-based productivity is now attributed to the
activity of phytoplankton including ocean-dwelling cyanobacteria. In response to
inherent restrictions on the rate of CO2 supply in the aquatic environment,
cyanobacteria have evolved a very efficient means of capturing inorganic carbon (Ci),
as either CO2 or HCO3-, for photosynthetic carbon fixation. This capturing mechanism,
known as a CO2 concentrating mechanism (CCM), involves the operation of active
CO2 and HCO3- transporters and results in the concentration of CO2 around Rubisco in
a unique microcompartment called the carboxysome. The CCM exhibits two basic
physiological states – a constitutive, low affinity state and a high affinity state that is
induced in response to Ci limitation. Many of the genetic components of the CCM,
including genes encoding Ci transporters, have been identified. It is apparent that the
expression of genes encoding the inducible, high-affinity Ci transporters is particularly
sensitive to Ci availability and we are now interested in defining how cyanobacterial
cells sense and respond to Ci limitation at the transcriptional level. Current theories
include direct sensing of external Ci, sensing of internal Ci-pool fluctuations or
detection of changes in photorespiratory intermediates, carbon metabolites or redox
potential. Presently there is no consensus view. We have investigated the
physiological and transcriptional response of CCM mutants and wildtype strains to
pharmacological treatments and various light, O2 and Ci regimes. Our data suggests
that perception of Ci limitation by a cyanobacterial cell is either directly or indirectly
related to the size of the internal Ci pool within the cell.
*corresponding author
61
Session VI Oral Presentation Abstracts
Regulation of sucrose metabolism in salt-treated cells of Nostoc sp. PCC 7120:
towards the understanding of the role of sucrose in cyanobacteria
1
A.C. CUMINO, 1L. CURATTI, 1W.A. VARGAS, 1L. GIARROCCO, 1C. MARCOZZI, 1* G.L.
SALERNO. 1Centro de Investigaciones Biológicas, FIBA, CONICET, Vieytes 3103, 7600 Mar
del Plata, Argentina
One of the physiological responses for salt adaptation of cyanobacteria consists in the
accumulation of organic osmoprotectants, low-molecular mass solutes that do not interfere with
cell metabolism. As no link has been found between the kind of osmotic protectant accumulated
and taxonomic grouping or the habitats of origin, the strains have been grouped according to
their tolerance to salt concentration. Most Nostoc spp. and Anabaena spp. strains are filamentous
heterocystic cyanobacteria usually growing in fresh water habitats. This group of cyanobacteria
is classified as low salt tolerant strains that accumulate sucrose (Suc) as a response to NaCl.
Recently, it was elucidated that sucrose synthesis in these strains occurs through the sequential
action of sucrose-phosphate synthase (SPS-A and SPS-B) and sucrose- phosphate phosphatase
(SPP) activities, and its degradation can be catalyzed by either sucrose synthase (SuS-A) or
alkaline-neutral invertases (Inv-A and Inv-B) [1]. When we investigated the effect of NaCl on
the regulation of sucrose metabolizing enzymes in several Anabaena spp. or Nostoc spp. strains,
we found that not only SPS but also SuS and Inv activities increased after salt addition.
Additionally, an increase in polypeptide levels of SPS, SuS and Inv was shown by
immnoanalysis after separating the polypeptides by SDS-PAGE. In Nostoc sp. PCC 7120, RTPCR and reporter-gene expression analyses showed a differential transcriptional regulation
between the two sps genes, and between the two inv genes. Putative promoters, specifically
activated by NaCl, were identified for some of these genes after primer extension analysis. These
results indicate that in Nostoc spp. and Anabaena spp. strains, not only sucrose levels but sucrose
turn-over, as well are stimulated by NaCl, suggesting that in these cyanobacteria sucrose
metabolism may display a more complex function than the synthesis of an osmolite. A complex
set of enzymes related to sucrose metabolism may be necessary to sensibly tune cellular sucrose
levels according to additional roles of sucrose metabolism that may be coordinated.
Supported by CONICET, ANPCyT, FIBA and UNMdP.
(1)
Salerno G., Curatti L. (2003) Origin of sucrose metabolism in higher plants: when, how and why? Trends Plant Sci. 8, 6369.
62
Session VI Oral Presentation Abstracts
Keynote Talk
Cyanobacterial photosynthetic CO2 concentrating mechanisms: solutions employing many
Ci transporters, two carboxysomes types and diverse carbonic anhydrases
MURRAY BADGER
Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, PO
Box 475, Canberra City, ACT, Australia
Photosynthetic CO2 concentrating mechanisms in cyanobacteria are essential for efficient
photosynthesis in the diverse array of aquatic environments in which cyanobacteria are found.
With the emergence of a diverse array of cyanobacterial genomes a wide array of genetic
diversity has been uncovered in the manner in which different cyanobacteria are able to achieve
a functional outcome. This diversity includes at least four bicarbonate transporters and two CO2
uptake mechanisms, two distinct types of carboxysomes and significant creativity in which
carbonic anhydrases are employed. This talk will summarize our current understanding and
research in this area.
63
Session I Poster Presentation Abstracts
Poster Presentations Session I
Physiology, Metabolism and Global Responses (I)
Session Chair: Günter Peschek, University of Vienna
64
Session I Poster Presentation Abstracts
Isolation and characterization of Nostoc punctiforme ATCC 29133 mutants
unable to differentiate into hormogonia.
AKIKO TOMITANI1,2,3*, PAULA S. DUGGAN2 and DAVID G. ADAMS2
1 The Kyoto University Museum, Kyoto University, Yoshida-hon-machi, Sakyo-ku, Kyoto
6068501, Japan
2 School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT. U.K.
3 Institute for Frontier Research on Earth Evolution, Japan Agency for Marine-Earth Science
and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
Cyanobacteria form a morphologically diversified group. Particularly in filamentous
cyanobacteria of subgroups Nostocales and Stigonematales, vegetative cells can mature in four
developmental directions (vegetative cells, heterocysts, akinetes, and hormogonia), in response
to environmental conditions. Hormogonia are transiently differentiated, small-celled filaments
lacking heterocysts and are often capable of gliding and/or buoyant motility1. The function of
hormogonia is to provide immotile strains with a means of dispersal in response to
environmental triggers2. They also play an important role as infective units in the establishment
of symbiotic association with various plant hosts. In hormogonia formation, cell division
occurs rapidly and synchronously in all cells without cell growth and DNA replication3. To
identify the genes involved in hormogonia differentiation, transposon mutants of Nostoc sp.
ATCC 291334 were prepared and screened. About a thousand mutant clones were induced to
form hormogonia by plant exudates, and then transferred to the dark in the presence of
penicillin. Mutants unable to differentiate into hormogonia do not divide in the dark and are
expected to survive the penicillin treatment. Following several rounds of penicillin selection in
the dark, the transposon and flanking DNAs were recovered from surviving mutants, cloned and
sequenced. The predicted proteins encoded by the identified genes include a membrane protein,
and proteins involved in sugar transport and signaling pathways (Histidine kinase,
Serine/Threonine kinase). The transposon mutants of those genes neither produce hormogonia,
nor establish a symbiotic association when co-cultured with the host plant Blasia pusilla. Most
mutants show similar growth rate to wild type, while a mutant of a two component regulatory
pathway does not grow at all in medium without combined nitrogen. Isolation of multiple genes
involved in hormogonia formation and their detailed characterization will provide us with a
novel insight into the mechanisms that control bacterial cell division.
1
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979) J. Gen. Microbiol.
111:1-61.
2
Adams, D. G. (2000). In Prokaryotic Development, (eds. Y. V. Brun and L. J. Shimkets. ASM Press,
Washington,) pp 51-81.
3
Tandeau de Marsac. N., Mazel, D., Bryant, D. A. & Houmard, J. (1988) Photosynth. Res. 18:99-132.
4
Cohen, M. F., Wallis, J. G., Campbell, E. L. and Meeks, J. C. (1994) Microbiol. 140:3233-3240.
65
Session I Poster Presentation Abstracts
Identification of a cis-acting antisense RNA potentially regulating furA expression
in Anabaena sp. PCC 7120.
1
J.A. HERNÁNDEZ, 2A.M. MURO-PASTOR, 2E. FLORES, 1M.T. BES, M.L. 1PELEATO AND
1
M.F. FILLAT.
(1) Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad
de Zaragoza, Pedro Cerbuna, 12. 50009-Zaragoza, Spain.
(2) Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones CientíficasUniversidad de Sevilla, Sevilla, Spain.
Ferric uptake regulation (Fur) proteins are prokaryotic transcriptional regulators that integrate iron
metabolism with several environmental stress responses. In the cyanobacterium Anabaena sp. PCC
7120 three open reading frames (all1691, all2473 and alr0957) containing the histidine-rich region
characteristic of the Fur (ferric uptake regulation) protein family have been identified (1). FurA is the
product of open reading frame all1691 that is located between sigC and alr1690, the latter encoding a
putative cell wall-binding protein. Anabaena FurA is a constitutive, moderately autoregulated protein
that controls the transcription of flavodoxin, the product of the isiB gene (2). Northern blot analysis of
furA showed an unexpected transcription pattern that consists of two hybridization bands of
approximately 1.8 and 0.7 kb. Iron depletion caused a similar effect on the abundance of both RNAs,
whose amount increased, after 48 h, about 1.9- and 1.7-fold, respectively. Hybridization of Anabaena
RNA samples with dsDNA probes corresponding to the furA homologues all2473 and alr0957, as well
as with the furA flanking genes, all1691 (sigC) and alr1690, showed that the short transcript
corresponded to the furA mRNA, whereas the longer transcript contained the alr1690 mRNA and a
large region that overlaps the furA gene. RT-PCR assays using RNA from Anabaena sp. PCC 7120
and insertional mutant strains containing the C.S3 cassette in different points of the furA genomic
region indicated that the 1.8-kb transcript is complementary to the furA mRNA. Derepression of FurA
in an alr1690 null mutant suggests that this transcript acts as a cis-acting antisense RNA (-furA RNA)
interfering with furA transcript translation thus contributing to determine cellular levels of the FurA
protein. These results state the importance of post-transcriptional control in Fur proteins and describe a
new mechanism of modulation of these regulators.
(1) http://www.kazusa.or.jp/cyano/link.html
(2) Hernández, J. A., Bes, M. T., Fillat, M. F., Neira, J. L. and Peleato, M. L. (2002) Biochem J 366, 315-22.
66
Session I Poster Presentation Abstracts
Differential circadian regulation of psbA gene expression in Synechococcus
elongatus PCC 7942
SHANNON R. CANALES and SUSAN S. GOLDEN*
Department of Biology, Texas A&M University, College Station, TX 77843
The psbA gene family in Synechococcus elongatus PCC 7942 encodes two forms of the D1
protein, which is an essential component of the photosystem II reaction center. Form I is encoded
by the psbAI gene and Form II is encoded by both the psbAII and psbAIII genes. To examine the
circadian transcriptional patterns of these photosynthesis genes, the upstream promoter regions
(PpsbA) were fused to luxAB and the levels of bioluminescence were measured over time. In
constant light conditions (LL), expression of bioluminescence from PpsbAII peaks 12 h out of
phase from both PpsbAI and PpsbAIII. Interestingly, when cultures are maintained in a cycle of
12 h light/12 h dark (LD), conditions that mimic daily external cues, peak transcription from
PpsbAII occurs 4 h before the other two genes experience their highest level of expression; the
latter occurs at the light to dark transition. To further examine the circadian regulation of these
promoters, expression patterns are being tested in the absence of the other two psbA genes. The
functional components of the promoters, previously defined in the context of light-responsive
regulation, are being tested to identify the region through which the circadian clock affects
transcriptonal patterning. Of particular interest are the enhancers of the upstream untranslated
regions of psbAII and psbAIII, which have been shown by others to bind the LysR-type
regulatorCmpR, and the negative regulator in the upstream untranslated region of psbAI.
*
Corresponding author
67
Session I Poster Presentation Abstracts
Novel expression regulation and biochemical activities of a redox-regulated
RNA helicase
L.M. PATTERSON-FORTIN, D. CHAMOT, and G.W. OWTTRIM*
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9
Photosynthetic organisms must sense and respond to changes in their light environment.
Our lab has previously shown that light regulated expression of the cyanobacterial DEAD-box
RNA helicase, CrhR, is mediated through light-driven electron flow. Specifically the reduced
redox poise of plastoquinone induces crhR expression in Synechocystis sp. strain PCC 6803. The
mechanism by which crhR transcription is differentially regulated in response to redox potential
is not known. DNA affinity chromatography and mass spectrometry identified the protein
responsible for regulating crhR expression as a LexA-related regulator. Northern analysis of
lexA and crhR transcript levels indicates LexA functions as a repressor of crhR expression in the
dark. Association of LexA with the E. coli SOS response warranted analysis of recA, lexA, and
crhR expression under UV-induced DNA damaging conditions. While Synechocystis recA is
DNA damage inducible, lexA and crhR expression is not affected. Furthermore, although
Synechocystis LexA lacks two of the three conserved residues required for the E. coli LexA selfcleavage reaction it is cleaved in E. coli in the absence of DNA damage. The results suggest
LexA represses expression of redox-responsive genes in Synechocystis rather than those required
for DNA repair with the potential for derepression by LexA cleavage. Biochemical analysis of
recombinant CrhR in vitro indicates that it is a bona fide RNA helicase possessing RNAdependent ATPase and ATP-dependant RNA unwinding activities. CrhR also performs ATPdependant RNA annealing, an activity which is unique among RNA helicases. The biochemical
properties provide the potential for CrhR to effect RNA secondary structure rearrangements via a
coupling of RNA unwinding and annealing. The data are discussed in a functional context with
respect to a role for CrhR in cyanobacterial gene expression in response to alterations in redox
potential.
68
Session I Poster Presentation Abstracts
RNA Structural Rearrangements by the Synechocystis RNA Helicase, CrhR
G.W. OWTTRIM AND D. CHAMOT
Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada, T6G 2E9
Rearrangement of RNA secondary structure is crucial for numerous biological
processes.
RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We
report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA
helicase possessing bidirectional ATP-dependent RNA helicase activity. We have also found
that CrhR catalyzes the ATP-dependent annealing of complementary RNAs into both intra- and
inter-molecular duplexes. Uniquely, and in contrast to other proteins that perform RNA
annealing, the CrhR catalyzed reaction requires ATP hydrolysis. Through a combination of the
unwinding and annealing activities, CrhR also catalyzes RNA strand exchange reactions
resulting in the formation of RNA secondary structures which are too stable to be resolved by
the helicase activity. RNA strand exchange most likely occurs through the CrhR-dependent
formation and resolution of an RNA branch migration structure. Demonstration that a second
cyanobacterial RNA helicase, CrhC, does not catalyze annealing supports the suggestion that this
activity is not a biochemical characteristic universally possessed by RNA helicases.
Biochemically, CrhR is therefore similar to RecA and related proteins that catalyze strand
exchange and branch migration on DNA substrates, a characteristic which is reflected in the
recently reported structural similarities between these proteins. The data indicates the potential
for dynamic RNA secondary structure rearrangements via CrhR through a combination of RNA
helicase and annealing activities.
69
Session I Poster Presentation Abstracts
Transcriptional regulation of the bidirectional NiFe-hydrogenase in
Synechocystis sp. PCC 6803
KIRSTIN GUTEKUNST*, SARANYA PHUNPRUCH, RÜDIGER SCHULZ-FRIEDRICH,
and, JENS APPEL
Botanical Institute, Christian-Albrechts-University, Am Botanischen Garten 1-9, D-24118 Kiel,
Germany
e-mail:[email protected]
The bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes
(hoxEFUYH) that are organized in one gene cluster. It was shown through RT-PCR that all hox
genes are transcribed as one unit. A mutant with a deletion in the supposed promotor region
upstream of hoxE exhibits as expected a decreased hydrogenase activity. Several DNA fragments
from this region were ligated into a promotor probe vector carrying the reporter genes luxAB.
The corresponding mutants were measured to further characterize the promoter. The luciferase
measurements as well as a band-shift-assay revealed a region binding a protein which is thought
to be an transcription factor. Further characterizations of the protein and the binding site are in
progress.
70
Session I Poster Presentation Abstracts
Novel Interaction between Two CheA-like Molecules Involved in Gliding
Motility of Cyanobacterium Synechocystis sp. PCC 6803
SOO YOUN KIM, YOUNG HYE KIM, JONG-SOON CHOI, YOUNG-HO CHUNG AND
YOUNG MOK PARK
Proteome Analysis Team, Korea Basic Science Institute, Daejeon 305-333, Korea
The unicellular cyanobacterium Synechocystis sp. PCC 6803 displays gliding motility that
depends on the type IV-like thick pili. All disruptants of chemotaxis-like gene locus (slr1041slr1044, called Tax3 by Bhaya et al) did not show gliding motility. Predicted proteins of slr1041,
slr1042, slr1043 and slr1044 are homologous to PatA, CheY, CheW and MCP, respectively. The
missing cheA-like gene in this cluster was identified, as novel split genes, slr0073 and slr0322.
The two disruptants of cheA-like genes did not show gliding motility on the agar surface. To
elucidate functional relationship between two CheA-like molecules, we examined possible
phosphorelay cascade between histidine kinase domain of Slr0322 and Hpt domain of Slr0073
using yeast two-hybrid and co-immunoprecipitation analyses. We detected the strong and
specific interactions between Slr0322 and Slr0073. These results suggest that the phosphorelay
signal of Slr0322-HK to Slr0073-Hpt exists in Synechocystis sp. PCC 6803. Also, we detected
the interactions between each of two CheAs (Slr0322 & Slr0073) and a CheW (Slr1043) and a
CheY (Slr1042). We will discuss the possible working model for a signal transduction pathway
of the gliding motility.
71
Session II Poster Presentation Abstracts
Poster Presentations Session II
Heterocysts and nitrogen metabolism
Session Chair: Karl Forchammer, Justus-Liebig
Universität Giessen
72
Session II Poster Presentation Abstracts
Characterization of Anabaena sp. strain PCC 7120 genes alr4311 and all4312
REBECCA E. THAYER AND STEPHANIE E. CURTIS*
Department of Genetics, Box 7614, North Carolina State University, Raleigh, NC 27695-7614
A screen to identify sequences up-regulated at the transcript level during heterocyst development
in Anabaena sp. strain PCC 7120 identified adjacent loci alr4311 and all4312 (1). The
transcripts of these genes are expressed at very low levels in vegetative cells, and increase in
abundance after nitrogen starvation and the induction of heterocyst development. The sequence
of alr4311 suggests it encodes the ATP-binding protein of an ABC transporter complex, while
that of all4312 suggests it encodes the response regulator of a two-component regulatory system.
A preliminary inactivation of each of the genes by interruption with plasmid sequences resulted
in strains that are inviable in the absence of fixed nitrogen. Characterization of the expression
profiles of the genes after nitrogen starvation is being conducted, as well as more detailed
phenotypic analyses of the alr4311 and all4312 mutant strains.
(1) Curtis, S. E. and P. B. Hebbar. 2001. A screen for sequences up-regulated during heterocyst development in
Anabaena sp. strain PCC 7120. Arch. Micrbiol. 175:313-322.
73
Session II Poster Presentation Abstracts
Nitrogen control of the glutamyl-tRNA synthetase in Tolypothrix sp. PCC
7601
IGNACIO LUQUE1,2,3,*, LIN JIA3, GÉRALD ZABULON2, NICOLE TANDEAU DE
MARSAC3, ENRIQUE FLORES4 AND JEAN HOUMARD2.
(1) Dpto Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, Campus de San
Vicente, Alicante 03080 Spain. (2) Unité des organismes photosynthetiques et environnementCNRS/Ecole Normale
superieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France. (3) Unité des cyanobacteries, Intitut Pasteur, 28 rue du Dr.
Roux F-75724 Paris France. (4) Intituto de Bioquímica Vegetal y Fotosíntesis. C.S.I.C.-Universidad de Sevilla, E41092 Seville, Spain.
Aminoacyl-tRNA synthetases are the enzymes responsible for charging the tRNAs with their
cognate amino acid (1). Although it has long been assumed that every cell should contain an
aminoacyl-tRNA synthetase for each amino acid this has recently been shown not to be
universal. Thus, the archaea, most bacteria and the eukaryotic organelles do not have a complete
set of aminoacyl-tRNA synthetases. In most bacteria, including the cyanobacteria, the glutamyltRNA synthetase (GltX or GluRS) is the enzyme that charges both tRNAglu and tRNAgln with
glutamic acid (2). The misacylated glu-tRNAgln is subsequently transformed to gln-tRNAgln in a
transamidation reaction in which glutamine acts as the amido donor. The main route for nitrogen
assimilation in cyanobacteria is the glutamine synthetase/glutamate synthase cycle (GS/GOGAT)
where both amino acids, glutamate and glutamine, are involved. We have estimated the
intracellular concentration of glu and gln in the cyanobacterium Tolypothrix sp. PCC 7601 (also
known as Calothrix sp. PCC 7601 or Fremyella diplosyphon) and observed that they
experimented dramatic alterations upon changes in the nitrogen source supplied to cells. In order
get some information on how GltX can adapt to these changes in the concentration of glu and
gln, we have analyzed the expression of the gltX gene under different nitrogen regimes. The gltX
transcript levels exhibited drastic transient alterations following changes in the nitrogen source
made available to cells. Strikingly the regulatory pattern significantly differed from that reported
for gltX from the unicellular cyanobacterium Synechococcus sp. PCC 7942 (3). We have also
analyzed the post-translational modifications of GltX in Tolypothrix under changing nitrogen
conditions to observe that the electrophoretic mobility of this protein in native acrylamide gels
varies concomitantly to changes in the nitrogen regime. Some of the mobility alterations seem to
result from interactions with other proteins. Thus, we have observed that the apparent molecular
mass of GltX varies under the conditions tested, and we have identified a 20 kDa protein that
specifically interacts with GltX. Our present efforts are focused to elucidate the effects of such
interactions on the function of GltX and to check if this protein also suffers some covalent posttranslational modifications.
(1) Ibba M, Decker HD, Sthatopoulos C, Tumbula DL & Söll D (2000) Trends Biochem Sci 25:311-316
(2) Freist W, Gauss DH, Söll D & Lapointe J (1997) Biol. Chem. 378:1313-1329.
(3) Luque I, Contreras A, Zabulon G, Herrero A and Houmard J (2002).Mol. Microbiol. 46: 1157-1167.
*Corresponding author
74
Session II Poster Presentation Abstracts
Nitrogen regulation in cyanobacteria: new insights in responses mediated by
the PII signal transduction protein
KARL FORCHHAMMER*, M. FADI AL-DEHNI, ANNETTE HEINRICH, NICOLE KLOFT,
MANI MAHESWARAN and ULRIKE RUPPERT
Institut für Mikrobiologie und Molekularbiologie der Justus-Liebig Universität Giessen, Heinrich-Buff-Ring 26-32;
D-35392 Giessen, Germany
PII signal transduction plays pervasive roles in microbial nitrogen control. Among all of the
various bacterial PII signalling systems, that in cyanobacteria is so far unique: in unicellular
strains, the mode of covalent modification is by serine phosphorylation and the interpretation of
the cellular nitrogen status occurs by measuring the cellular 2-oxoglutarate levels (1). Recent
advances have been the identification of the phospho-PII phosphatase (2), the resolution of the
crystal structure of PII proteins from Synechococcus and Synechocystis strains (3) and the
identification of novel functions of PII regulation. PII is required for the control of nitrate/nitrite
uptake as well as for the induction of NtcA-dependent gene expression under conditions of
nitrogen deprivation(4). Phosphorylated PII signals nitrogen deficiency towards NtcA thereby
greatly enhancing its activity under conditions of nitrogen deprivation. Dephosphorylation of
PII-P (upon nitrogen-excess or carbon-limited conditions) is specifically catalysed by a protein
phosphatase of the 2C family (PphA) (2). Phenotypic analysis of the PphA-deficient mutant
demonstrated a requirement for PphA-dependent PII dephosphorylation to optimise the
utilization of nitrate as N-source. In the absence of non-phosphorylated PII (corresponding to the
high phosphorylation state of PII in the PphA deficient mutant), the cells are unable to adjust the
activities of nitrate- and nitrite reductases: under conditions of limiting PSI-reduced ferredoxin,
the activity of nitrate reduction exceeds that of nitrite reduction, leading to excess formation and
excretion of nitrite. In agreement with its requirement for nitrate utilization, the levels of the
PphA increase with the nitrate and nitrite concentration in the medium.
An additional role of non-phosphorylated PII could be identified recently by yeast-two hybrid
screening: N-acetyl glutamate kinase (NAGK), the key enzyme of the arginine biosynthesis
pathway, forms a tight complex with PII (5). Upon complex formation, the catalytic activity of
NAGK is greatly enhanced and arginine feedback control is alleviated (details of PII-NAGK
complex formation will be shown in the presentation by M. Maheswaran). In vivo NAGK
activity is controlled by the phosphorylation status of PII and therefore, arginine synthesis is the
first amino acid biosynthetic pathway, which is under global carbon/nitrogen control. These
findings highlight the role of PII to coordinate a concerted cellular response of key steps of
nitrogen metabolism according to the physiological needs of the cells.
(1)Forchhammer, K. (2004) FEMS Microbiol. Rev. 28: 319-333.
(2) Ruppert, U., Irmler, A., Kloft, N. und Forchhammer, K. 2002. Mol. Microbiol. 44: 855-864
(3) Xu, Y., Carr, P.D., Clancy, P., Garcia-Dominguez, M., Forchhammer, K., Florencio, F., Tandeau de Marsac, N.,
Vasudevan, S. and Ollis, D. (2003) Acta Cryst.D 59: 2183-2190.
(4) Aldehni, M.F., Sauer, J., Spielhaupter, C. Schmid, R. und Forchhammer, K. (2003) J. Bacteriol. 185: 2582-2591
(5) Heinrich, A., Maheswaran, M. Ruppert, U. and Forchhammer, K. (2004) Mol. Microbiol.52:1303-1314.
* corresponding author
75
Session II Poster Presentation Abstracts
Possible role of a noncoding RNA in the initiation of heterocyst differentiation
ANDREY V MATVEYEV, YUE ZHAO, JEN FETTWEIS, and JEFF ELHAI*
Dept. of Biology, Virginia Commonwealth University, Richmond VA 23284
From the onset of nitrogen starvation of
Anabaena PCC 7120, a series of events unfold
culminating about 18 hours later in the appearance of mature, N2-fixing heterocysts. Hundreds of
genes are induced at different times over the course of differentiation (1). Several specific genes
important to the process have been identified, most notably hetR (2), whose expression is
necessary and (in some circumstances) sufficient to trigger heterocyst differentiation. However,
despite much effort, no global genetic circuitry has been elucidated of comparable explanatory
power to the cascade of sigma factors governing the temporal and spatial expression of genes in
during sporulation by Bacillus subtilis (3). Whatever circuitry is eventually found promises to
represent something new to biology. Serendipity may work where a rational search has failed.
In an attempt to understand the role of DNA methyltransferases in the control of cell cycle and
heterocyst differentiation, we cloned and
inactivated dmtB, encoding a GGCCspecific methyltransferase. Surprisingly,
the resulting mutant was unable to initiate
heterocyst differentiation. Loss of the
methyltransferase itself, however, was not
responsible for this remarkable phenotype,
as complementation of the mutant with
intact dmtB restored DNA methylation but
not the ability to differentiate. Moreover, when the interrupting C.K3 cassette (4) was inserted in
the opposite orientation (antiparallel to dmtB), methylation was lost but heterocyst differentiation
was normal. These results pointed to activation by the strong PpsbA promoter of C.K3 of a
downstream gene, perhaps t r p D 2 , highly similar to genes encoding anthranilate
phosphoribosyltransferase (Anabaena possesses another similar gene in its trp operon).
Activation of trpD2 with the PpsbA promoter had no effect on differentiation, nor did inactivation
of the gene by C.K3 placed in parallel to trpD2. However, C.K3 placed antiparallel to the gene
produced the same Het- phenotype as did the original dmtB knockout. Taken together, these
results indicated that expression of some element in or near the small intergenic region blocked
heterocyst differentiation. The intergenic sequence is quite interesting. A 17-bp segment flanked
by inverted repeats matches almost exactly a segment immediately upstream from hetRI, the
transcriptional start site closest to hetR (5). Furthermore, while the corresponding 17-bp segment
in the dmtB/trpD2 intergenic region of Nostoc punctiforme differs in three nucleotides, but two
of these differences are found also in Nostoc's hetRI region. These and other findings are most
readily explained by the existence of a noncoding RNA transcribed from the intergenic region
that regulates the expression of hetR.
1. Ehira S, Ohmori M, Sato N (2003). DNA Research 10: 97–113.
2. Buikema WJ, Haselkorn R (1991). Genes Develop 5:321-330.
3. Errington J (2003). Nat Rev Microbiol 1:117-26.
4. Elhai J, Wolk CP (1988). Gene 68:119-138.
5. Buikema WJ, Haselkorn R (2001). Proc Natl Acad Sci USA 98:2729-2734.
*
Jeff Elhai, Dept. of Biology, 1000 W. Cary St., Virginia Commonwealth University, Richmond VA 23284.
E-mail:[email protected]; Tel: 1-804-828-0794
76
Session II Poster Presentation Abstracts
Construction of a nitrate responsive Synechocystis sp. strain PCC 6803
bioreporter for estimating nitrate bioavailability in freshwater
NATALIA V. IVANIKOVA*, R. MICHAEL L. McKAY AND GEORGE S. BULLERJAHN, Department of
Biological Sciences, Bowling Green State University, BOWLING GREEN OH 43403
A recently developed approach for the quantification of nutrient
bioavailability in aquatic
ecosystems is the use of cyanobacterial whole-cell bioreporters (eg. 1). Indeed, previous studies have
successfully employed recombinant bioluminescent cyanobacterial strains to monitor Fe and P
availability in freshwater. In this study, we constructed a Synechocystis sp. PCC 6803 bioluminescent
reporter for the assessment of nitrate bioavailability. Specifically, a 380 base pair DNA fragment
containing the NtcA/B-dependent nitrate-activated nirA promoter was fused to the bacterial luciferase
genes, luxAB, and introduced into Synechocystis by genetic transformation (2). Characterization of this
strain yielded dose-dependent increased bioluminescence as nitrate increased in the medium from 1-100
micromolar. This biosensor will be deployed in Summer 2004 in an effort to determine the factors
limiting nitrate drawdown in Lake Superior, an ecosystem whose nitrate levels have increased 6-fold in
the last century to approximately 30 µM. Pilot experiments performed on pelagic Lake Superior water
samples suggest that the bioreporter luminescent response is attenuated, indicating nutrient and/or light
limitation constraining nitrate utlilization, Indeed, amendment of water samples with Fe and P together
yielded luminescence appropriate for the nitrate levels detected by chemical means (3). These data
suggest that picophytoplankton are co-limited by P and Fe in the lake. Supported by NSF grants OCE0327738 and 0352274 awarded to R.M.L.M. and G.S.B.
1. Porta, D., G.S. Bullerjahn, K.A. Durham, S.W. Wilhelm, M.R. Twiss and R.M.L. McKay. 2003. Physiological
characterization of a Synechococcus sp. (Cyanophyceae) strain PCC 7942 iron-dependent bioreporter for freshwater
environments. J. Phycol. 39: 64-73.
2. Kunert, A., M. Hagemann and N. Erdmann. 2000. Construction of promoter-probe vectors for Synechocystis sp. PCC
6803 using the light-emitting reporter systems Gfp and LuxAB. J. Microbiol. Methods 41: 185-194.
3. Ivanikova, N.V., R.M.L. McKay and G.S. Bullerjahn. 2004. Construction and characterization of a freshwater
nitrate-sensing bioreporter. Limnol. Oceanogr: Methods, submitted.
*corresponding author, [email protected]
77
Session II Poster Presentation Abstracts
Characterization of the DNA-binding activity of Anabaena 7120 devH protein
MARTHA E. RAMIREZ AND STEPHANIE E. CURTIS*
Department of Genetics, Box 7614, North Carolina State University, Raleigh, NC 27695-7614
The Anabaena sp. strain PCC 7120 devH gene is essential for heterocyst function (1). A
devH mutant is capable of nitrogen fixation but only under anaerobic conditions, due in part to
reduced glycolipid gene expression and absence of the heterocyst glycolipid layer (2). The
precise role of DevH in heterocyst development and function is unknown, but the structure of the
protein suggests it is a transcriptional regulator. A recently identified DevH target is the
promoter of the devH gene. DevH binds a region of the devH promoter that contains two
adjacent 12-bp palindromes. An in vitro binding-site selection (SELEX) study identified 46
related sequences that bind DevH with high affinity and are similar to the binding sites identified
in the devH promoter. The consensus DevH binding sequence is very similar to that of the
cyanobacterial transcriptional activator NtcA.
(1) Hebbar, P. B., and S. E. Curtis. 2000. Characterization of devH, a gene encoding a putative DNA binding
protein required for heterocyst function in Anabaena sp. strain PCC 7120. J. Bacteriol. 182:3572-3581.
(2) Ramirez, M. E., Hebbar, P. B., Zhou, R., Wolk, C. P. and S. E. Curtis. Anabaena sp. strain PCC 7120 gene
devH is required for synthesis of the heterocyst glycolipid layer. Submitted.
78
Session II Poster Presentation Abstracts
Testing whether insertion sequences within putative regulatory
genes affect the phenotype of Anabaena sp. strain PCC 7120
SIGAL LECHNO-YOSSEF1, KARIN JÄGER1, C. PETER WOLK1* SATOSHI TABATA2,
AND TAKAKAZU KANEKO2, 1MSU-DOE Plant Research Laboratory, Michigan State
University, E. Lansing, MI 48824, U.S.A. and 2Kazusa DNA Research Institute, 2-6-7 Kazusakamatari, Kisarazu, Chiba, 292-0818, Japan
Regulatory protein kinases in eukaryotes normally phosphorylate hydroxyl amino acids such
as serine, threonine and tyrosine, whereas those in bacteria more commonly phosphorylate
histidine residues. Histidine kinases are involved in such different signaling processes as host
recognition in symbiosis and pathogenesis, sensing of the availability of carbon and nitrogen,
chemotaxis, and differentiation, including sporulation. Insertion sequences (ISs) are transposable
elements 0.8 to 2.5 kb in size that normally bear genes required for their transposition. Active ISs
are present in Anabaena 7120 (1-4). Analysis of the Anabaena 7120 genome showed three
instances in which presumptively encoded kinases may have been interrupted by IS891:
chromosomally encoded His kinases All3985 (extended) and Alr4105 (extended) and _
megaplasmid-encoded Ser/Thr kinase Alr7232 (extended). Gliding motility and the formation of
gas vesicles and akinetes are common in other members of the Nostocaceae, and orthologs of
requisite genes are present in Anabaena 7120. Perhaps these processes do not occur in Anabaena
7120 because of IS-transposition. To test this idea, we cured the ISs from the genomic sequences
by PCR and introduced the cured sequences back into Anabaena 7120. First, the portions of each
IS-interrupted ORF to either side of the IS were amplified separately by PCR with pairs of
primers, one outer and one ORF-internal. Each ORF-internal primer was comprised of sequences
contiguous with both ends of the IS. The resulting, PCR-amplified fragments were then used as
templates with the two original external primers to amplify the gene without the IS. The cured
form of the gene was introduced into an appropriate, pUC-based sequencing clone from the
Anabaena sequencing project, and the insert from the modified clone was transferred into
derivative pRL2833b (5) of Nostoc replicon pDU1. The pDU1 construct was then introduced
into Anabaena 7120 to generate a strain bearing both the IS-interrupted, chromosomal ORF and
the pDU1-borne, cured form of the ORF with its native promoter. To date, when grown under
nitrogen-replete, nitrogen-deficient, or nitrogen- and phosphorus-deficient conditions, no
derivative strain bearing any of the cured genes has shown a phenotype observable by light
microscopy that differs from that of the original Anabaena. In addition, RT-PCR of alr4105
(extended) showed no transcription of that gene. Plasmids were recovered from exconjugants to
which the three pDU1-derivatives had been transferred. Those from two resembled what had
been transferred. However, three plasmids recovered from the strain to which uninterrupted
(extended) alr7232 had been transferred provided evidence suggestive of IS891 having been reintroduced into the pDU1 derivative in its original position, consistent with double
recombination having taken place.
1. Cai, Y. and C.P. Wolk, J. Bacteriol., 1990. 172: 3138-3145. 2. Bancroft, I. and C.P. Wolk, J. Bacteriol., 1989.
171: 5949-5954. 3. Kaneko, T., et al., DNA Res., 2001. 8: 205-213. 4. Alam, J., et al., J. Bacteriol., 1991. 173: 57785783. 5. Huang, G., and C.P. Wolk, unpubl.
79
Session II Poster Presentation Abstracts
The response of Synechocystis sp. PCC 6803 to nitrogen starvation:
transcriptomics versus proteomics
VLADIMIR KRASIKOV 1*, HENK L. DEKKER 2, and HANS C. P. MATTHIJS 1
1
Aquatic Microbiology, Institute of Biodiversity and Ecosystem Dynamics, Universiteit van
Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands
2
Mass Spectrometry Group, Swammerdam Institute for Life Sciences, University of Amsterdam,
Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
Nitrogen is one of the nutrients that is playing an essential role in metabolism and ultimately
is needed for the growth of cyanobacteria. To survive in nutrient limited conditions, cells should
mobilise systems for high affinity uptake of limiting nutrients, adapt physiological processes to
enable more economic usage of the nutrient concerned, and strive to be able to utilise nontraditional resources. Recently, an effective technique called DNA microarray has been
developed and used to monitor gene expression in cyanobacteria. Classical proteomics including
two-dimensional gel electrophoresis for separation and mass spectrometry for qualitative
analysis of proteins in complex mixtures has been successfully applied to evaluate stress
responses in cyanobacteria. However, overlaying of DNA array results with proved expression of
proteins has not been reported so far. Protein expression profiles in Synechocystis sp. PCC 6803
cells, in response to relatively short nitrogen starvation, were determined using the cleavable
isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the
mixed protein samples by SDS-PAGE (total proteins isolated from cells growing 12 hours in
nitrogen free medium were mixed in proportion 1:1 with proteins isolated from normally grown
culture), followed by excision of regions from an entire gel lane. Proteins were subjected to ingel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid
chromatography coupled to tandem mass spectrometry. The comparison of the global
transcriptome analysis with qualitative and quantitative proteomics of cyanobacterial cells
exposed to nitrogen starvation will be discussed.
This work was supported by a Dutch Science Foundation Pioneer grant to Prof. dr. J. Huisman,
and by a NWO grant.
* Corresponding author:
Vladimir Krasikov
Aquatic Microbiology,
Institute of Biodiversity and Ecosystem Dynamics,
Universiteit van Amsterdam,
Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands
E-mail: [email protected]
Tel.: +31205257070,
Fax: +31205257064
80
Session III Poster Presentation Abstracts
Poster Presentations Session III
Physiology, Metabolism and Global Responses (II)
Session Chair: Francis X. Cunningham, Jr., University
of Maryland
81
Session III Poster Presentation Abstracts
Genetic analysis of nonribosomal peptide synthetase genes in cyanobacteria
AKITO NISHIZAWA1, TAKAKAZU MIURA1, ARIZAL BIN ARSHAD1, TOMOYASU
NISHIZAWA1, MUNEHIKO ASAYAMA1, TOMOYO NAKANO2, KIYONAGA FUJII2,
KENICHI HARADA2 AND MAKOTO SHIRAI1
1
College of Agriculture, Ibaraki University, Inashiki Ibaraki 300-0393, Japan
2
Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan
Cyanobacteria are known to produce a wide rage of secondary metabolites, including
nonribosomal peptides. Toxic cyanobacterial waterblooms are found worldwide in eutrophic
lakes, ponds and dams. Microcystis species are some of the most common waterbloom-forming
species of caynobacteria. Microcystis aeruginosa K-139, which was isolated from Lake
Kasumigaura, produces nonribosomal peptides, hepatotoxic microcystin and neurotoxin
micropeptin. We have identified the complete synthetase gene structures for microcystin and
micropeptin. The mcirocystin synthetase gene (mcy) cluster of M. aeruginosa K-139, is
composed of two nonribosomal peptide synthetase (NRPS)-polyketide synthase hybride genes,
three NRPS genes, one polyketide synthase (PKS) gene, and four genes for modification of Mcy
proteins. The micropeptin synthetase gene (mip) was consisted of seven NRPS genes.
Furthermore, we identified two non-ribosomal peptide synthetase genes, psm3 and psm4, from a
strain K-139. The psm3 gene was a cluster spanning 30kb, including 14 bidiretionally transcribed
open reading frame arranged in two operon. Primer extension and QRT-PCR analyses revealed
the transcriptional expression of psm3. Alignment analysis in the binding pocket of adenylation
domains in NRPS suggested that Psm3C activate Asp. ATP-PPi exchange experiment revealed
that Psm3B activate Tyr. Partial DNA sequence analysis revealed that the psm4 gene is involved
in nonribosomal peptide synthesis. In addition to microcystin and micropeptin, we isolated two
nonribosomal peptides, aeruginosin and microviridin, from M. aeruginosa K-139. Disruption of
psm3 did not revealed disappearance both of aeruginosin and microviridin production. These
results indicated that a strain K-139 produces at least five nonribosomal peptides. To understand
a molecular mechanism of non-ribosomal peptide synthesis and perform genetic engineering, we
attempt to produce nonribosomal peptides of Cyanobacteria in heterologous hosts.
82
Session III Poster Presentation Abstracts
Ribonucleotide reduction in the cyanobacteria
FLORENCE K. GLEASON, Dept. of Plant Biology, 250 Biological Sciences Center, University
of Minnesota, St. Paul, MN 55108 USA.
Ribonucleotide reductase reduces ribonucleotides to the corresponding deoxyribonucleotides
and supplies the precursors for DNA synthesis and repair. Unlike most essential enzymes, the
amino acid sequence of ribonucleotide reductases is not conserved among different organisms
except for a few critical residues in the active site required for thiyl radical formation during
catalysis. Ribonucleotide reductases are divided into three classes based mainly on the
mechanism used to generate the catalytically important thiyl radical. Class I reductases utilize a
separate protein cofactor containing an iron stabilized free radical that is transferred to the active
site during turnover. This is the most wide-spread type of reductase found in some bacteria and
most eukaryotic organisms. Class II enzymes utilize coenzyme B12 to generate the enzyme
radical and are found in a variety of bacteria and archaea. Class III enzymes also require an iron
cofactor protein and are active only under strictly anaerobic conditions (1). Cyanobacteria seem
to be unique in that most of these organisms have a class II enzyme. We have cloned the gene
for the ribonucleotide reductase from Anabaena sp. PCC7120. The gene codes for a 1,172
amino acid protein that contains a 407 amino acid intein. The gene has been expressed in E. coli
and the intein removes itself, yielding an active reductase. The protein reduces all four common
ribonucleoside triphosphates and is absolutely dependent on the presence of coenzyme B12 (2).
An external reducing agent is also required, either the artificial reductant, dithiothreitol, or the
disulfide-containing redox protein, thioredoxin. The enzyme is relatively inactive but can be
stimulated by adding deoxynucleotide triphosphates or high concentrations of ATP. Sequence
comparisons to other reductases suggest that the cyanobacterial proteins are all related to the
Anabaena enzyme, showing 75-90% sequence similarity. However, the cyanobacterial
reductases show only modest similarity to class II enzymes from other bacteria such as
Lactobacillus or Geobacter. The cyanobacterial enzymes show almost no sequence conservation
with Class II enzymes found in photosynthetic bacteria. Also in contrast to many other groups of
bacteria, the cyanobacteria do not have genes for other classes of reductases. It is suggested that
their occupation of iron-poor environments provides a strong selection for the maintaining only
the B12-dependent ribonucleotide reductase in the cyanobacteria.
1. Jordan, A., and Reichard, P. (1998) Ribonucleotide reductases. Annu. Rev. Biochem. 67:71-98.
2. Gleason, F.K. and Olszewski, N. (2002) Isolation of the gene for the B12-dependent ribonucleotide reductase from
Anabaena sp. Strain PCC7120 and expression in Escherichia coli, J. Bacteriol. 184:6544-6550.
83
Session III Poster Presentation Abstracts
Protein trans-splicing of the -subunit of the DNA-polymerase III of
Synechocystis sp. PCC 6803: does it exert a regulatory role?
Monika Klissenbauer, Rüdiger Schulz-Friedrich and Jens Appel*
Botanisches Institut, Christian-Albrechts-Universität, Am Botanischen Garten 1-9,
D-24118 Kiel, Germany
*e-mail: [email protected]
Inteins are peptide sequences that self splice out of completely translated proteins by joining the flanking
sequences to yield a new peptide bond between the so called exteins (1). Since these events are reminiscent of
introns splicing themselves out of transcribed RNAs and linking the exons to a complete translatable ORF these
names were given accordingly. Recent years saw the rapid development of many protein modifying techniques
using inteins and much effort has been placed on the elucidation of the underlying mechanisms (2). In contrast the
functional significance of inteins has been studied only rarely. Up to now protein trans-splicing is known only from
the cyanobacterial DnaE protein that forms the -subunit of the DNA-polymerase III (3). The split DnaE was first
found in the complete genome sequence of Synechocystis sp. PCC 6803. It is encoded in two different ORFs that are
separated more than 745 kb on the chromosome. In contrast to other bacteria, cyanobacteria are known to contain
several genome equivalents per cell. It also has been shown that the number of chromosomes varies with culture
age, cell type and other conditions (4). The regulatory mechanisms underlying these variations have not yet been
characterized. We therefore set out to investigate the split dnaE of Synechocystis by eliminating the intein sequences
and joining the two separate dnaE genes to one continuous ORF. Several independent clones of this mutant strain
were tested for their growth characteristics and genome equivalents under different conditions in comparison to the
wild type. The results are discussed concerning a regulatory role of DnaE trans-splicing on cell division and the
copy number of chromosomes.
1. Paulus, H (2000) Annu. Rev. Biochem. 69, 447-496.
2. Noren, CJ, Wang, J, Perler, FB (2000) Angew. Chem. Int. Ed. 39, 450-466.
3. Wu, H, Hu, Z, Liu, XQ (1998) Proc. Natl. Acad. Sci. USA 95, 9226-9231
4. Lee, MH, Scherer, M, Rigali, S, Golden JW (2003) J. Bacteriol. 185, 4315-4325
84
Session III Poster Presentation Abstracts
Characterization of group 2 sigma factors of RNA polymerase and their roles
in the cyanobacterium Synechocystis sp. strain PCC 6803
SOUSUKE IMAMURA, MUNEHIKO ASAYAMA*, MAKOTO SHIRAI
Ibaraki University, Laboratory of Molecular Genetics, Ami, Ibaraki 300-0393, Japan
The RNA polymerase (RNAP) holoenzyme of eubacteria consists of a core enzyme and a
sigma factor. The core enzyme catalyzes RNA synthesis and the sigma factor is required for the
initiation of transcription from a specific promoter sequence. A unicellular cyanobacterium
Synechocystis sp. strain PCC 6803 possesses nine sigma factors, group 1, SigA; group 2, SigB to
SigE; and group 3, SigF to SigI, by its whole genome sequence information (1). However, the
clear functions and roles of individual sigma factors remain to be resolved. Here we present the
functions of group 2 sigma factors in PCC 6803. We identified dark-/light-induced sigma factor
SigB/SigD, of which expression was accelerated under opposite redox (oxidation/reduction)
states in an electron transport chain of photosynthesis. Furthermore, expression of the darkinduced lrtA and light-induced psbA2/3 transcript was significantly reduced in the sigB and sigD
knockout strains, respectively. These findings clearly showed that SigB/SigD contribute to
transcription for a subset of dark-/light-responsive genes in the cyanobacterium (2). On the other
hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the
exposure of cells to heat-shock, and specific promoter recognition by SigB on the heat-shock
hspA promoter were observed. These findings clearly indicated that SigB is also a heat-shock
responsive sigma factor (1). In analyses of transcript and protein levels using the sigC knockout
strain, it was revealed that the glnB, which encodes for a nitrogen regulatory protein PII, nitrogen
promoter (P2) was specifically recognized by SigC in the stationary phase under conditions of
nitrogen deprivation. In vitro studies with purified enzymes also indicated effective transcription
from P2 by RNAP-SigC with NtcA, a global nitrogen regulator that belongs to the Crp family.
These results clearly suggested that SigC is a sigma factor that regulates nitrogen related gene
expressions in the stationary phase (3).
(1) Imamura, S. et al. (2003) J Mol Biol 325, 857-872.
(2) Imamura, S. et al. (2003) FEBS Lett 554, 357-362.
(3) Asayama, M. et al. (2004) Biosci Biotechnol Biochem 68, 477-487.
85
Session III Poster Presentation Abstracts
The composition and dynamics of the phytoplankton assemblage in Lake Kinneret
is strongly affected by cyanobacterium - dinoflagellate communication
1
SCHATZ DANIELLA, 1VARDI ASSAF, 2SUKENIK ASSAF, 1LEVINE ALEX and AARON
KAPLAN1*
1
Institute of Life Sciences, The Hebrew University of Jerusalem, Israel.
2
Oceanographic & Limnological Research, P.O.Box 447, Migdal 14950 Israel.
The reasons for annual variability in composition of phytoplankton assemblages and toxic
cyanobacterial blooms are poorly understood but may include allelopathic interactions. We show that
a bloom of a toxic cyanobacterium, Microcystis sp. or alternatively domination by the dinoflagellate,
Peridinium gatunense, in Lake Kinneret, may be accounted for by mutual density-dependent
allelopathic interactions. Over the last 30 years the abundance of these species in the lake displayed
strong negative correlation. Laboratory experiments showed reciprocal, density-dependent but
nutrient-independent inhibition of growth (1). Application of spent P. gatunense medium induced
sedimentation and subsequently massive lysis of Microcystis cells, within 24 hr, concomitantly with a
large rise in the level of McyB, which is involved in microcystin biosynthesis. The older was the
culture of Peridinium the more effective was its spent media in promoting the level of McyB in
Microcystis. We show that the induction of expression of mcyB was mediated by a factor released
from Microcystis cells during their lysis. Spent media from Microcystis inhibited internal carbonic
anhydrase in Peridinium which than became CO2-limited. The consequent accumulation of reactive O2
species resulted in activation of a MAPK cascade which led to apoptosis-like process, mediated by
specific proteases, in some of the Peridinium cells and cell division of others. We propose that a crosstalk via allelochemicals may explain the composition of the phytoplankton assemblage in Lake
Kinneret in the spring, including presence or absence of Peridinium or Microcystis blooms.
1. Vardi, A., Schatz, D., Beeri, K., Motro, U., Sukenik, A., Levine, A. and A. Kaplan (2002) Dinoflagellatecyanobacterium communication may determine the composition of phytoplankton assemblage in a mesotrophic
lake. Current Biology 12: 1767-1772
* Corresponding author
86
Session III Poster Presentation Abstracts
Investigation of carbon and light on cyanobacterial toxin production
Douglas Graham School of life sciences, The Robert Gordon University, St. Andrews street,
Aberdeen, UK, AB25 1HG
Linda A. Lawton* School of life sciences, The Robert Gordon University, St. Andrews street,
Aberdeen, UK, AB25 1HG
Cyanobacteria (blue green algae) are a widely distributed and diverse group of unicellular and
multicellular photosynthetic prokaryotes. They are known to produce toxic secondary
metabolites that fall into two main classes, hepatotoxins and neurotoxins the function of which is
still unclear. Globally the most commonly occurring toxins are hepatotoxins, microcystin and
nodularin often found in the bloom forming genera Microcystis, Anabeana, Nostoc and
Oscillatoria. Typically found in eutrophic water bodies and slow moving rivers, they pose a
significant hazard to both human and animal health following ingestion of contaminated water.
The implication of toxic bloom formation is a major concern for water management; therefore
understanding the natural function of these secondary metabolites may help in treatment.
Research has primarily focused on the effect of environmental factors like light, temperature, pH,
limited nutrients and micronutrients, but all the findings have produced no clear indication of
these factors being responsible for the regulation of toxin production. Our research into the
effects of increasing inorganic carbon and light intensity on hepatotoxin production in
Microcystis aeruginosa, Microcystis sp. and Nodularia spumigenia, found significant changes in
the levels of toxin produced. Increasing inorganic carbon reduced levels of biomass and cause
significant reductions in the level of intracellular microcystin and nodularin. In the most
profound case an 80% reduction in the intracellular level of microcystin was observed when
grown in the presence of 40mM sodium bicarbonate. Also previous studies have suggested that
toxins are only released after call lysis, but in the presence of increased inorganic carbon and
light the extracellular toxin level exceeded the intracellular levels in cultures of N. spumigenia.
* Corresponding author, e mail address [email protected]
87
Session III Poster Presentation Abstracts
“Chlorophyll Pasteur point”, a critical atmospheric oxygen level for ancestral
chlorophyll biosynthesis
YUICHI FUJITA* and SHOJI YAMAZAKI
Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, JAPAN
The advent of oxygenic photosynthesis in ancestral cyanobacteria led to the most
important biologically driven change in the Earth’s environment. Cyanobacteria became
ubiquitous in all environments containing water, an unlimited source of electron donors, and
transformed the Earth’s atmosphere from anoxic to oxic.
To survive in the oxidative
environments they generated, however, the creation of an oxygen-tolerant enzyme in the
penultimate step of the chlorophyll biosynthesis pathway appears to have been necessary. Here
we provide evidence that the rise of atmospheric oxygen level in Proterozoic era may have been
a major selective pressure to create an oxygen-tolerant protochlorophyllide (Pchlide) reductase
(light-dependent Pchlide oxidoreductase; LPOR) that compensated for the existing oxygensensitive Pchlide reductase (dark-operative Pchlide oxidoreductase; DPOR). A mutant, YFP12,
of an extant cyanobacterium Plectonema boryanum lacking LPOR could not grow
photoautotrophically under aerobic conditions, but grew under anaerobic conditions. The
maximal oxygen level in which YFP12 was able to grow was 3% (v/v). The contents of three
subunits, ChlL, ChlN and ChlB, of DPOR were greatly increased in the YFP12 cells grown in
the anaerobic conditions compared with the wild-type cells. These results suggest that 3%
oxygen in the environment is the upper limit for protecting the DPOR activity from oxygen. The
oxygen level 3% is coincident with a proposed atmospheric oxygen level at 2.2-2.0 gigayears
ago (Gya), implying that the oxygen-tolerant Pchlide reductase, LPOR, evolved no later than
2.2-2.0 Gya from NAD(P)(H)-accepting oxidoreductases family.
We propose to call
the critical oxygen level for the probable ancestral chlorophyll biosynthesis “Chlorophyll Pasteur
point”.
*Corresponding author
88
Session III Poster Presentation Abstracts
Construction of cyanobacterial bioreporters for detecting nutrient deficiency
in marine waters.
R. BOYANAPALLI, G. S. BULLERJAHN, R. M. L. MCKAY*
Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio,
43403 USA.
Picoplankton (0.2 – 2 µm) serve as the dominant phytoplankton assemblage in most oligotrophic
waters. A long held paradigm in marine science is that phytoplankton are limited by nitrogen. However,
numerous reports in recent years have demonstrated that both phosphorus and iron deficiency are
widespread, particularly associated with oligotrophic oceanic gyres and “high nutrient, low chlorophyll”
regions. Whereas concentrations of dissolved nutrients can serve as a first-order proxy for nutrient
deficiency, chemical speciation can represent an obstacle in the application of this proxy. Nutrient
bioavailability could be understood better if a biological system were to be used to estimate nutrient
supply. We have been working on the development and characterization of cyanobacterial bioreporters
to monitor bioavailable phosphorus and iron in marine waters. The cyanobacterium used in this study is
the coastal isolate Synechococcus sp. PCC 7002. In developing these strains, we have used expression
vectors capable of integrating into the cyanobacterial chromosome by homologous recombination. The
iron bioreporter we are developing features the promoter element of the Synechococcus iron stress
inducible gene isiAB fused to promoterless bacterial luciferase genes, luxAB, from Vibrio harveyi. The
phosphate bioreporter we are constructing features the promoter for phoH, a gene up-regulated under
phosphate stress. Both reporters feature chloramphenicol selectable markers. Expression of luciferase
regulated by isiAB or phoH promoters will be quantified with a luminometer and the response calibrated
using Aquil growth medium containing known additions of phosphate or iron. We plan to test the
bioreporters on samples collected using clean sampling methods from the Southern Ocean (FeCycle
cruise; January, 2003) and from the central North Pacific gyre region (RoMP III, August/September,
2003). For each of these cruises, we have complementary measures of phosphate (alkaline phosphatase
activity) and iron bioavailability (ferredoxin index, cellular elemental stoichiometry) with which we can
compare the bioreporter response. Supported by NSF grant OCE-0327738 awarded to R.M.L.M. and
G.S.B.
*corresponding author, [email protected]
89
Session IV Poster Presentation Abstracts
Poster Presentations Session IV
Photosynthesis and responses to light
Session Chair: John Cobley, University of San
Francisco
90
Session IV Poster Presentation Abstracts
Expression and mutagenesis of mapA, a Synechococcus sp. PCC 7942 iron
responsive gene: evidence for oxidative stress protection
ARMERIA VICOL1*, CHRISTEL S. HASSLER2 AND GEORGE S. BULLERJAHN1, 1Department of Biological
Sciences, Bowling Green State University, Bowling Green OH 43403, and 2Department of Biology, Clarkson
University, Potsdam NY 13699
Our lab has recently focused on the construction of cyanobacterial bioreporters suitable for use as
sensors for nutrient deficiency and stress in natural habitats (1,2). Previous work by Sherman’s group
has identified a Synechococcus sp. PCC 7942 gene, mapA, expressed under low iron conditions (3), and
more recent work has shown that mapA transcription is rapidly activated upon treatment of cultures
with peroxide (4). We have constructed PmapA::luxAB fusions to identify both the elements of the
promoter and Fe concentration yielding low iron-dependent transcription. Additionally, we have
constructed a mapA mutant that expresses a truncated MapA protein that likely interferes with wild type
mapA function. Our studies reveal that low Fe dependent expression occurs at Fe concentrations (pFe
18.9) over 10-fold higher than those triggering transcription from the Fur-regulated isiA promoter, and
secondly, promoter fusion deletions suggest the involvement of multiple transcription factors
responsible for mapA expression under conditions of low Fe growth, peroxide treatment and growth
phase. Lastly, the mapA construct expressing the truncated MapA protein exhibits a high light lethal
phenotype consistent with MapA playing a role in oxidative stress. Owing to the pattern of Low Fe
expression, we are currently using the luxAB promoter fusions in concert with other promoter fusions to
determine bioavailable Fe in fresh water environments (Lake Superior). Supported by NSF award
0327738.
1.
Porta, D., G.S. Bullerjahn, K.A. Durham, S.W. Wilhelm, M.R. Twiss and R.M.L. McKay. 2003. Physiological
characterization of a Synechococcus sp. (Cyanophyceae) strain PCC 7942 iron-dependent bioreporter for freshwater
environments. J. Phycol. 39: 64-73.
2.
Ivanikova, N.V., R.M.L. McKay and G.S. Bullerjahn. 2004. Construction and characterization of a
freshwater nitrate-sensing bioreporter. Limnol. Oceanogr: Methods, submitted.
3.
Webb, R., T. Troyan, D. Sherman and L.A. Sherman, 1994. MapA, an iron-regulated, cytoplasmic
membrane protein in the cyanobacterium Synechococcus sp. PCC 7942. J. Bacteriol. 176: 4906-4913.
4.
Yousef, N., E.K. Pistorius and K.-P. Michel. 2003. Comparative analysis of idiA and isiA transcription
under iron starvation and oxidative stress in Synechococcus elongatus PCC 7942 wild type and selected
mutants. Arch Microbiol. 180: 471-483.
*corresponding author
91
Session IV Poster Presentation Abstracts
Cyanobacterial respiratory terminal oxidases
G. SCHMETTERER, D. PILS, C. TRAUTNER, C. WILKEN, B. WIESER
Institute of Physical Chemistry, Vienna University, UZA2, Althanstrasse 14, A-1090 Vienna,
Austria
Respiratory terminal oxidases (RTO's) are the key enzymes of cyanobacterial respiration,
since they are probably the only components of the cyanobacterial repiratory electron transport
chain not directly involved in photosynthesis. Except for Gloeobacter violaceus PCC 7421 that
contains no thylakoids, all cyanobacteria probably have two independent respiratory chains, one
in the cytoplasmic membrane and one - linked to photosynthetic electron transport - in the
thylakoids. All cyanobacterial RTO's belong to only two groups of enzymes, the relatives of the
heme-copper enzymes and those related to cytochrome bd quinol oxidase (Qox) of Escherichia
coli. (Quite recently the existence of genes related to the cyanide-insensitive terminal oxidase of
chloroplasts in a few cyanobacteria was discovered on the basis of sequence similarities;
however, no biochemical or genetic evidence for their function in cyanobacteria is available so
far, and their involvement in respiration must remain uncertain for the time being.) Heme-copper
RTO's are either genuine cytochrome c oxidases (Cox) or related enzymes called ARTO
(Alternate Respiratory Terminal Oxidase), whose electron donor is uncertain. A striking result of
the total genomic sequences of a number of cyanobacteria is that the number of RTO's present in
the different strains is by far not constant. Indeed, all strains contain at least one cytochrome c
oxidase, but ARTO or Qox is not present in all strains. It is our aim to characterize the function
of each set of genes encoding RTO's in cyanobacteria. Due to the difference of occurrence of
such genes in different cyanobacteria, it is impossible to generalize results obtained in one strain
to another one or cyanobacteria in general. Heterocyst forming strains appear to contain more
RTO's than simpler cyanobacteria (possibly up to five). We have analyzed especially
Synechocystis sp. PCC6803, Nostoc(Anabaena) sp. PCC7120 and Anabaena variabilis
ATCC29413. PCC6803 and ATCC29413 are facultative heterotrophs and one and only one RTO
(a Cox) is essential for heterotrophic growth in these strains. A highly related Cox exists in the
obligate autotroph PCC7120, where its function remains uncertain. There is now good evidence
that in PCC6803 the single ARTO probably functions as the terminal respiratory oxidase of the
respiratory chain in the cytoplasmic membrane. In PCC7120, however, one of the Cox's and an
ARTO are involved in protecting the heterocysts from the deleterious action of dioxygen on
nitrogenase. Producing mutants lacking one or more of RTO'S in cyanobacteria invariably leads
to a curious result: the total respiratory activity cannot be predicted in any mutant, and the
respiratory rates of strains containing more than one RTO are highly non-additive when
compared to the respiratory rates of those strains containing only a sinlge RTO. The possible
reasons for this will be discussed. A detailed current model of the function of the at least four,
possibly five RTO's in the complicated hetercyst forming strain ATCC29413 will also be
presented.
92
Session IV Poster Presentation Abstracts
Circadian rhythm of gene expression and cloning of clock genes in the
facultative filamentous cyanobacterium Plectonema boryanum
1*
1
2
KAZUKI TERAUCHI , MITSUNORI KATAYAMA , YUICHI FUJITA and TAKAO
1
KONDO
1 Division of Biological Science, Graduate School of Science, Nagoya University, and CREST,
JST, Nagoya 464-8602, Japan , 2 Laboratory of Molecular Plant Physiology, Graduate School
of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Cyanobacteria are the simplest organisms known to exhibit circadian rhythms. We have
studied the mechanisms of circadian rhythms using the cyanobacterium Synechococcus
elongatus PCC 7942 and demonstrated that kaiABC gene cluster is essential for the clock
functions (1). The obligate photoautotrophic organism S. elongatus PCC 7942 is not a suitable
model to address the questions that photosynthesis is involved in sustaining the expression of
circadian rhythms and that light is necessary for the oscillation of the circadian clock. In order to
focus on these aspects, we tried to monitor gene expression in the facultative cyanobacterium
Plectonema boryanum. This organism exhibits heterotrophic growth using glucose in complete
darkness and various genetic tools are also available (2). A promoterless segment of luciferase
genes was introduced downstream of the promoter for the P. boryanum chlB gene, which
encodes a subunit B of light-independent protochlorophyllide reductase, or petF gene encoding
ferredoxin. These reporter constructions were introduced in P. boryanum. We monitored the
bioluminescence of reporter strains by an automated monitoring system as promoter activity.
Bioluminescence of the reporter strains oscillated with a period of about 24 h in continuous
light. In addition, we cloned kaiABC genes from P. boryanum, showing that a circadian clock
gene cluster kaiABC was conserved as well as other cyanobacteria. These data indicate that this
organism has the same mechanism controlling circadian rhythms as S. elongatus PCC 7942.
When bioluminescence was monitored in continuous dark, it oscillated with a period about 21 h.
This observation suggests that photosynthetic metabolism is not necessary for the oscillation of
the circadian clock to persist and that light signal is needed for the clock to cycle in circadian
periodicity. Results of the effect of DCMU, an inhibitor of the photosystem II activity, on the
oscillation of bioluminescence will be presented.
1) Ishiura et al. 1998, Science 281, 1519-1523.
2) Fujita et al. 1996, Plant Cell Physiol. 37, 313-323.
*Corresponding author
93
Session IV Poster Presentation Abstracts
Unveiling the presence of more than one oscillator in S. elongatus PCC7942
EUGENIA M. CLERICO1, JAYNA L. DITTY2 AND SUSAN S. GOLDEN1*
1
2
Department of Biology, Texas A&M University, College Station TX 77843-3258 USA
Department of Biology, University of St. Thomas, 2115 Summit Ave, St. Paul, MN 55105 USA
Cyanobacteria possess intrinsic circadian clocks that allow cells to coordinate physiological
processes with the Earth’s day-night cycles. The circadian oscillator of Synechococcus elongatus
PCC 7942 (comprised of at least the KaiA, KaiB and KaiC proteins) generates daily rhythms of
expression from genes throughout its genome. One of our goals is to understand how the
cyanobacterial cell organizes its internal oscillator and transmits temporal information to clockcontrolled genes. We monitor the period, amplitude, and phasing of the circadian rhythm from
any cyanobacterial promoter by using luciferase gene fusions (Vibrio harveyi "luxAB", or firefly
"luc"), such that light production reports transcription. Inactivation of any of the group two
sigma factors genes of S. elongatus PCC 7942 (rpoD2, rpoD2, rpoD4 and sigC) singly or
pairwise alters circadian expression from the psbAI promoter, changing amplitude, phase angle,
waveform or period. However, only the rpoD2 mutation and rpoD3rpoD4 and rpoD2rpoD3
double mutations affected expression from kaiB promoter. When sigC is inactivated we see a
remarkable effect of a 2 h lengthening of circadian period of expression from the psbAI
promoter, but not of that from kaiB or purF. These data suggest that searate timing circuits with
different periods can be present in a cell. Both of the oscillations in a sigC strain are dependent
on the period dictated by alleles of the kai genes. Taking advantage of different substrate
specificities of the Luc and Lux luciferases, we have created dually-reporting strains in which the
two reporters are driven by promoters that behave differently in the sigC mutant background.
Bioluminescence from either Luc or Lux will depend on exogenous application of the
appropriate substrate (luciferin or n-decanal, respectively). This way we have created S.
elongatus strains AMC1114 and AMC1127, both bearing PkaiB::luc and PpsbAI::luxAB reporter
systems, with sigC inactivated in AMC1127. Bioluminescence of these two strains was measured
by a cooled-CCD camera system from entrained cultures with the proper substrates added. The
data collected show that it is possible to monitor two different periods simultaneously from the
same strain, and that multiple timing circuits with different periods can be operating in S.
elongatus cells.
*
Corresponding author
94
Session IV Poster Presentation Abstracts
Role of kaiBC transcriptional timing in the circadian clock mechanism of
Synechococcus elongatus PCC 7942
JAYNA L. DITTY1*, SHANNON R. CANALES2, and SUSAN S. GOLDEN2
Department of Biology, The University of St. Thomas, St. Paul, MN 55105
Department of Biology, Texas A&M University, College Station, TX 77843
Central to models for animal and fungal circadian clock timing is negative feedback loops
involving clock genes that negatively regulate their own expression [1]. The single-celled
cyanobacterium, Synechococcus elongatus PCC 7942, has a circadian pacemaker comprised of
the products of at least three genes, kaiA, kaiB, and kaiC. The kai locus is expressed from two
promoters, one upstream of kaiA (monocistronic message) and one upstream of kaiB (dicistronic
kaiBC message), that are expressed in the same circadian phase in wild-type cells. Previous work
has shown that KaiA is required for expression from the kaiBC promoter, and that
overexpression of kaiA enhances expression from kaiBC, suggesting that KaiA is a positive
activator of the kaiBC promoter. KaiC is required for normal levels of expression from its own
promoter; however, overexpression of kaiC blocks expression from kaiBC, suggesting a role in
negative autoregulation [2]. These data were interpreted to be consistent with the animal and
fungal circadian timing models. However, mutants of S. elongatus have been identified that
change the phase relationship between kaiA and kaiBC expression without disrupting circadian
timing, suggesting that the relative transcriptional activity of expression from the kaiA and kaiBC
promoters is not important for generating circadian rhythms [3, 4]. The role of transcriptional
timing of the kaiBC gene locus for circadian timekeeping in S. elongatus was investigated. The
natural transcriptional regulation of the kaiBC genes (peak expression at dusk) was by-passed by
expressing the kaiBC dicistron from a heterologous promoter whose peak expression is 12 h out
of phase from the norm (peak expression at dawn). Expressing kaiBC from this heterologous
promoter showed no effect on the timing capabilities of the S. elongatus circadian clock,
suggesting that the timing mechanism of the circadian clock in cyanobacteria may be based upon
a post-transcriptional mechanism.
1.
2.
3.
4.
Harmer, S.L., S. Panda, and S.A. Kay, Molecular bases of circadian rhythms. Annu. Rev. Cell Dev. Biol.,
2001. 17: p. 215-53.
Ishiura, M., et al., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria.
Science, 1998. 281(5382): p. 1519-23.
Katayama, M., et al., cpmA, a gene involved in an output pathway of the cyanobacterial circadian system.
J. Bacteriol., 1999. 181(11): p. 3516-24.
Nair, U., et al., Roles for sigma factors in global circadian regulation of the cyanobacterial genome. J.
Bacteriol., 2002. 184(13): p. 3530-8.
95
Session IV Poster Presentation Abstracts
Homotypic interactions of central oscillator components in the cyanobacterial
circadian clock
GUOGANG DONG, SUSAN S. GOLDEN*
Department of Biology, Texas A&M University, College Station, TX 77843
The cyanobacteri a are thus far the simplest organism s and the only prokaryote s known to
sustain circadian rhythms. The unicellular strain Synechococcus elongatus PCC 7942 has been
developed as a model organism in which to explore circadian mechanism. The products of three
clustered genes kaiA, kaiB and kaiC are considered central oscillator components, as disruption
of any of these genes abolishes the circadian rhythm. KaiA, KaiB and KaiC interact with one
another in all possible combinations, as well as with themselves, to form higher order complexes.
Both the heterotypic and homotypic interactions are believed to play critical roles in circadian
timing. Interaction of KaiA with KaiC stimulates the autophosphorylation of KaiC, while the
presence of KaiB antagonizes this effect. However, much less is known regarding the function of
homotypic interactions of Kai proteins. In this study we employed a lambda repressor assay
system that simplifies the identification and analysis of homotypic interactions. We made
constructs by replacing the C-terminal (dimerization) domain of lambda repressor with either
full-length or individual domains of Kai proteins and introduced them into Esherichia coli.
Modified lambda phages that lack the repressor protein were cross-streaked over the recombinant
E. coli. The phages will remain in the lysogenic state if the chimeric lambda repressor protein
dimerizes and binds to the lambda operator; otherwise they will enter the lytic cycle and kill the
recombinant E. coli cells. This technique provides both selection for homotypic interaction and
counter-selection (with a different E. coli host) for conditional mutations that specifically disrupt
such interactions. However, some proteins that are known to be involved in homotypic
interactions, KaiA and KaiC, proved negative in the selection. Full-length KaiB, known to form
homodimers and possibly tetramers, was positive in the selection. We are now developing the
selection for temperature-sensitive mutations that specifically interrupt the homotypic
interaction. KaiB is the smallest of the three Kai proteins, and its function is the least understood.
This project will allow us to investigate how the circadian clock is affected when KaiB
interactions are disrupted at different points in the circadian cycle.
*
Corresponding author. Email: [email protected]
96
Session IV Poster Presentation Abstracts
Alterations in the gene expressions by the disruption of genes encoding
phytochrome-related protein in Synechocystis sp. PCC 6803
MITSUNORI KATAYAMA1, MINORU KANEHISA2 and MASAHIKO IKEUCHI1
1
Department of Life Sciences (Biology), University of Tokyo, komaba 3-8-1, Meguro, Tokyo,
153-8902, Japan, 2Bioinformatics Center, Institute for Chemical Research, Kyoto University,
Uji, Kyoto 611-0011, Japan
As completion of the determination of genomic sequences, it has been revealed that genes
encoding protein, which is structurally related to phytochrome of higher plant (phytochromerelated protein, Prp) widely distribute in various bacteria. Terrestrial cyanobacteria often possess
multiple genes for Prp. For example, Synechocystis sp. PCC 6803 carries as many as nine
candidates (sll0041, sll0821, sll1124, sll1473, slr0473, slr1212, slr1393, slr1805, slr1969).
Among these, sll0041 and sll0821 are involved in phototactic motility. sll1124 is involved in
growth under the blue light. In contrast to the phenotypic characterization, regulation of gene
expression by Prp is largely unknown except RcaE of Fremyella diplosiphon, which is involved
in the regulation of the transcription of cpcB2A2 and cpeBA during complementary chromatic
adaptation. To obtain information about regulation of gene expression by Prp in Synechocystis
sp. PCC 6803, we compared gene expression patterns between wild type strain and prp
disruptants using the technique of DNA microarray analysis. In consequence, we found out that
inactivation of sll1473 and slr1212 caused prominent change in the expression level of several
genes. Inactivation of sll1473 led to severe reduction of the expression of cpcG2 (sll1471) and
the adjacent gene sll1472 under constant illumination. The transcript of cpcG2 was undetectable
in the darkness and robustly induced by illumination of orange to red color of light. This
induction was almost completely eliminated in the sll1473 disruptant suggesting that Slr1473
functions as a sensor that perceives light signal and activates the expression of cpcG2.
Inactivation of slr1212 markedly reduced the accumulation of transcript of a series of genes
including ftsH (slr0228 and slr1604), sds (slr0611) and hliA (ssl2542) in the darkness. Gene
expression pattern in slr1212 disruptant was the same as wild type strain under constant
illumination. It is suggested that Slr1212 is involved in the recognition of the absence of
environmental light. On the other hand, some genes whose expression levels were altered by
disruption of slr1212 have been also reported as high-light responsive gene. We are investigating
the effect of disruption of slr1212 on gene expression patterns under high-light condition.
97
Session IV Poster Presentation Abstracts
Activation of photosynthesis and resistance to photoinhibition in
cyanobacteria within biological desert crust.
YARIV HAREL, ITZHAK OHAD and AARON KAPLAN*
Avron-Evenari Minerva Center of Photosynthesis Research, The Hebrew University of
Jerusalem, Jerusalem, 91014, Israel.
Filamentous cyanobacteria are the main primary producers in biological desert sand crusts.
The cells are exposed to extreme environmental conditions including temperature, light and
diurnal desiccation/rehydration cycles. We have studied the kinetics of activation of
photosynthesis during rehydration of the cyanobacteria, primarily Microcoleus sp., within crust
samples collected in the Negev desert, Israel. We also investigated their susceptibility to
photoinhibition. Activation of the photosynthetic apparatus, measured by fluorescence kinetics,
thermoluminescence and low temperature fluorescence emission spectra, did not require de
novo protein synthesis. Over 50% of the PSII activity, assembled phycobilisomes and PSI
antennae were detected within less than 5 min of rehydration. Energy transfer to PSII and PSI
by the respective antennae was fully established within 10-20 min of rehydration. The
activation of a fraction of PSII population (about 20-30%) was light and temperaturedependent but did not require electron flow to plastoquinone (was not inhibited by DCMU).
The cyanobacteria within the crusts are remarkably resistant to photoinhibition in either the
presence or absence of protein synthesis. The rate of PSII repair increased with light intensity
and with time of exposure. Consequently, the rate of photosynthesis in high-light-exposed
crusts reached a constant, relatively high, level. This is in contrast to model organisms such as
Synechocystis sp. strain PCC 6803 where PSII activity declined continuously over the entire
exposure to high illumination. Ability of the crust’s organisms to rapidly activate
photosynthesis upon rehydration and withstand photoinhibition under high light intensity may
partly explain their ability to survive in this ecosystem.
*
Corresponding author
98
Session V Poster Presentation Abstracts
Poster Presentations Session V
Structural aspects
Session Chair: Cheryl Kerfeld, UCLA
99
Session V Poster Presentation Abstracts
Photosystem I cyclic electron transfer pathways and function
HANS C. P. MATTHIJS1*, NATALIYA YEREMENKO1, PEERADA PROMMEENATE2,
WOLFGANG SCHIEFER3, ROBERT JEANJEAN4, PETER NIXON2 AND MICHEL
HAVAUX5
1
Aquatic Microbiology, University of Amsterdam Institute for Biodiversity and Ecosystem
Dynamics Nieuwe Achtergracht 127 Amsterdam 1018 WS the Netherlands
2
Dept. Biol. Sciences, Imperial College London Wolfson Laboratories South Kensington campus
London United Kingdom
3
Lehrstuhl Biochemie der Pflanzen Gebäude ND, Rm 3/133 Ruhr-Universität Bochum
Universitätsstraße 150 Bochum D-44801 Germany
4
LCB-CNRS, 31 Chemin Joseph Aiguier Marseille Cedex 20 Marseille F-13402 France
5
CEA/Cadarache, DSV, DEVM Laboratoire d’Ecophysiologie de la Photosynthèse UMR 163
CNRS CEA, Univ. Méditerranée-CEA 1000 Saint-Paul-lez-Durance 13108 France
New results from gene array display prompted the design of a series of deletion mutants to
clarify pathways and to evaluate the physiological significance of cyclic electron flow around
Photosystem I in the cyanobacterium Synechocystis PCC 6803. Mutant characterization showed
that products of the ORFs slr1208 and ssr 2016 (both till present known to encode so-called
hypothetical proteins) were involved in cyclic electron flow in cyanobacteria and participate
independently from the established constitutive NADH-dehydrogenase I, succinate
dehydrogenease and the inducible ferredoxin:NADP+ mediated pathways. Both the slr1208 and
ssr2016 knock-out mutants exhibited an antimycin A insensitive phenotype in Photosystem I
(PS1) cyclic flow. This suggested the involvement of the gene products in the still poorly
documented ferredoxin:quinone reductase mediated pathway. We studied a whole set of different
knockout mutants with lesser pathways available and arrived at the conclusion that PSI cyclic
electron flow attributed about 10% to the growth rate of cells incubated at optimal light intensity,
and that the function of cyclic flow became more important at low light, with 20 to 30%
reduction of the growth rate in the absence of PS1 cyclic flow and similar up to 70% at high light
conditions. Mechanism and function of Photosystem I cyclic electron flow will be reviewed.
* Corresponding author:
E-mail: [email protected]
Phone: +31205257070
Fax: +31205257064
100
Session V Poster Presentation Abstracts
In situ effects of mutations of the extrinsic cytochrome c550 of Photosystem II
in Synechocystis sp. PCC6803‡
ZHAOLIANG LI1, HEATHER ANDREWS1, JULIAN J. EATON-RYE3 AND ROBERT L.
BURNAP1*
1
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater,
OK 74078; 3Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New
Zealand
The H2O oxidizing domain of the cyanobacterial photosystem II (PSII) complex contains
a low potential, c-type cytochrome termed c550 that is essential for the in vivo stability of the PSII
complex. A mutant lacking cytochrome c550 (psbV) in Synechocystis sp. PCC 6803 has been
further analyzed together with a construct in which the distal axial heme iron ligand, histidine 92,
has been substituted with a methionine (C550-H92M). Heme staining of SDS-PAGE showed
that the C550-H92M mutation did not disturb the accumulation and heme binding properties of
the cytochrome. In psbV cells, the number of charge separating PSII centers was estimated to
be 56% of the wild-type, but of the existing centers, 33% lacked photooxidizable Mn ions.
C550-H92M did not discernibly affect the intrinsic PSII electron transfer kinetics compared to
the wild type nor did it exhibit a significant fraction of centers lacking photooxidizable Mn,
however, the number of charge separating PSII centers in mutant cells was 69% of the wild type.
C550-H92M lost photoautotrophic growth ability in the absence of Ca2+, but its growth was not
affected by depletion of Cl-, which differs from psbV. Taken together, the results suggest that in
the absence of cytochrome c550, electron transfer on the donor side is retarded, perhaps at the
level of Yz to P680+ transfer; the heme ligand, His92, is not absolutely required for assembly of
functional PSII centers, however, replacement by methionine prevents normal accumulation of
PSII centers in the thylakoid membranes and alters the Ca2+ requirement of PSII. The results are
discussed in terms of current understanding of the Ca2+ site of PSII.
101
Session V Poster Presentation Abstracts
Progress in sequencing, assembly and annotation of the genome of the marine
unicellular cyanobacterium Synechococcus sp. PCC 7002
Tao Li1,4, Jürgen Marquardt1, Gaozhong Shen1, Christopher Nomura1, Søren Persson 1, Chris
Detter2, Christa Lanz3, Stephan Schuster3, Jindong Zhao4, and Donald A. Bryant1*
1
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, PA 16802 USA; 2DOE Joint Genome Institute, 2800 Mitchell Drive, B400,
Walnut Creek, CA 94598 USA; 3AG Genomics and Signal Transduction, Max-Planck-Institute
for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany; 4College of Life
Science, Peking University, Beijing, China
The unicellular marine cyanobacterium Synechococcus sp. PCC 7002 has long served as a model
organism for the genetic and biochemical characterization of genes involved in photosynthesis,
respiration and biosynthetic pathways. The sequencing of the genome of this organism is now
nearly complete. Primary sequence data were collected from a combination of 23,279 reads from
whole-genome shotgun sequencing and about 1000 reads from targeted sequencing of cosmid
and BAC libraries. After assembly, the connecting of >800 contigs was pursued through primer
walking with templates from cosmid libraries and the sequencing of PCR products. Recently,
paired-end sequencing of 1,536 fosmids with average inserts of 37.5 kb facilitated the
scaffolding of the chromosome and the largest plasmids. The chromosome has been assembled
into a single scaffold that contains one remaining physical gap and that presently includes ~3.0
Mb. Additionally, our data confirm the presence of five of the six plasmids identified by Roberts
and Koths (1976): pAQ1 (4,809 bp), pAQ3 (16,073 bp), pAQ4 (32,035 bp), pAQ5 (38,516 bp),
and pAQ6 (~115 kb). These sizes closely match the sizes measured by electron microscopy and
gel electrophoresis: 4.6 kb, 15.9 kb, 31.0 kb. 38.6 kb, and 115.6 kb. We have shown that plasmid
pAQ2 (~10 kb) is actually a dimer of pAQ1 and that trimers of pAQ1 also occur. Interestingly,
our most recent assembly data suggest the existence of a sixth plasmid, which we have named
pAQ7 (~125 kb). A preliminary annotation of the genome has been performed using TIGR’s
Annotation Engine for Prokaryotic Annotation and Analysis. The Synechococcus sp. PCC 7002
genome model encodes 3,498 ORFs; the coding percentage is 88% and the genome has a mol%
G+C content of 49.6. Results of the preliminary analysis and some aspects of comparative
genomics will be presented.
Roberts TM and Koths KE 1976. The blue-green alga Agmenellum quadruplicatum contains covalently closed DNA
circles. Cell 9:551-557.
102
Session V Poster Presentation Abstracts
First fruits of the Synechococcus elongatus PCC 7942
functional genomics project
C. KAY HOLTMAN, YOU CHEN, and SUSAN S. GOLDEN*, Department of Biology, Texas A&M
University, College Station, TX 77843-3258
We are engaged in a functional genomics project that aims to inactivate each locus in the genome
of Synechococcus elongatus PCC 7942 and assay the resulting mutants for defects in circadian
rhythms. The goal is to identify all loci that contribute to the function of the circadian clock,
while producing and archiving a genetic resource for the cyanobacterial community. S. elongatus
PCC 7942 is the model organism for cyanobacterial circadian rhythms, which can be monitored
as 24-h rhythms of bioluminescence produced by the circadian expression of luciferase reporter
genes. Our approach is to isolate Mu or Tn5 insertions in cloned genomic DNA, determine the
positions of insertions by nucleotide sequencing, and transfer the mutations to the chromosome
in reporter strains by homologous recombination. Each mutant is then screened for defects in
circadian rhythmicity. We have developed high throughput methods for both transformation and
circadian screening. The Joint Genome Institute (JGI) has finished the complete S. elongatus
genome sequence using plasmid and fosmid libraries. Integration of our insertion data with the
JGI sequence has facilitated the functional genomics project by reducing our sequencing burden
and revealing the specific clones to target to complete global mutagenesis. Among the first
approximately 200 mutagenized loci we found mutants that exhibit an altered circadian
phenotype. All previous insertions in kaiABC were found to cause an arrhythmic circadian
phenotype; however, a Mu insertion that truncates kaiA exhibited a short period phenotype and
has implications for protein-protein interactions of the clock. Mu insertions in genes that encode
a putative response regulator and a heat shock protein have also been found that give altered
circadian phenotypes. Insertions in genes clpP2 and clpX resulted in cells with a long circadian
period. However, the phenotypes were detected in merodiploids, as the mutant alleles did not
segregate, suggesting that the genes are essential. We have developed an antisense method that is
effective for controlling expression of these essential genes, in which segments of the target gene
are expressed in antisense from an IPTG-inducible promoter. Titration of antisense expression
via IPTG concentration gives a range of phenotypes extending from no effect, through a
phenocopy of the merodiploid inactivation phenotype, to loss of bioluminescence that we
interpret as cell death. The methods, clones, and mutagenesis templates of the project will be
important resources for the S. elongatus research community.
*Author for correspondence; email: [email protected]
103
Session V Poster Presentation Abstracts
Deletion of large chromosomal fragments of Anabaena sp. PCC 7120 with a
Cre/loxP system
YinZhang and Jindong Zhao, College of Life Sciences, Peking University, Beijing 100871,
China
[email protected]
The Cre/loxP system has been widely used in gene manipulation of eukaryotic cells as
well as prokaryotic cells. It is based on the recombination system of bacteriophage P1 for site
specific crossover. We have constructed a Cre/loxP system for manipulation of the genome of
the heterocystous cyanobacterium Anabaena sp. PCC 7120. This system consists of two suicidal
plasmids containing loxP sequence and a shuttle vector pRL25C containing the cre gene under
control of the copper inducible petE promoter. To delete a fragment from the genome, the
flanking regions of the target fragment were PCR-amplified and inserted into the suicidal
plasmids. The suicidal plasmids were then transformed into Anabaena cells by conjugal transfer.
Appropriate antibiotics selection allows site-specific insertion of the loxP sequence into the
flanking regions of the target fragment. The shuttle vector pRL25C/cre is then transformed into
the cells. In the presence of copper in growth medium, the expression of cre is induced, leading
to specific deletion of the target fragment. This system has been tested for four fragments of
different sizes: 1 kb (glnB), 2 kb (hetR), 10 kb (cpcBA operon) and 22 kb (unknown gene
clusters). Our results show that this system could be used effectively for deleting both small and
large fragments from the genome of Anabaena 7120. Other possible applications of this system
in genetic manipulation of cyanobacterial genomes will be discussed.
104
ParticipantAddressess
Poster Presentations Session VI
Carbon metabolism
Session Chair: Dean Price, Australian National
University
105
ParticipantAddressess
IDENTIFICATION OF A NEW CLASS OF BICARBONATE
TRANSPORTER FROM THE MARINE CYANOBACTERIUM,
SYNECHOCOCCUS PCC7002
PRICE, G.D., WOODGER, F.J., TUCKER, L., BADGER, M.R. and 1HOWITT, S.M.
Molecular Plant Physiology, Research School of Biological Sciences, Australian
National University, ACT, 0200, Canberra, Australia; 1. Biochem & Molec Biol,
Science Faculty, ANU.
In aquatic environments the CO2 supply rate for photosynthesis can be severely
restricted, compared to terrestrial environments, and in order to maintain the efficiency
of photosynthetic carbon fixation cyanobacteria have evolved an efficient mechanism
for accumulation of inorganic carbon (Ci; CO2 & HCO3-,) known as a CO2
concentrating mechanism (CCM). This CCM involves the operation of active CO2 and
HCO3- transporters and results in the concentration of CO2 around Rubisco in a unique
microcompartment called the carboxysome. In freshwater strains of cyanobacteria
there are two CO2 uptake systems and at least two HCO3- transporters employed to
provide effective accumulation of Ci within the cell – the genes involved have been
identified. However, in marine cyanobacteria the physiological characteristics and
genetic identification of Ci transporters are less well understood. We have used the
marine cyanobacterium, Synechococcus PCC7002 as a model marine cyanobacterium
to characterize the properties of HCO3- transporters. We have confirmed thorough gene
disruptions, and gain-of-function analyses, that the sbtA homolog codes for a HCO3transporter, and significantly, we have identified a new HCO3- transporter, named bicA,
that codes for a low affinity HCO3- transporter with a high flux property. Close
homologs of bicA are present in the genome databases of all sequenced marine
cyanobacteria. The potential importance of the BicA transporter in marine systems will
be discussed.
106
ParticipantAddressess
Pleiotropic regulation of carbohydrate metabolism by Hik8 (a SasA
orthologue) in Synechocystis 6803
ABHAY K SINGH AND LOUIS A SHERMAN*
Department of Biological Sciences, Purdue University, West Lafayette, IN
Organisms respond to changes in environmental pertubations by regulating the expression of
genes that are crucial for growth and survival under stress conditions. Histidine kinases are often
involved in sensing these pertubations and the transduction of signals to trigger the responses.
We have used full genome microarrays of the cyanobacterium Synechocystis sp PCC 6803 to
study the global gene expression in response to environmental stresses such as nutrient
deficiency or oxidative stress (1). We have focused on one such histidine kinase (sll0750, Hik8)
which is related to SasA of Synechococus sp PCC 7942 (2) and which was found to be
differentially regulated under some stress conditions. A deletion mutant (hik8) was analyzed for
differential gene expression relative to the wild-type when grown photoautotrophically and after
1 h in the dark. Preliminary analysis of the microarray data indicated that two main functional
categories were affected by the absence of hik8; (a) genes involved in carbohydrate metabolism;
and (b) genes coding for ribosomal proteins, especially in dark-adapted hik8. Additionally, the
cyanobacterial phytochrome (cph1) and its cognant response regulator (rcp1), which are cotranscribed in Synechocystis 6803, were also regulated by hik8. Northern blot analysis of genes
encoding key enzymes of carbohydrate metabolism demonstrated that phosphofructokinase,
glyceraldehyde 3 phosphate dehydrogenase, fructose bisphosphate aldolase, glucose 6 phosphate
dehydrogenase, 6 phosphogluconate dehydrogenase, phosphoenolpyruvate synthase, ADPglucose pyrophosphorylase and glycogen phosphorylase were differentially regulated in hik8
grown in the presence or absence of glucose. In some cases, differential expression was
dependent on growth conditions (photoautotrophic vs. photoheterotrophic). The hik8 strain was
conditionally lethal; it had a comparable doubling time to wild-type in photoautotrophic and
photoheterotrophic growth conditions in continuous light, whereas it grew poorly compared to
the wild-type under photoheterotrophic conditions with different light and dark cycles. Growth
was completely stopped and cells eventually died when the light duration was less than 6 h on a
24-h regime. The determination of glycogen content indicated that hik8 strain had the ability to
accumulate glycogen, but was unable to properly utilize these reserves for growth. Enzyme
activities of G6PDH, 6PGDH and PFK was significantly reduced in hik8 compared to WT. In
contrast, there was no change in activity of G3PDH. These results suggest that the conditionallethal phenotype of hik8 is due to the inability to metabolize glucose for generation of reducing
power and substrates for biosynthesis. The results demonstrated that Hik8 has a pleiotropic
control of genes involved in central carbohydrate metabolism.
1. Singh AK, McIntyre LM, Sherman LA.2003. Plant Physiol. 132:1825-1839.
2. Iwasaki H, Williams SB, Kitayama Y, Ishiura M, Golden SS, Kondo T. 2000. Cell. 101:223-233.
107
ParticipantAddressess
Towards resolving the Glucose sensing in Synechocystis PCC 6803
1
KAHLON, S., 1BEERI K., 2OHKAWA, H., 1MURIK, O., 3HIHARA, Y., 4OGAWA, T., 5SUZUKI, I.
and A. KAPLAN1*
1
Dept. Plant and Environmental Sciences, The Hebrew University of Jerusalem, Israel
Dept. Biology, Washington University, St. Louis, USA.
3
Dept. Biochemistry and Molecular Biology, Saitama University, Japan
4
Bioscience Center, Nagoya University, Japan
5
National Institute for Basic Biology, Okazaki, Japan
2
There is a large diversity among cyanobacteria with respect to carbon nutrition. While some are
obligate photoautotrophs others can switch between photoautotrophic, photomixotrophic and
heterotrophic modes of metabolism. Glucose sensitive and tolerant strains of Synechocystis PCC 6803
are available but little is known about the regulation between photoautotrophic and photomixotrophic
growth. Inactivation of both genomic and plasmid copies of sll0790 (encoding Hik31) in
Synechocystis PCC 6803 resulted in a mutant unable to grow in the presence of D-glucose. The extent
of glucose-dependent death of hik31 was affected by the light intensity and ambient CO2 level. We
are investigating the role of Hik31 in the acclimation of Synechocystis PCC 6803 to photomixotrophic
growth by additional mutations and analysis of glucose-related processes and transcript levels in the
wild type and the mutants. Inactivation of the glucose transporter in hik31 rescued the cells
indicating that the glucose signal is sensed inside the cells or that the transporter, as in certain
eukaryotic cells, mediates the signal. The glucose sensitive (PCC) and hik31 strains do not show
glucokinase activity suggesting inability to convert glucose to glucose 6-phosphate. The levels and
activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased
significantly in WT cells exposed to glucose but were constitutively high in hik31 cells regardless of
the presence of glucose. A 105 kDa protein, the nature of which is not known but which is recognized
by a G6PD antibody, is present in WT but not in hik31. We propose that Hik31 is involved in the
regulation between photoautotrophic and photomixotrophic growth of Synechocystis 6803.
* Corresponding author
108
ParticipantAddressess
A proteomic study of carboxysomes from -cyanobacteria
BEN M. LONG, G. DEAN PRICE & MURRAY R. BADGER*
Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National
University, PO Box 475, Canberra ACT 2601.
Carboxysomes are protein-bound, polyhedral structures within cyanobacteria, containing the
key enzyme for photosynthetic CO2-fixation, namely Rubisco. Sequencing of cyanobacterial
genomes has revealed that cyanobacteria possess one, or other, of two types of carboxysomes.
Cyanobacteria containing Form-1A Rubisco (e.g. Prochlorococcus marinus MED4) possess carboxysomes, while those with Form-1B Rubisco possess -carboxysomes (e.g. Synechococcus
PCC7942). Given the central importance of carboxysomes in the CO2 concentrating mechanism
(CCM) of cyanobacteria, understanding the nature and composition of these structures is of
considerable importance. In an effort to characterise the structure of -carboxysomes,
particularly the outer protein shell, we have undertaken a proteomic analysis of these structures
from the freshwater cyanobacterium Synechococcus sp. PCC7942. Both MALDI-TOF analysis
of excised SDS-PAGE bands and MudPIT analysis of complex mixtures of digested proteins
have been used in an attempt to identify the proteins constituting -carboxysomes. We report
here some preliminary data on the identity of proteins associated with carboxysomes from
Synechococcus sp. PCC7942.
*
Corresponding author
109
ParticipantAddressess
Global patterns of gene expression in Synechocystis sp. PCC 6803 in response
to inorganic carbon limitation and the inactivation of ccmR, a LysR family
regulator
HONG-LIANG WANG, BRADLEY L. POSTIER and ROBERT L. BURNAP*
Department of Microbiology & Molecular Genetics, 307 Life Sciences East, Oklahoma State
University, Stillwater, OK 74075
The cyanobacterium Synechocystis sp. PCC 6803 possesses multiple inorganic carbon
(Ci) uptake systems that are regulated according to Ci availability in the environment. The
control mechanisms of these systems and their integration with other cell functions remain to be
clarified. Full genome microarrays and RT-PCR techniques were used to analyze the changes in
global gene expression in response to Ci downshift and the inactivation of ccmR (sll1594,
formerly, ndhR), a LysR family regulator of Ci uptake. A relatively mild Ci-limitation [3% CO2
(v/v) in air to air alone] induced a dramatic up-regulation of genes encoding both inducible CO2
and HCO3- uptake systems in the cyanobacterial cells grown in Na2CO3-free BG-11 medium
buffered at pH 7.0. The expression of slr1513 (designated sbtB, more recently) and sll1735,
physically clustered with sbtA and ndhF3/ndhD3/cupA, respectively, were also coordinated with
their upstream genes encoding the essential components for HCO3- and CO2 uptakes. Analyses of
RT-PCR and DNA microarray hybridization revealed the expression of ndhD5/ndhD6,
physically forming a probable transcriptional unit with downstream genes with homologies to
antiporter proteins. This leads us to propose that these genes encode in sodium efflux system,
driven by redox energy and used to drive the sodium dependent bicarbonate uptake transporter,
SbtA, identified by Ogawa. An opposite regulation of the acquisition and thence assimilation of
carbon and nitrogen occurred, demonstrating a striking expression coordination of relevant genes
and operons. Based on the analyses of RT-PCR and DNA microarray hybridization, ndhR
inactivation up-regulated the expressions of sbtA/sbtB, ndhF3/ndhD3/cupA/sll1735 and slr200613 including ndhD5 and ndhD6, indicating a vital role of the regulatory gene in both CO2 and
HCO3-.
Based upon the current information, we suggest that ndhR is renamed ccmR to better
represent its broader regulatory characteristics and that the regulated genes encode an integrated
system of proteins operating to concentrate inorganic carbon using redox energy transduced by
Type I dehydrogenases directly as with the CUP subsystem or indirectly via a sodium gradient
generated by the NdhD5/NdhD6 subsystem.
*
Corresponding author
110
Participant List
Printed 8/12/2004
Title: 8th Cyanobacterial Molecular Biology Workshop
Area: CALS
Number: 172
Schedule #: 561059
Subtitle: 00
Term: F2005
Section: 1
Meeting Dates: August 24 - 29, 2004
Name
Title
Company
City, State
Allen, Mary
Prof
Wellesley College
Wellesley, MA
(781) 283-3068
Kiel, Germany
49 431 880 4237
Botanical Institute
Kiel, Germany
49 4318504
The Australian National University
Canberra City, Australia 61 2 61253741
Appel, Jens
Backasch, Ninja
Badger, Murray
Prof
Work Phone
Work Email
[email protected]
[email protected]
[email protected]
Baranova, Maria
Grad Student
Bowling Green State University
Bowling Green, OH
(419) 372-4890
[email protected]
Boyanapalli, Ramakrishna
Grad Student
Bowling Green State University
Bowling Green, OH
(419) 494-1445
[email protected]
Brown, Jessica
Grad Student
University of Alberta
Edmonton, AB Canada
(780) 492-1805
[email protected]
Bowling Green State University
Bowling Green, OH
Bullerjahn, George
Burey, Suzanne
Vienna, Austria
(419) 372-8527
43 14277 52810
Burnap, Robert
Oklahoma State University
Stillwater, O
(405) 744-7445
Cadoret, Jean C.
Ecole Normale Superieure
Paris, France
33 144 323534
Canales, Shannon
Texas A & M University
College Station, TX
University of Tennessee
Durham, United
Kingdom
Knoxville, TN
University of Leeds
Leeds, United Kingdom 44 0113 3435651
Cann, Martin
Carberry, Matthew
Chapman, Karen E.
Mrs
(979) 845-9821
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
(865) 974-4014
[email protected]
[email protected]
Chen, You
Texas A&M University
College Station, TX
(979) 845-9821
[email protected]
Clerico, Eugenia
Texas A & M University
College Station, TX
(979) 845-9821
[email protected]
University of San Francisco
San Francisco, CA
(415) 422-6450
[email protected]
Cunningham, Jr., Francis X.
University of Maryland
College Park, MD
(301) 405-1035
[email protected]
Curtis, Stephanie
NC State
Raleigh, NC
(919) 515-5747
[email protected]
Cobley, John
Prof
Ditty, Jayna
Assistant Professor
University of St. Thomas
St. Paul, MN
(651) 962-5245
[email protected]
Dong, Guogang
Grad Student
Texas A&M University
College Station, TX
(979) 845-9821
[email protected]
VCU
Richmond, VA
(804) 828-0794
[email protected]
(517) 353-6641
Elhai, Jeff
Michigan State University
East Lansing, MI
Fernandez-Pinas, Francisca Dr.
Fan, Qing
Universidad Autonoma de Madrid
MADRID, Spain
Forchhammer, Karl
Professor
University Giessen
Giessen, Germany
Fromme, Petra
Prof. Dr.
Arizona State University
Tempe, AZ
Fujita, Yuichi
Assistant Professor
Nagoya University
Nagoya, Japan
Form Modified: 5/212003
Post Research Assoc
Page 1 of 3
349-1497 Ext. 8176
49 641 9935545
(480) 965-9028
81 52 7894105
[email protected]
[email protected]
karl.forchhammer@mikro,bio,uni-giessen.de
[email protected]
[email protected]
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Participant List
Printed 8/12/2004
Name
Title
Company
City, State
Gleason, Florence
Professor
University of MInnesota
Saint Paul, MN
(612) 625-4275
[email protected]
Golden, James
Professor
Texas A&M University
College Station, TX
(979) 845-9823
[email protected]
Golden, Susan
Prof
Texas A&M University
College Station, TX
(979) 845-9824
[email protected]
Graham, Douglas
PhD Student
The Robert Gordon University
Aberdeen, United
Kingdom
Kiel, Germany
44 07966 966 292
Hagemann, Martin
Dr.
Universtiy Rostock
Rostock, D Germany
49 3814986
Haselkorn, Robert
Dist. Serv. Prof
Mol. Genetics & Cell BIol., U of Chi
Chicago, IL
Hihara, Yukako
Dr
Saitama University
Saitama, Japan
Holtman, Carolyn K.
Assistant Reserach Scientist Texas A&M University
Gutekunst, Kirstin
Horn, Darryl
Houmard, Jean
Dr
Ivleva, Natalia
(773) 702-1069
81 48 8583396
[email protected]
[email protected]
[email protected]
(979) 845-4388
[email protected]
(920) 424-7084
[email protected]
Ecole Normale Superieure
Paris, France
33 144 323519
Texas A & M University
College Station, TX
University of Wisconsin-Oshkosh
Oshkosh, WI
Jerusalem, Israel
Grad Student
49 431 8804237
College Station, TX
Kaplan, Aaron
Kappell, Anthony
[email protected]
Oshkosh, WI
Marseille, France
Prof
Work Email
University of Wisconsin Oshkosh
Jeanjean, Robert
Kallas, Toivo
Work Phone
Univ. of Texas at Arlington
Arlington, TX
[email protected]
(979) 845-9821
[email protected]
33 49 1164298
[email protected]
(920) 424-7084
972 2658523
(817) 938-6823 Ext.
[email protected]
[email protected]
[email protected]
Katayama, Mitsunori
Assistant Prof
Department of LIfe Sciences
Meguro, Japan
Kennelly, Peter
Professor
Virginia Tech
Blacksburg, VA
(540) 231-4317
[email protected]
UCLA
Los Angeles, CA
(310) 825-7417
[email protected]
University of Amsterdam
Kerfeld, Cheryl
Krasikov, Vladimir V.
Msc.
Kufryk, Galyna
Arizona State University
Amsterdam,
Netherlands
Tempe, AZ
Lechno-Yossef, Sigal
Michigan State University
East Lansing, MI
Leganes, Francisco
Dr.
Liu, Jinjie
Universidad Autonoma de Madrid
MADRID, Spain
MIchigan State University
East Lansing, MI
81 5454 6647
[email protected]
34 205 257070
[email protected]
(480) 965-3698
[email protected]
(517) 353-6641
349-1497 Ext. 8176
[email protected]
(517) 353-6641
Long, Ben
Dr
Australian National University
Canberra, Australia
61 261 254213
[email protected]
Luque, Ignacio
Dr
Universidad de Alicante
Alicante, Spain
34 965 909587
[email protected]
49 641 9935554
[email protected]
Mani, Maheswaran
Mr
IMMB
Giessen, Germany
Martin, Miriam
Post-Doc Researcher
Universtiy fo California-Davis
Davis, CA
University of Amsterdam
Matthijs, Hans
McLean, Debra
Admin Assist
BGSU
Amsterdam,
Netherlands
Bowling Green, OH
Meeks, Jack
Prof
University of California
Davis, CA
Form Modified: 5/212003
Page 2 of 3
(530) 752-7769
[email protected]
31 205 257070
[email protected]
(419) 372-8550
[email protected]
(530) 752-3346
[email protected]
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Participant List
Printed 8/12/2004
Name
Title
Mukhopadhyay, Archana
Company
City, State
Virginia Tech
Blacksburg, VA
(540) 231-3062
[email protected]
(709) 737-8529
[email protected]
Work Phone
Work Email
Mulligan, Martin
Memorial University of Newfoundland
St. John's, NL Canada
Muramatsu, Masayuki
University of Tokyo
Saitama, Japan
81 48 8583396
[email protected]
Muro-Pastor, Alicia
CSIC-University of Sevilla
Sevilla, Spain
34 954 489578
[email protected]
Nagoya University
Nagoya, Japan
81 52 789 2963
[email protected]
Ibaraki University
Inashiki Ami, Japan
81 29 888 8649
[email protected]
49 234 3223633
[email protected]
Nakajima, Masato
Division of Biological
Science
Nishizawa, Akito
Nowaczyk, Marc
Dipl. Biol.
Ruhr-University Bochum
Bochum, Germany
Owttrim, George
Associate Professor
University of Alberta
Edmonton, AB Canada
Patterson-Foctin, Laura
Grad Student
University of Alberta
Edmonton, AB Canada
Peschek, Gunter A.
Professor
University of Vienna
Vienna, Austria
43 1427752
[email protected]
Price, G Dean
Dr
Australian National University
61 281 258423
[email protected]
Ramirez, Martha
Researcher
NC State
Acton-Canberra,
Australia
Raleigh, NC
Salerno, Graciela
Dr
FIBA
54 223 474 8257
Schluchter, Wendy
Assistant Professor
University of New Orleans
Mar del Plata,
Argentina
New Orleans, LA
Schmetterer, Georg
Prof.
Inst. Physical Chemistry, Vienna Univers Vienna, Austria
Schwarz, Rakefet
Ramat-Gan, Israel
Shea, Katherine
Wellesley College
Wellesley, MA
(780) 492-1803
[email protected]
(780) 492-1805 Ext.
[email protected]
(919) 515-5730
(504) 280-7194 Ext.
[email protected]
[email protected]
[email protected]
43 1 4277 52548
[email protected]
972 3 531 7790
[email protected]
(781) 283-3068
[email protected]
Shen, Gaozhong
Dr
Penn State
University Park, PA
(814) 863-7405
[email protected]
Sherman, Louis
Professor
Purdue University
West Lafayette, IN
(765) 494-8106
[email protected]
Shirai, Makoto
Dr
Ibaraki Univ.
Inashiki, Japan
81 29 888 8652
[email protected]
Six, Christopher
PhD Student
Station Biologique de Roscoff
Roscoff, ZZ France
81 28 888 8649
[email protected]
Sousuke, Imamura
Ibaraki University
Inashiki Ami, Japan
Summers, Michael
California State University Northride
Northridge, CA
(818) 677-7146
[email protected]
81 52 7892495
[email protected]
Terauchi, Kazuki
Dr
Nagoya University
Nagoya, Japan
Thayer, Rebecca
Grad Student
North Carolina State University
Raleigh, NC
Thiel, Teresa
Dr
Tomitani, Akiko
Dr
University of Leeds
Yokosuka, Japan
Wolk, C. Peter
Professor
Michigan State University
E. Lansing, MI
Woodger, Fiona
Dr
Australian National University
Canberra, Australia
Xu, Yao
REs. Assist. Prof.
Vanderbilt University
Nashville, TN
(615) 343-1350
Yeremenko, Nataliya
Msc.
University of Amsterdam
Amsterdam,
Netherlands
34 205 257070
Form Modified: 5/212003
St. Louis, MO
(919) 515-5730
Page 3 of 3
(314) 516-6208
81 46 8679781
(517) 353-2049 Ext. 51735391
61 261 254213
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
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