Coordination of Carbon Dioxide and Nitrogen Metabolism in Rhodobacter sphaeroides
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Ryan Michael Farmer
Graduate Program in Microbiology
The Ohio State University
2013
Dissertation Committee:
Professor F. Robert Tabita, Advisor
Associate Professor Birgit E. Alber
Professor Charles Daniels
Professor Joseph Krzycki
Copyright by
Ryan Michael Farmer
2013
Abstract
Studies of metabolism usually characterize a system in isolation without a wholecell focus on interactions with other metabolic pathways. In this work, two different
metabolic pathways of the nonsulfur purple bacterium Rhodobacter sphaeroides were
studied simultaneously in vivo to determine their influence upon each other.
Rb.
sphaeroides is capable of nitrogen fixation via nitrogenase catalysis and carbon dioxide
fixation via the Calvin-Benson-Bassham (CBB) cycle.
When the CBB cycle was
inactivated through gene deletions, strains developed that deregulated nitrogenase.
Genomic sequencing and comparative analyses of these mutant strains revealed multiple
mutations that could account for the nitrogenase active phenotype. A mutation in the
gene that encodes for glutamine synthetase and another in the gene that encodes for one
subunit of nitrogenase were shown be sufficient to derepress nitrogenase synthesis and to
affect its activity, respectively.
Further studies of nitrogenase regulation led to the
observation that the N-terminal GAF domain of the transcriptional regulator of
nitrogenase, NifA, contributed to its post-translational response to cellular nitrogen
status. Finally, the reciprocal response of the CBB cycle to nitrogenase activity was
investigated determining that the expression of the genes of the CBB cycle was repressed
when nitrogenase was active. The CBB enzyme phosphoribulokinase was found to
mediate this repression. This study thus explored the coordination of carbon dioxide and
nitrogen metabolism in Rb. sphaeroides.
ii
Dedication
This document is dedicated to Penny for her perpetual enthusiasm.
iii
Acknowledgments
I would like to acknowledge the U.S. Department of Energy and The Ohio State
University for providing funds that have supported me and this research. I would also
like to thank Dr. Tabita for effectively and efficiently managing the funds and the
research.
iv
Vita
2008................................................................B.S. Biochemistry,
Allegheny College
2012................................................................M.S. Microbiology,
The Ohio State University
2008 to present ..............................................Graduate Research and Teaching Associate,
Department of Microbiology,
The Ohio State University
Fields of Study
Major Field: Microbiology
v
Table of Contents
Abstract ............................................................................................................................... ii
Dedication .......................................................................................................................... iii
Acknowledgments.............................................................................................................. iv
Vita...................................................................................................................................... v
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
Introduction ......................................................................................................................... 1
Chapter 1 : Characterization of the PHC Phenotype ......................................................... 7
Introduction ..................................................................................................................... 7
Materials and Methods .................................................................................................. 10
Results ........................................................................................................................... 21
Discussion ..................................................................................................................... 38
Chapter 2 : Alterations to NifA Influence Nitrogenase Synthesis ................................... 45
Introduction ................................................................................................................... 45
Materials and Methods .................................................................................................. 49
Results ........................................................................................................................... 54
vi
Discussion ..................................................................................................................... 64
Chapter 3 : Nitrogenase-Mediated Repression of cbb Transcription............................... 69
Introduction ................................................................................................................... 69
Materials and Methods .................................................................................................. 72
Results ........................................................................................................................... 76
Discussion ..................................................................................................................... 87
Conclusions and Future Directions ................................................................................... 93
References ......................................................................................................................... 98
vii
List of Tables
Table 1.1 Strains and plasmids used in Chapter 1 ............................................................ 12
Table 1.2 Oligonucleotides used in Chapter 1 .................................................................. 13
Table 1.3 Additional requirements for photoheterotrophic growth .................................. 22
Table 1.4 Metrics for genomic sequences......................................................................... 24
Table 1.5 Unique mutations in strain 16PHC ................................................................... 28
Table 1.6 Glutamine synthetase activity ........................................................................... 32
Table 1.7 Nitrogenase activities of glnA allelic strains..................................................... 34
Table 1.8 Phenotypic effects of nifK alleles ..................................................................... 39
Table 2.1 Strains and plasmids used in Chapter 2 ............................................................ 50
Table 2.2 Oligonucleotides used in Chapter 2 .................................................................. 51
Table 2.3 Nitrogen-fixing growth of nifA complementation strains ................................. 57
Table 2.4 Nitrogenase activities of nifA complementation strains ................................... 59
Table 3.1 Strains and plasmids used in Chapter 3 ............................................................ 73
Table 3.2 Oligonucleotides used in Chapter 3 .................................................................. 74
Table 3.3 Growth of PRK complementation strains ......................................................... 83
Table 3.4 NADH levels..................................................................................................... 88
viii
List of Figures
Figure 1.1 Schematic for chromosomal gene deletions .................................................. 14
Figure 1.2 Genome alignments of strains 16 and 16PHC ............................................... 25
Figure 1.3 Venn diagram of high confidence differences in genomes of strains compared
to strain 2.4.1 ................................................................................................. 26
Figure 1.4 Logo diagram of a partial consensus GlnA sequence .................................... 29
Figure 1.5 Crystal structure of glutamine synthetase ...................................................... 31
Figure 1.6 Western immunoblot of crude extracts from cultures of glnA allelic strains
using antisera to NifH.................................................................................... 33
Figure 1.7 Logo diagrams of partial NifK sequences ..................................................... 36
Figure 1.8 Crystal structure of nitrogenase ..................................................................... 37
Figure 2.1 Oligomeric state of bEBPs ............................................................................. 47
Figure 2.2 SWISS-MODEL of NifA from Rb. sphaeroides ........................................... 55
Figure 2.3 Western immunoblot of crude extracts from cultures of nifA complementation
strains using antisera to NifH ........................................................................ 58
Figure 2.4 Homology between NifA and NtrC ............................................................... 61
Figure 2.5 Purification of recombinant His-tagged NifA................................................ 62
Figure 2.6 Solubility of recombinant His-tagged NifA ................................................... 63
Figure 3.1 Western immunoblot of crude extracts from cultures of strain 2.4.1 grown
with different nitrogen sources ...................................................................... 78
ix
Figure 3.2 Plasmid based cbb promoter fusion activities from lysates of strains 2.4.1 and
NK10 ............................................................................................................. 79
Figure 3.3 Western immunoblot of crude extracts from cultures of strain 145 .............. 81
Figure 3.4 Western immunoblot of crude extracts from cultures of strain 2.4.1 grown
with tungsten ................................................................................................. 82
Figure 3.5 Western immunoblot of crude extracts from cultures of strain 15165 .......... 85
Figure 3.6 Chromosomal based cbbI promoter fusion activities from lysates of strains
2.4.1C1 and 15165C1 .................................................................................... 86
x
Introduction
Life is a complex and ever changing process; yet its continuance depends upon
balance. For the simplest chemical reaction, the amount of reactants and products are in
a balance that is governed by the reaction’s equilibrium constant. Multiple consecutive
reactions that are catalyzed by enzymes form the basis of an organism’s metabolic
pathway.
Such a pathway is still governed by the equilibriums of the individual
reactions, but the pathway can be controlled by regulating the flux through it. This can
occur by affecting the activity of the enzymes or their abundance.
Disruptions in
metabolic pathways lead to conditions where homeostasis, the cumulative, regulated
balance of an organism, can no longer be maintained. These disruptions can be caused
by unexpected changes in the constituents of a reaction, for example the loss of a reactant
or the buildup of a product, or aberrant regulation, thereby changing the flux through the
pathway. Once metabolic disruptions occur, illness or death usually follows. This
project began by observing how an organism dealt with an imbalance.
The organism of study is Rhodobacter sphaeroides, which belongs to the
nonsulfur purple (NSP) bacteria. The NSP bacteria are free-living, aquatic and soil,
Gram negative rods that possess unique metabolic capabilities, many of which can have
redundant functions (Madigan and Jung 2009). Redundancies occur in pathways of both
energy and carbon metabolism that allow for the creation of conditional lethal mutants.
For energy production, Rb. sphaeroides can grow under aerobic chemotrophic conditions
1
by utilizing aerobic respiration and also anaerobic anoxygenic phototrophic conditions by
utilizing cyclic photophosphorylation.
Phototrophic growth requires anaerobic
conditions because the light reactions can generate destructive oxygen species and also
requires oxidation and reduction (redox) homeostasis because redox imbalance can arrest
cyclic photophosphorylation (Krieger-Liszkay 2004; Ziegelhoffer and Donohue 2009).
The redox sensitivity occurs because reduced electron carriers must transfer their
electrons to oxidized carriers, which establishes a flow of electrons that is harnessed for
energy (Lavergne et al. 2009; McEwan 1994). Upon imbalance, increasing number of
carriers become reduced (or oxidized) thereby slowing the electron flow of the cycle; if
left uncorrected, the cell would no longer generate energy and would eventually die
(McEwan 1994; Falcone and Tabita 1991; Hallenbeck et al. 1990a; Zannoni et al. 2009).
For phototrophic growth to occur the cell must maintain redox balance.
Carbon metabolism in Rb. sphaeroides can also occur through two growth modes.
Under heterotrophic conditions the cell consumes already reduced carbon, especially
acidic fermentative end products (Madigan and Jung 2009). Conversely, for autotrophic
growth the cell must reduce its own carbon from carbon dioxide (CO2) by using the
Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle (reviewed in Tabita
1988). The CBB cycle converts CO2 to cell material and begins through the catalysis of
three enzymes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is one of
two unique enzymes of the CBB cycle and performs the actual carbon-fixation step by
catalyzing the carboxylation of ribulose-1,5-bisphosphate (RuBP) to form two molecules
of 3-phosphoglycerate. Phosphoglycerate kinase then hydrolyzes ATP to produce 1,32
bisphosphoglycerate
from
3-phosphoglycerate.
Lastly,
3-phosphoglycerate
dehydrogenase then reduces 1,3-bisphosphoglycerate to form glyceraldehyde-3phosphate, which leaves the cycle for cellular metabolism.
To complete the cycle,
stoichiometrically, 5 molecules of glyceraldehyde-3-phosphate remain in the cycle for
every 1 that leaves.
Multiple enzymatic reactions then convert those remaining
glyceraldehyde-3-phosphates into 3 molecules of RuBP, which is the substrate for
RubisCO. The final step of RuBP creation is catalyzed by the other unique enzyme of
this cycle: phosphoribulokinase (PRK).
Classically, this pathway is used to supply
carbon to the cell, but it can also function in energy metabolism as a fermentative
pathway to maintain cellular redox homeostasis through the reducing activity of 3phosphoglycerate dehydrogenase (reviewed in Tabita 1995).
The necessity of the fermentative aspect of the CBB cycle for photoheterotrophic
growth is dictated by the oxidation states of the growth substrates. When the oxidation
state of the carbon source is equal to or more than that of the cell the requirement for the
CBB cycle is dependent upon how the carbon source is metabolized (Laguna et al. 2011).
For the organic acids malate and succinate, the first steps in their metabolism are
oxidative, which generate reductant (NADH) and CO2. More NADH is produced in
these steps than are later consumed, so Rb. sphaeroides requires the reductive processes
of the CBB cycle to oxidize NADH and recycle NAD+, ultimately using the
metabolically-derived CO2 as the electron acceptor (Laguna et al. 2011). Therefore,
when the CBB cycle is rendered non-functional, a phototrophic growth defect when
malate or succinate were the carbon source has been observed (Falcone and Tabita 1991;
3
Hallenbeck et al. 1990a; Hallenbeck et al. 1990b; Romagnoli and Tabita 2006; Tichi and
Tabita 2000; Chapter 1). When the oxidation level of the carbon source is less than that
of the cell, then electron acceptors must be supplied to the growth medium. These
acceptors could be additional CO2 or alternatives such as dimethylsulfoxide (DMSO)
(Joshi et al. 2009; Jouanneau and Tabita 1986; Rey et al. 2007). Though the CBB cycle
is the only redox balancing pathway that is naturally expressed photoheterotrophically,
other systems to relieve extra reductant can be induced; for example nitrogenase can be
expressed under nitrogen depleted conditions.
Nitrogenase is the enzyme complex that catalyzes nitrogen fixation, which is the
reduction of nitrogen gas to ammonia. In nature there are three types of nitrogenases
which are classified according to the metal cofactor that resides at the active site, but Rb.
sphaeroides only encodes for one copy of the most common type: the molybdenum (Mo)
nitrogenase (Masepohl and Kranz 2009). This nitrogenase is encoded by three genes,
nifHDK, whose products forms the structure of nitrogenase, but many more proteins are
needed for its activity. There are two complex and unique metal clusters that require
accessory proteins for their assembly and maturation. The P cluster is an [8Fe-7S] cluster
that is involved in the relay of electrons to the active site containing the ironmolybdenum cofactor (Burgess and Lowe 1996; Dixon and Kahn 2004; Seefeldt et al.
2012). A unique attribute of the Mo-nitrogenases is that the Mo can be substituted by
tungsten (W). These W-nitrogenases lack the ability to reduce acetylene, which is a
convenient alternative substrate used to measure nitrogenase activity; such Wnitrogenases still retain limited proton reducing (hydrogenase) activity (Siemann et al.
4
2003). Even under optimal conditions, hydrogen production is an obligate product of
nitrogen fixation, but it can also be the only product if no nitrogen is present (Hillmer and
Gest 1977).
Both the nitrogenase and hydrogenase activities are energetically
demanding, requiring the hydrolysis of two molecules of ATP per electron delivered to
the active site; therefore, nitrogenase is only synthesized under conditions that lack an
easily assimilated sources of nitrogen, such as ammonia (reviewed in Dixon and Kahn
2004; Masepohl and Kranz 2009).
To utilize nitrogenase catalysis to maintain redox balance under nitrogen replete
conditions, normal ammonia-induced repression must be overcome.
This can be
achieved through genetic mutations. Photoheterotrophic competent (PHC) strains arose
through spontaneous mutations that restored growth when the original strain was redox
imbalanced. In the NSP bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides,
Rhodospirillum rubrum, and Rhodopseudomonas palustris, the PHC strains arose through
adaptive growth of CBB null strains (Falcone and Tabita 1993; Joshi and Tabita 1996;
Laguna 2010; Tichi and Tabita 2000; Wang et al. 2010; Wang et al. 1993). Additionally
in Rps. palustris, PHC strains arose from the wild-type (WT) strain grown with substrates
more reduced than that of the cell (Rey et al. 2007). Observations of the PHC strains
confirmed that they synthesized the nitrogenase complex under normally repressive
conditions and also showed that they repressed the CBB genes (Rey et al. 2007; Smith
and Tabita 2002). Results presented in the first chapter will analyze a PHC strain from
Rb. sphaeroides to determine how it altered its nitrogen metabolism. In the second
chapter a nitrogen regulatory protein will be further characterized. The last chapter will
5
address results leading to the determination of a molecular mechanism for the CBB gene
repression. Through its metabolic versatility and redundancy, Rhodobacter sphaeroides
can maintain homeostasis in ways that other organisms cannot, thereby allowing the
elucidation of metabolic interactions that otherwise would have remained a mystery.
6
Chapter 1: Characterization of the PHC Phenotype
INTRODUCTION
Redox balance is maintained through the CBB cycle when Rb. sphaeroides is
grown photoheterotrophically.
Disruptions to the CBB cycle can be made under
chemotrophic growth where the redox environment is maintained through aerobic
respiration, but under anaerobic, phototrophic conditions CBB disrupted strains
eventually mutate into PHC strains (Falcone and Tabita 1993; Joshi and Tabita 1996;
Laguna 2010; Tichi and Tabita 2000; Wang et al. 2010; Wang et al. 1993). These strains
can synthesize nitrogenase under ordinarily repressive conditions, thereby dissipating
accumulated reducing equivalents as the nitrogenase-catalyzed product, hydrogen (Joshi
and Tabita 1996; Laguna 2010; Tichi and Tabita 2000; Wang et al. 2010). In strain
16PHC, a PHC strain in Rb. sphaeroides, the mutation(s) that cause this deregulation and
constitutive activity of nitrogenase remain to be elucidated but have been hypothesized to
occur in gene(s) of the nitrogen regulatory cascade (Laguna 2010).
The nitrogen regulatory cascade is a complex signaling system that controls the
expression of genes encoding for the nitrogenase complex. The cascade is initiated by
the activity of glutamine synthetase (GS) (reviewed in Forchhammer 2007).
This
enzyme catalyzes the incorporation of ammonia onto glutamate (Glu) to produce
glutamine (Gln). The newly acquired amino group is then transferred to the central
7
carbon metabolite, α-ketoglutarate (αKG) via glutamine oxoglutarate aminotransferase
(GOGAT). Therefore this cycle converts one molecule of ammonia and one molecule of
αKG to net one molecule of Glu through a Gln intermediate. The enzymatic activities of
the GS-GOGAT system influence the cellular concentrations of Gln and αKG.
These metabolites then control the activities of additional proteins within the
signaling cascade, as has been extensively studied in E. coli, Salmonella enterica, and the
NSP bacterium Rb. capsulatus (reviewed in Dixon and Kahn 2004; Forchhammer 2007;
Masepohl and Kranz 2009). During nitrogen depletion Gln levels are low whereas αKG
levels are high. This lowered ratio of Gln:αKG leads to the stimulation of the kinase
activity of NtrB through the actions of two signaling proteins, GlnD and GlnB. The
cascade concludes by activating the transcription factor NtrC by phosphorylation via
NtrB.
NtrC is a global transcription factor that when phosphorylated activates the
expression of genes whose products are involved in the metabolism of alternative
nitrogen containing compounds, i.e. compounds other than ammonia.
activates the expression of nifA.
Also NtrC
NifA is the transcriptional regulator of the entire
nitrogenase regulon, which includes the genes that encode for the structural, assembly,
maturation, and activation proteins. The nitrogen regulatory cascade is in place to limit
alternative nitrogen metabolism under growth conditions where ammonia is available, but
under conditions when nitrogen gas or glutamate is the nitrogen source, the signaling
cascade activates NtrC to activate transcription of the genes necessary for the assimilation
of these nitrogen sources.
8
Previously, PHC strains were shown to circumvent this regulatory cascade and
synthesized nitrogenase even in the presence of ammonia. For certain PHC strains,
single point mutations in nifA were shown to be sufficient for deregulation of nitrogenase
(Laguna 2010; Paschen et al. 2001; Rey et al. 2007; Zou et al. 2008). Sequencing of the
nifA gene from Rb. sphaeroides strain 16PHC also revealed a mutation, but this allele
was not sufficient to derepress nitrogenase in an otherwise WT background (Laguna
2010). Another interesting observation of strain 16PHC was that it had appreciable
nitrogenase activity when its progenitor strains did not (Joshi and Tabita 1996). Strain
2.4.1 is the type strain of Rb. sphaeroides and through spontaneous mutations, a
streptomycin resistant strain was selected from strain 2.4.1 called strain HR (Weaver and
Tabita 1983). Strain 16 was constructed as a targeted RubisCO deletion strain of strain
HR and therefore could not grow photoheterotrophically (Falcone and Tabita 1991).
When cultured photoheterotrophically, strain 16 spontaneously mutated into the PHC
strain 16PHC (Joshi and Tabita 1996; Wang et al. 1993). During strain evolution, strain
HR, and subsequently strain 16, lost the ability to grow under nitrogen-fixing conditions
and lost an appreciable amount of nitrogenase activity even when grown under the
derepressing conditions of growth on glutamate, which has been observed before in other
NSP bacteria (Joshi and Tabita 1996; Wall et al. 1984). Therefore, it is remarkable that
the nitrogenase null phenotype of strains HR and 16 was reverted during the evolution to
strain 16PHC and also that strain 16PHC retains high levels of nitrogenase activity even
during growth on ammonia (Joshi and Tabita 1996).
9
In this chapter, these two phenotypes of strain 16PHC, nitrogenase activity and
derepression, were characterized. First, analyses of strains derived directly from strain
2.4.1 were performed to gain a more complete understanding of the growth requirements
of CBB null strains. Then full genome sequences were analyzed to confirm known
genotypes, and strain-based comparisons were made to identify the genetic basis for the
deregulation and activity of nitrogenase in strain 16PHC. Genetic experiments were
performed to confirm the involvement of mutant alleles to produce the observed
phenotypes.
Lastly, enzyme activities were assayed to devise a hypothesis for a
molecular mechanism describing the derepression and activity of nitrogenase in strain
16PHC. Contributions to work in this chapter include those made by Dr. Rick Laguna
for cloning glnA and performing the γ-glutamyl transferase reaction, undergraduate
Trung Ho for sequence confirmations, and the Plant Microbe Genomics Facility for
sequencing reactions.
MATERIALS AND METHODS
Growth conditions. Routine maintenance of Escherichia coli was achieved by
growth either in liquid or on solid Luria Bertani (LB) medium (1% tryptone, 0.5% yeast
extract, 0.5% NaCl) at 37 °C. Chemoheterotrophic growth of Rhodobacter sphaeroides
was used for routine maintenance in liquid or on solid complex peptone yeast extract
(Weaver and Tabita 1983) or LB media at 30°C. Experimental cultures were grown
10
photoheterotrophically in 30 ml sealed tubes with 10 ml of defined Ormerod’s medium
supplemented with 30 mM malate as the carbon source at 30 °C and illuminated by
incandescent light bulbs (Ormerod et al. 1961).
The atmospheric headspace was
exchanged for either argon or nitrogen for all photoheterotrophic cultures; argon was
used when fixed nitrogen, 15 mM ammonium sulfate or 25 mM potassium glutamate,
was supplied in the media. In some instances dimethylsulfoxide (DMSO) was added to a
final concentration of 40 mM, or in other cases Ormerod’s media lacked the normal
complement of molybdenum and was supplemented with 5 μM sodium tungstate.
Antibiotics were used at the following concentrations (μg per ml): for E. coli, kanamycin,
30; spectinomycin, 50; chloramphenicol, 12.5; for Rb. sphaeroides, kanamycin, 30;
streptomycin, 25-50; spectinomycin, 5 in complex media or 25 in defined media.
Bacterial strains and plasmids. The bacterial strains and plasmids used in this
chapter are listed in Table 1.1. Standard molecular biology techniques, unless otherwise
stated, were used for gene cloning and construction. Oligonucleotides used are listed in
Table 1.2, and a schematic for chromosomal deletions is shown in Figure 1.1. E. coli
strain JM109 was used for maintenance and construction of all plasmids, and strains S171 and SM10 were used to conjugate the plasmids into Rb. sphaeroides. For conjugations,
a 1 ml suspension of an approximate 1:1 ratio of exponential phase cultures of E. coli to
Rb. sphaeroides was washed with LB media. Cells were suspended in approximately 100
μl, spotted onto LB plates, and grown overnight at 30°C in the dark. The spot was
11
Table 1.1 Strains and plasmids used in Chapter 1.
Strain or Plasmid
E. coli
JM109
S17-1
SM10
Rb. sphaeroides
2.4.1
HR
16
16PHC
321
NK10
NK10K
NK10H
NK10P
1323
1551
B214
15165
193
Plasmids
pCR-Blunt IITOPO
pK18mobsacB
pJQ200mp18Km
pJQdsac
pSUP::FII::DI
pSUP::E25Δ::Km
Description
Cloning strain
Conjugation strain, Smr
Conjugation strain, Kmr
Source or Reference
Yanisch-Perron et al. (1985)
Simon, Priefer, Pühler (1983)
Simon, Priefer, Pühler (1983)
Type strain
Smr spontaneous mutant of strain 2.4.1
ΔcbbLS::km, ΔcbbM::tm derivative of
strain HR
PHC strain derived from strain 16
Strain HR containing the glnA allele
from strain 16PHC
ΔnifK strain derived from strain 2.4.1
Strain NK10 containing the nifK allele
from strain 2.4.1
Strain NK10 containing the nifK allele
from strain HR
Strain NK10 containing the nifK allele
from strain 16PHC
ΔcbbM derivative of strain 2.4.1
ΔcbbLS derivative of strain 1323
ΔcbbPII derivative of strain 2.4.1
ΔcbbPI derivative of strain B214
ΔnifA derivative of strain 1551
van Niel (1944)
Weaver and Tabita (1983)
Falcone and Tabita (1991)
Cloning vector
Invitrogen
Allelic exchange vector harboring
sacB; Kmr
Allelic exchange vector harboring
sacB; Kmr
ΔsacB vector derived from plasmid
pJQ200mp18Km
ΔcbbM::tm allelic exchange vector
ΔcbbLS::km allelic exchange vector
Schafer et al. (1994)
12
Wang et al. (1993)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Laguna (2010)
This study
Falcone and Tabita (1991)
Falcone and Tabita (1991)
Table 1.2 Oligonucleotides used in Chapter 1. Each oligonucleotide pair was used to
clone the indicated construct.
5’-3’ sequence
Name
F nifD in spe
R dnifD bcl
Construct
Upstream HRSa for
CGAAGGCAAACTAGTGATGCTCTATGTGG
CGCGCCGCTCGGGCTGATCAGGCCGTCTTCTTCCAGGG nifK deletion
F dnifK bcl
R dnifK pst
CCCTGGAAGAAGACGGCCTGATCAGCCCGAGCGGCGCG
ACGTCGGTGATGGGCTGCAG
Downstream HRS
for nifK deletion
F nifD in spe
R nifK RIV
CGAAGGCAAACTAGTGATGCTCTATGTGG
GATATCGAATTCAGCGGGTCAGGTC
nifK chromosomal
complementation
F PRKa in spe
R PRKa in xba
CGCCACTAGTCGAAGCCGATCC
GAGGTCTAGACGTGCCGGATCG
cbbFPA genetic
region from cbbI
F PRKa ds fus
R PRKa us fus
GGAGCTGATCCGGACCCGGGGC
GTCCGGATCAGCTCCGGAACAGGCC
Inverse primers for
cbbPI deletion
F PRKb in spe
R PRKb in xba
TCGTACTAGTTTGCGATCAACGCCTC
CCGGTCTAGACCTCGATCCCCTC
cbbFPT genetic
region from cbbII
F PRKb ds fus
R PRKb us fus
GGAGTTAAGGCGCGACAGACAGACGGAG
TCGCGCCTTAACTCCGGAACAGGCCCCG
Inverse primers for
cbbPII deletion
F Rs 548 T speI
R nifA pro ncoI
GTACTAGTGCAAGGTCCTGCAGGAG
GTCCATGGCCAGACCTCCGT
Upstream HRS for
nifA deletionb
F nifA T sacI
Downstream HRS
GCGAGCTCGAGAACTGCATC
for nifA deletion
R nifA T xbaI
GGCTCTAGATTTGTCGCACCCC
a
HRS, homologous recombination site
b
an incomplete amplification product was ultimately used in which the 3’ primer
sequence plus an additional 3 base pairs were missing
13
Figure 1.1 Schematic for chromosomal gene deletions. Genomic regions that were
sites of engineered disruptions in coding regions are shown (green).
14
streaked onto defined media and incubated at 30°C in the dark to enrich for Rb.
sphaeroides; single colonies were then subcultured onto complex media to visualize E.
coli contaminants.
Strain 321. To construct a strain in which the glnA allele from strain 16PHC
occurs in a clean background, a 1000 bp region containing the glnA mutation was
amplified from strain 16PHC genomic DNA. The product was sequenced to ensure that
the mutated region of glnA was amplified and was cloned into plasmid pJQ200mp18Km.
The mutated glnA replacement constructs were then transformed into E. coli strain S17-1
and conjugated into strain HR. Upon single recombination, the chromosomal glnA gene
was disrupted resulting in a strain that was a glutamine auxotroph; therefore double
homologous recombination strains were selected for the ability to grow without added
glutamine. The glnA locus was then sequenced to confirm the loss of the integrated
plasmid and for the appropriate allele.
Strain NK10. To construct a nifK deletion strain, about 500 bp upstream of the
stop codon for nifD and about 500 bp downstream of the stop codon for nifK were
amplified from strain 2.4.1 genomic DNA and fused via overlapping amplification. This
1000 bp product was then cloned into the pCR-Blunt II-TOPO vector, sequenced then
sub-cloned into plasmid pK18mobsacB. This construct was transformed into strain S171 and conjugated into strain 2.4.1. Exconjugates were selected for kanamycin resistance,
grown overnight, and plated on defined media supplemented with 10% sucrose. The nifK
region from sucrose resistant colonies was sequenced to confirm the deletion resulting in
strain NK10.
15
Strains NK10K, NK10H, and NK10P. To chromosomally complement the nifK
deletion strain NK10 with nifK alleles, a region from about 500 bp upstream of the stop
codon for nifD to the end of nifK was amplified from strains 2.4.1, HR, and 16PHC to
construct strains NK10K, NK10H, and NK10P respectively, and then cloned into the
pCR-Blunt II-TOPO vector. This region was then sub-cloned into plasmid pJQdsac,
transformed into strain S17-1, and conjugated into strain NK10. The plasmid pJQdsac
was constructed after digestion of plasmid pJQ200mp18Km with EcoRI and KpnI
followed by ligating the large fragments together resulting in the excision of the sacB
gene. Kanamycin resistance colonies were selected and sequenced to confirm that the
plasmid integration occurred to repair the nifHDK operon and to confirm the nifK allele.
Strain 1551. To construct strain 1551, a nonpolar RubisCO deletion strain derived
from strain 2.4.1, the gene encoding form-II RubisCO, cbbM, was deleted from the
genome of strain 2.4.1 then the genes encoding form-I RubisCO, cbbLS, were also
deleted.
Plasmid pSUP::FII::DI containing a deletion-insertion of cbbM with a
trimethoprim (Tm) cassette was digested with PstI to remove the Tm cassette. The
resulting nonpolar deletion fragment of cbbM was then cloned into plasmid pCR-Blunt
II-TOPO, sequenced to confirm its identity, then cloned into the suicide vector
pJQ200mp18Km, transformed into strain S17-1, and conjugated into strain 2.4.1.
Exconjugates were selected for kanamycin resistance, subcultured until kanamycin
sensitive colonies appeared, and then sequenced to confirm the deletion of the cbbM
gene. This resulted in the creation of strain 1323.
16
The upstream and downstream homologous recombination sites from plasmid
pSUP::E25Δ::Km containing the deletion-insertion construct of cbbLS were cloned into
pCR-Blunt II-TOPO to construct a deletion of the cbbLS genes. This construct was then
cloned into the suicide vector pJQ200mp18Km, transformed into strain S17-1, and
conjugated into strain 1323. Double recombinants were screened for as above to isolate
strain 1551.
Strain 15165. To construct a nonpolar PRK deletion strain from strain 2.4.1, the
cbbPII gene was deleted from the genome of strain 2.4.1 then the cbbPI gene was deleted.
The cbbFPT genetic region from the cbbII operon from strain 2.4.1 was cloned into
plasmid pCR-Blunt II-TOPO. Inverse amplification and infusion reactions (Clontech,
Mountain View, CA) were performed to construct a nonpolar deletion of the cbbPII gene
by removing the entire sequence from the stop codon of cbbF to the stop codon of cbbP.
This region was then cloned into the suicide vector pJQ200mp18Km, transformed into
strain S17-1, and conjugated into strain 2.4.1. Double recombinants were screened for as
above to isolate the cbbPII deletion strain B214. Similar methods were employed to
construct the cbbPI nonpolar deletion construct. This was then conjugated into strain
B214 and screened as above to isolate the double PRK deletion strain 15165.
Strain 193. To construct a strain harboring a nonpolar deletion of nifA derived
from strain 1551, a nifA deletion fragment was constructed by ligating approximately 400
bases upstream of a putative ribosome binding site of nifA to approximately 400 bases of
the 3’ coding region into plasmid pCR-Blunt II-TOPO. This construct was then cloned
17
into the suicide vector pJQ200mp18Km, transformed into strain S17-1, and conjugated
into strain 1551. Double recombinants were screened as above to isolate strain 193.
Enzyme assays and biochemical procedures.
Acetylene reduction assays. The acetylene reduction assay was used to determine
nitrogenase activity and was adapted from Tichi and Tabita (2000) and Heiniger et al.
(2012). 500 μl of culture and 2.5 ml of acetylene were injected into 25 ml argon flushed
vials and incubated at 30°C for about 20 min in the light. To detect ethylene formed, 200
μl of head space was injected with a gastight syringe (Hamilton, Reno, NV) into a GC2014 gas chromatograph (Shimadzu, Columbia, MD) installed with a Flame Ionization
Detector and an RT-Alumina BOND/Na2SO4 column (30m, ID 0.53mm, df 10μm)
(Restek, Bellefonte, PA). Helium was supplied as the carrier gas at 47 cm/s with a split
ratio of 10.3. The temperatures of the injector, column, and detector were 150°C, 130°C,
and 150°C respectively. Time points were sampled over the course of 1 h. The rate of
ethylene formation was generated by plotting the area under the ethylene peak verses
time and was converted to moles of ethylene by comparison to a standard curve, using the
density of ethylene at 22.5°C and 1 atm as 0.04146 mol/L obtained from the National
Institute of Standards and Technology, http://webbook.nist.gov/chemistry, accessed
January 2012. Finally, the rate of ethylene produced was normalized to the dry cell
weight (DCW) of the assayed culture. The DCW was calculated from the optical density
at 660 nm (OD660) using a hyperbolic regression plotted from experimentally measured
data of OD660 vs. DCW. The limit of detection for this assay was about 0.5 nmol of
18
ethylene generated/min/mg of DCW. Unless otherwise stated, triplicate cultures were
assayed in duplicate and the values are reported as averages of the cultures ± standard
deviations.
Western immunoblot determinations. Approximately 5 ml of culture was
harvested by centrifugation at max speed in a tabletop microcentrifuge; cell pellets were
stored at -80°C. Cell pellets were suspended in TEM buffer (50 mM Tris-Cl, pH 7.5, 1
mM EDTA, 5 mM β-mercaptoethanol) and lysed by sonication (Heat Systems
Ultrasonics Inc. W385) for 5 sec cycle time, 50 % duty cycle, 2 min run time, and 2.5
output control. The cellular lysate was clarified by centrifugation (13,000 x g, 10 min,
4°C). The Bradford method was used to determine protein levels with bovine serum
albumin as the standard (Bio-Rad Laboratories, Hercules, CA). A 5 μg aliquot of soluble
protein was subjected to SDS-PAGE using 10% acrylamide gels and then transferred to
an Immobilon-P membrane (Millipore, Billerica, MA) by a Trans-Blot SD semi-dry
electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA) for 15 min at 20 V.
Western immunoblot analysis was performed according to standard procedures
(Ausubel et al. 2001). Antiserum raised in rabbits directed against Rhodospirillum
rubrum dinitrogenase reductase (NifH), Rb. sphaeroides form-I PRK, form-I RubisCO,
or form-II RubisCO was used as the primary antibody. The secondary antibody was an
alkaline phosphatase labeled goat anti-rabbit immunoglobulin G (Bio-Rad Laboratories,
Hercules, CA). The immunoblot membranes were developed according to AttoPhos
Fluorescent Substrate System (Promega, Madison, WI) and visualized with a Storm 840
imaging system (Molecular Dynamics, Sunnyvale, CA).
19
Glutamine synthetase assay. Glutamine synthetase activity was measured using
the γ-glutamyl transferase reaction (Johansson and Gest 1977; Shapiro and Stadtman
1970; Stadtman et al. 1979). Cell pellets were lysed as described above. 20 μg of soluble
protein extract, as determined by the Bradford method, described above, was added to the
assay buffer (80 mM Hepes, 80 mM imidazole, 20 mM glutamine, 0.8 mM MnCl2, 0.4
mM ADP, 40 mM KAsO4, 40 M NH2OH, pH 7.6). The amount of γ-glutamyl
hydroxamate was determined spectrophotometrically at 540 nm over a 15 min time
period at 30 °C, terminated by addition of 1 ml stop solution (10 % FeCl3, 24 %
trichloroacetic acid, 6 N HCl), and compared to a standard curve. Unless otherwise
stated, triplicate cultures were assayed and the values are reported as averages of the
cultures ± standard deviations.
Data Analysis
Genome comparisons. Chromosomal DNA was isolated from Rb. sphaeroides
using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and
sequenced using a 454 FLX Titanium Genome Sequencer (Roche, Indianapolis, IN). The
reads were mapped to the annotated genome of strain 2.4.1 (National Center for
Biotechnology Information, ftp://ftp.ncbi.nih.gov/genomes, accessed 2011) using the GS
Mapper software (Roche, Indianapolis, IN). Additional analysis and annotation was
performed with Microsoft Office (Microsoft, Redmond, WA) and visualized with
Integrated Genomics Viewer (Robinson et al. 2011; Thorvaldsdottir et al. 2012).
20
Logo diagrams. Protein sequences were retrieved and aligned using the Integrated
Microbial
Genomes
database
(http://img.jgi.doe.gov/cgi-bin/w/main.cgi
accessed
November 2012 - January 2013). Logo diagrams of aligned sequences were generated
using the website http://weblogo.berkeley.edu/.
Protein structures. Pymol (http://pymol.org/educational/) was used to visualize all
structural diagrams.
RESULTS
Photoheterotrophic growth. Different strains of Rb. sphaeroides were tested for
photoheterotrophic growth with malate in the presence or absence of the terminal electron
acceptor dimethylsulfoxide (DMSO) and ammonia or glutamate as the nitrogen source.
Certain strains needed the alternative electron acceptor DMSO for photoheterotrophic
growth, but this requirement sometimes depended on the nitrogen source employed
(Table 1.3). Strains 2.4.1, HR, and 16PHC never required DMSO, whereas DMSO was
required for strain 16 grown with both nitrogen sources. Since strain 16 was derived
from strain HR, other CBB null strains were constructed directly from strain 2.4.1: strain
1551 contains deletions of segments of the coding regions of the genes that encode for
forms I and II RubisCO and strain 15165 contains nonpolar deletions of both genes that
21
Table 1.3 Additional requirements for photoheterotrophic growth. Strains were
inoculated into Ormerod’s minimal media with the indicated nitrogen source and
supplemented with 30 mM malate.
Nitrogen source
Strain
Ammonia Glutamate
2.4.1
-a
HR
b
16
DMSO
DMSO
16PHC
1551
DMSO
15165
DMSO
193
DMSO
DMSO
a
-, no additional substrates required
b
DMSO, dimethylsulfoxide required
22
encode for PRK. Additionally, strain 1551 was used to construct strain 193, which also
harbors a nonpolar deletion of nifA. All three of these strains required DMSO for growth
on ammonia, but only strain 193 also required DMSO for growth on glutamate. These
results indicate that CBB null strains derived from strain 2.4.1 can maintain redox
balance when grown with glutamate, as they do not require a terminal electron acceptor,
but those derived from strain HR cannot, unless they are adaptive PHC strains.
Genomic comparisons. The genomes of strains HR, 16, and 16PHC were
massively parallel pyrosequenced using a Roche GS FLX system and mapped against the
annotated genome of strain 2.4.1 using the GSMapper software. Upon completion, the
coverage of each strain’s genome was 98% and each base had an average depth of at least
9 reads (Table 1.4). Due to the large number of contigs, the genome for each strain could
not be closed (Table 1.4). The accuracy of these genomic sequences and assemblies was
confirmed by identifying known genotypes of each strain (Figure 1.2), and once a
particular mutation was identified for further analysis, its locus was amplified with a
high-fidelity polymerase and sequenced via Sanger Capillary DNA Sequencing.
When the genomes were analyzed for sequences that differed from strain 2.4.1,
hundreds of mutations were revealed (Figure 1.3). A mutation that encodes for a K43R
substitution in ribosomal protein S12 was found in strain HR and its derivative strains,
which might be responsible for the streptomycin resistance, as has been seen in
Mycobacterium tuberculosis (Finken et al. 1993). Alternatively, in order to identify key
mutations that could form the genetic basis of the strain 16PHC phenotype, only the 78
23
Table 1.4 Metrics for genomic sequences. Genomes from the indicated strains were
massively parallel pyrosequenced and aligned to the annotated genome of strain 2.4.1.
Total Number of reads
Average Read Length (bp)
Average Depth
Number of Large Contigsa
Coverage (%)
chromosome 1
chromosome 2
plasmid A
plasmid B
plasmid C
plasmid D
plasmid E
a
at least 500 bp in length
Strain
HR
16
16PHC
110786 107575 100381
447
439
439
10.1
9.7
9.0
189
112
175
24
98.31
98.21
85.80
0.28
95.30
81.34
98.45
98.55
98.52
89.15
0.27
96.26
82.94
98.68
98.39
98.34
85.17
0.27
94.54
79.89
97.30
Figure 1.2 Genome alignments of strains 16 and 16PHC. Reads obtained from the
pyrosequencing of the genomes of strain 16 (A) and strain 16PHC (B) were aligned
against the genome of strain 2.4.1 using GSMapper and visualized with Integrated
Genomics Viewer. For strain 16, no reads mapped to the segment of cbbL and cbbS that
corresponds to the gene deletion fragment. For strain 16PHC, a mismatch is found in all
reads containing base position 153 of nifA, which corresponds to the g153a nucleotide
mutation. Horizontal black bars with chevrons, genes with transcription direction
indicated; Gray bars, individual sequencing reads; within each read: Black dot, insertion
or deletion; Black bar, base mismatch.
25
Figure 1.3 Venn diagram of high confidence differences in genomes of strains
compared to strain 2.4.1. Genomes from strains HR (black), 16 (Blue), and 16PHC
(green) were massively parallel pyrosequenced and aligned to the annotated genome of
strain 2.4.1. The total number of loci that differed from strain 2.4.1 is indicated in the
parentheses.
26
mutations that were unique to strain 16PHC were further considered. Even though
GSMapper had algorithms to report only the high confidence differences, the loci of each
of these mutations were visually inspected to discount the 68 mutations that resulted from
poor sequences or poor alignments. Of the 10 remaining mutations (Table 1.5), one was
silent and two were in noncoding sequences upstream of genes for hypothetical proteins
and a 16S rRNA. Within the list of genes that contain the seven remaining mutations,
three are known to be involved in nitrogen metabolism, amtB, glnA, and nifA, and might
therefore affect the regulation of the nitrogenase complex.
A similar analysis was conducted to determine if any loci in strain 16PHC
contained WT sequences that were mutated in strains HR and 16; perhaps indicating a
reversion during the evolution from strain 16 to strain 16PHC. Of the 97 loci that
contained mutations that occurred in both strains HR and 16 but not in strain 16PHC, 68
were silent and only one was of a high confidence after a visual inspection: a1397g in
nifK encoding an H466R substitution in one subunit of the nitrogenase complex in strains
HR and 16. Upon analyzing nifK, a second mutation was noticed, which occurred in all 3
sequenced strains: a47g encoding a K16R substitution.
Analysis of glnA. Primary structure comparisons of position 255 in GlnA,
glutamine synthetase, from Rb. sphaeroides indicated that it is a highly conserved residue
(Figure 1.4). An alignment was generated of 100 GlnA sequences retrieved from the
Integrated Microbial Genomes database; to increase sequence diversity, each sequence
represents a unique genus. Only two residues were located at position 255 and both
27
Table 1.5 Unique mutations in strain 16PHC. Genomes from strains HR, 16, and
16PHC were massively parallel pyrosequenced and aligned to the annotated genome of
strain 2.4.1. The highly confident mutations that occurred only in strain 16PHC are
indicated.
Gene
amtB
glnA
nifA
nuoM
cycB
Mutation
Substitution
a128g
D43G
a763g
T255A
c153a
F51L
a973g
T325A
2 bp deletion
frame shift at
at position 440 position 147
RSP_0169
a167g
Y56C
RSP_0575
c391t
Q131*
RSP_3907
g24a
Silent
NCSa
1 bp insertion
NCS
Gene Product
ammonium transporter
L-glutamine synthetase
NifA subfamily transcriptional regulator
NADH dehydrogenase subunit M
cytochrome c553i
Na+/solute symporter
signal transduction protein
hypothetical protein
14 bp upstream of 16S rRNA and 65 bp
upstream of RSP_3837 (hypothetical)
1025 bp upstream of RSP_6250
(hypothetical) and 2500 bp downstream
of RSP_0002 (histone-line nucleoidstructuring protein)
23 bp deletion
*stop codon
a
NCS, noncoding sequence
28
Figure 1.4 Logo diagram of a partial consensus GlnA sequence. A logo diagram of
100 GlnA sequences, each from different genera, was created and centered on residue
255 from Rb. sphaeroides. The threonine at position 255 was highly conserved. None of
the sequences contained an alanine at position 255, which was found in strain 16PHC.
29
contained a hydroxyl functional group; 91% of those residues were threonine and the
other 9% were serine. The quaternary structure of glutamine synthetase is composed of
12 GlnA peptides which are arranged as a bilayer of 6-peptide rings (Eisenberg et al.
2000). Residues 253-255 are located at a bilayer interface near a conserved Tyr residue
(Figure 1.5); indicating the potential that these residues might interact with each other to
stabilize the quaternary structure.
The phenotypic effect of the glnA alleles may be observed when comparing
strains HR, 16PHC, and 321. Strain 321 was constructed by a chromosomal exchange of
the glnA allele of strain HR for that of strain 16PHC; therefore the only genetically
engineered difference between strains HR and 321 is the guanine to adenosine exchange
at position 763 of glnA. When the activity of glutamine synthetase was measured from
cellular lysates via a γ-glutamyl transferase assay, strains 16PHC and 321 had
approximately the same activity levels, which were lower than that for strain HR (Table
1.6). Another similarity occurred between strains 16PHC and 321 when nitrogenase
protein abundance was probed by Western immunoblots using antibodies targeted to
NifH (Figure 1.6).
The lysates of both strains contained detectable levels of NifH
regardless if the cells were cultured with ammonia or with glutamate as the nitrogen
source, whereas NifH was only present in the lysate of strain HR grown with glutamate.
Strains 16PHC and 321 not only had nitrogenase protein when cultured with both
nitrogen sources, they also had nitrogenase activity, as measured by acetylene reduction,
though their activity levels were different (Table 1.7). Additionally, the nitrogenase
30
Figure 1.5 Crystal structure of glutamine synthetase. The glutamine synthetase (GS)
from Salmonella enterica sv. Typhimurium (pdb accession, 1FPY) was used to analyze
position 255. GS is a homododecamer composed of a bilayer of hexameric rings (green
and black) that is stabilized by the C-terminal helix that protrudes from one layer into the
other; half of each layer is shown. Position 255 was located at a bilayer interface (inset)
in which threonyl side chains located at positions 255 and 253 were in close proximity to
a tyrosyl side chain from the helical C-terminus of a peptide from the opposite layer. The
numbering is according to Rb. sphaeroides.
31
Table 1.6 Glutamine synthetase activity. The glutamine synthetase (GS) activities
from cellular lysates were measured using the γ-glutamyl transferase reaction and
reported as the nmol of γ-glutamyl hydroxamate formed/min/mg of protein in crude cell
extract.
Strain GS activity
HR
502 ± 11
16PHC 200 ± 8
321
195 ± 29
32
Figure 1.6 Western immunoblot of crude extracts from cultures of glnA allelic
strains using antisera to NifH. Cell extract from strains HR (lanes 1 and 2), 16PHC
(lanes 3 and 4), and 321 (lanes 5 and 6) were blotted with antibodies directed against
NifH to visualize nitrogenase. All samples contained NifH when the cells were cultured
with glutamate (lanes 2, 4, and 6), but only strains 16PHC and 321 contained detectable
levels of NifH when the cells were cultured with ammonia (lanes 1, 3, and 5).
33
Table 1.7 Nitrogenase activities of glnA allelic strains. Each strain was cultured with
the indicated nitrogen source and assayed for acetylene reduction activity. Results are
reported in nmol of ethylene generated/min/mg of dry cell weight.
Nitrogen source
Strain Ammonia Glutamate
HR
NDa
7±2
16PHC
18 ± 4
97 ± 7
321
6±2
15 ± 1
a
ND, not detected
34
activity level of strain 321 with ammonia was the same as strain HR with glutamate.
These data indicate that, like strain 16PHC, strain 321 can derepress nitrogenase.
Analysis of nifK. The roles of the nifK alleles of strains 2.4.1, HR, and 16PHC
were first analyzed through sequence comparisons.
The 55 NifK sequences in the
database of the Integrated Microbial Genomes that correspond to one subunit of the
nitrogenase complex were aligned and logo diagrams were constructed of the residues
around positions that correspond to 16 and 466 of Rb. sphaeroides NifK (Figure 1.7).
The most common residue at position 16 was arginine while the lysine residue that is
found in the strain 2.4.1 sequence was the 3rd most abundant. Position 466 is the first
histidine in a DRHHXHR motif that occurs in 65% of the sequences. When these
positions were mapped to a crystal structure of nitrogenase from Azotobacter vinelandii,
position 16 was surface exposed while position 466 was buried and located near the NifK
dimer interface and surrounded by other highly conserved residues (Figure 1.8). Neither
position was located adjacent to any metal clusters, which participates in catalysis.
To determine the effect of the various nifK alleles, the phenotype of constructed
strains were compared to strain 2.4.1. Strain NK10 was derived from strain 2.4.1 and
contains a deletion of the nifK gene. This strain was complemented with nifK alleles
cloned into the suicide vector pJQdsac and designed so that single recombination into the
chromosome of strain NK10 would repair the nifHDK operon. Strains NK10K, NK10H,
and NK10P were the complemented strains and harbored the nifK alleles from strains
2.4.1, HR, and 16PHC respectively. When these strains were grown with nitrogen gas as
35
Figure 1.7 Logo diagrams of partial NifK sequences. Logo diagrams of 55 NifK
sequences were created and centered on residue 16 (A) or 466 (B) of NifK from Rb.
sphaeroides. Arginine, which occurred in NifK from strains HR, 16, and 16PHC, was
the most common residue at position 16 while lysine, which occurred in NifK from strain
2.4.1, was the third most abundant. Position 466 was the third position of the
DRHHXHR motif; no sequences contained an arginine at position 466, which occurred in
NifK from strains HR and 16.
36
Figure 1.8 Crystal structure of nitrogenase. The crystal structure of nitrogenase from
Azotobacter vinelandii (pdb accession 1M34) was used to highlight the positions that
were mutated in strains HR and 16. Position 16 (top inset) was a surface exposed Leu in
A. vinelandii and the His corresponding to position 466 (bottom inset) was buried and
was near the NifK dimer interface, surrounding residues include Ser, Trp, Arg, and the Cterminal Arg. Neither position was near the catalytic metal clusters, space-filled orange
and yellow. Black, NifH; Green, NifD; Gray, NifK.
37
the sole nitrogen source, strains 2.4.1, NK10K, and NK10P were all capable of growth at
the same rate, while strain NK10 did not grow (Table 1.8). For strains HR and NK10H,
growth was only observed after a significant lag phase, and upon sequencing of strains
that grew after this lag, all cultures contained nifK alleles resulting in a straight reversion
of the H466R substitution. When these conditioned cultures were again grown under
nitrogen-fixing conditions the lag time became comparable to those of strains 2.4.1,
NK10K, and NK10P. Since not all cultures were able to grow with nitrogen gas as the
nitrogen source, assays for nitrogenase activity were performed on cultures grown with
glutamate (Table 1.8).
Strains 2.4.1, NK10K, and NK10P all contained high rates of
acetylene reduction, while strains HR and NK10H had acetylene reduction rates that were
about 10% of strains 2.4.1, NK10K, and NK10P. Also, no acetylene reduction activity
was detected from cultures of strain NK10.
DISCUSSION
Though PHC strains have been partially characterized previously, only
preliminary studies of the PHC strain derived from Rb. sphaeroides have been reported
(Laguna 2010; Smith and Tabita 2002; Joshi and Tabita 1996; Wang et al. 1993). In this
study, strain 16PHC was shown to contain mutations in glnA and nifA, and although point
38
Table 1.8 Phenotypic effects of nifK alleles. Doubling times, reported in hours, were
calculated for strains that contain different nifK alleles grown under nitrogen-fixing
conditions, and nitrogenase activities, reported in nmol of ethylene generated/min/mg of
dry cell weight, were determined for strains grown with glutamate.
Strain
Doubling time
Nitrogenase activity
2.4.1
8.8 ± 0.8
70 ± 5
HR
*
7±2
NK10
NGa
NDb
NK10K
8.1 ± 0.2
83 ± 4
NK10H
*
6±1
NK10P
7.7 ± 0.5
86 ± 9
*growth occurred only after a lag of 10+ days; upon
sequencing, nifK was found to be mutated resulting in a
straight reversion of the H466R substitution.
a
NG, no growth
b
ND, not detected
39
mutations in the nifA genes from other PHC strains were sufficient to derepress
nitrogenase, this was not the case for the mutation in nifA from strain 16PHC (Laguna
2010). It is likely that derepression of nitrogenase synthesis in strain 16PHC still might
involve aberrant control of the nitrogen regulatory cascade, and sequence studies revealed
that the only other mutation in genes whose products are members of this cascade
mapped to glnA.
The product of glnA is glutamine synthetase (GS); this enzyme has been studied
in many bacteria and initiates the regulatory cascade by controlling the cellular levels of
Gln (Forchhammer 2007; Ikeda, Shauger, Kustu 1996; Li et al. 2010).
GS is a
dodecamer of GlnA peptides arranged in a bilayer of two 6-member rings (Eisenberg et
al. 2000). This bilayer is stabilized by numerous interactions, one of which is the Cterminal α-helix that protrudes from one peptide into a channel created by a peptide of the
adjacent layer (Yamashita et al. 1989). At the end of this helix is a conserved Tyr residue
that resides next to the Thr residues at positions 253 and 255 of the other peptide. When
an Ala is substituted for the Thr at position 255 in strain 16PHC the resulting change
from a polar to a hydrophobic side chain could disrupt noncovalent interactions that
contribute to stabilizing the bilayer. This could result in the observed decreased activity
for the enzyme because quaternary structure is essential for activity (Almassy et al.
1986). With decreased activity, the cellular levels of the product of its reaction, Gln,
would then also decrease, which could be sufficient to initiate the regulatory cascade. In
support of this hypothesis, transcript analysis of strain 16PHC revealed an increase in
transcripts of not only nitrogenase genes, but also of other genes which are induced
40
through the regulatory cascade (Laguna 2010). Also, studies of a ΔglnA strain of Rb.
sphaeroides showed complete derepression of nitrogenase (Li et al. 2010).
To test if GlnA T255A is sufficient to derepress nitrogenase, strain 321 was
constructed from strain HR. Strain 321 contains the glnA allele from strain 16PHC
instead of the WT allele; thus, any phenotypic differences would then be due to this
mutation. When GS activity was measured in extracts from strain 321, the same activity
was obtained as strain 16PHC, which was less than half the GS activity of extracts from
strain HR. These results indicate that the T255A substitution is sufficient to lower the GS
activity level to that of strain 16PHC.
To examine the effect upon nitrogenase
derepression, Western immunoblots were used to detect the appearance of nitrogenase
using antibodies raised against the NifH subunit.
The blot revealed that NifH was
present in extracts of strains 321 and 16PHC regardless of the nitrogen source. However,
NifH was only detected in extracts of strain HR grown with glutamate as the nitrogen
source. These results indicate that this GlnA substitution is also sufficient to derepress
synthesis of nitrogenase. This observation was supported by the presence of nitrogenase
activity from cultures of strain 321 grown with both nitrogen sources, whereas strain HR
only contained activity when grown with glutamate. These experiments also showed that
the GlnA substitution was not sufficient for restoring nitrogenase activity because the
activity levels from strain 321 were much lower than those from strain 16PHC.
Strain 16PHC of Rb. sphaeroides is unique among the PHC-like strains of NSP
bacteria because its single mutation in nifA is not sufficient to derepress nitrogenase
synthesis in the presence of ammonia (Laguna 2010).
41
Strain 16PHC developed a
mutation in glnA to derepress nitrogenase synthesis, but strain 16PHC also must evolve
changes to regain sufficient nitrogenase activity. Two substitutions in NifK were found;
the first resulted in a K16R substitution. Position 16 is found as a surface exposed
position in nitrogenase from A. vinelandii. Both Lys and Arg are hydrophilic residues,
therefore no gross structural changes would be necessary to accommodate this
substitution. This was supported by observations of no phenotypic differences among
strains that have the WT sequence or just this substitution; therefore it was a neutral
substitution.
The other substitution in NifK, H466R, was detrimental. Strains that harbor this
mutation had drastically reduced nitrogenase activities and were not able to grow under
nitrogen-fixing conditions. Though what threshold level of nitrogenase activity needed to
support nitrogen-fixing growth has not been determined, other mutations that decrease
nitrogenase activity have been shown to abolish growth (Brigle et al. 1987). After an
extended lag phase, strains HR and NK10K did mutate to restore growth, but the
mutation always repaired the substitution instead of adapting other residues. Therefore
the His at position 466 was a critical residue. This His was part of a conserved motif that
occurred near the NifK dimer interface. Residues that surround position 466 included
Ser, Trp, Arg, and the C-terminus of the other NifK peptide. With the cavity formed by
these residues, the imidazole ring of the His side chain might be the only moiety that can
adequately occupy the space while interacting with these residues, therefore, helping to
keep the dimer stable.
In the evolution of strain 16PHC, the repair of the H466R
42
substitution in NifK seemed to be necessary to regain nitrogenase activity, while the
K16R substitution was tolerated with no ill effect.
For cells to use the nitrogenase complex to maintain redox balance, nitrogenase
must be active enough to support efficient reductant turnover. Therefore it was not
surprising that the CBB null strain 16, which contains the detrimental H466R substitution
in NifK, required the exogenous electron acceptor DMSO when grown under the natural
nitrogenase-derepressing condition of growth with glutamate. This was not the case for
CBB null strains derived from strains that contain highly active nitrogenases as was also
observed in other species (Tichi and Tabita 2000; Tichi and Tabita 2001).
The
hypothesis of nitrogenase-catalyzed redox balance was also supported by the observation
that strain 193 required DMSO when grown with glutamate, because the deletion of nifA
prevented nitrogenase synthesis in this strain (Chapters 2 and 3). Though others have
stated that accumulation of the CBB metabolite RuBP is what represses the growth of
ΔRubisCO strains and not redox imbalance, the observation that strain 15165 and other
PRK deletion strains, which contains nonpolar deletions in both genes required for RuBP
production, still required DMSO when grown with ammonia refutes this hypothesis for
Rb. sphaeroides (Wang et al. 2011; Hallenbeck et al. 1990b). Therefore, for redox
balance to be maintained in photoheterotrophically grown Rb. sphaeroides, the CBB
cycle or nitrogenase must be active and synthesized.
In conclusion, to become competent in photoheterotrophic growth the evolution
of strain 16PHC must include mutations that cause derepression of the nitrogenase
complex, and if the CBB null strain was derived from strain HR, restore its activity. The
43
T255A substitution in GlnA was sufficient to derepress nitrogenase synthesis in Rb.
sphaeroides.
Moreover, the H466R substitution in NifK was sufficient to decrease
nitrogenase activity. To further enhance the PHC phenotype in Rb. sphaeroides, other
mutations may also prove to be important to derepress nitrogenase synthesis, such as
substitutions in the nitrogenase transcriptional regulator NifA or the ammonia transporter
AmtB, as discovered in the genome of strain 16PHC. Substitutions in such proteins were
shown to affect nitrogenase gene expression in other species (Paschen et al. 2001; Rey et
al. 2007; Yakunin and Hallenbeck 2002; Zhang et al. 2006). However, it is unlikely that
other mutations were responsible for the recovery of nitrogenase activity because no
further mutations at loci known to affect nitrogenase activity were found to be unique in
strain16PHC.
44
Chapter 2: Alterations to NifA Influence Nitrogenase Synthesis
INTRODUCTION
The nitrogen regulatory cascade balances the metabolism of nitrogen sources
based on the needs of the cell. When the cells are cultured in media deficient in the
biologically relevant nitrogen source ammonia, signals are relayed to activate the
expression of nitrogenase, which catalyzes the reduction of nitrogen gas into ammonia
(Masepohl and Kranz 2009). These signals are relayed from one tier of the cascade to the
next through post-translational modifications of regulatory proteins, which have been
extensively studied in E. coli, S. enterica, and the NSP bacterium Rb. capsulatus (Dixon
and Kahn 2004, Forchhammer 2007, Masepohl and Kranz 2009). The cascade terminates
with the transcription of genes in the nitrogenase regulon via the transcriptional activator
NifA (Dixon and Kahn 2004). Transcription initiation from promoters of genes whose
products are involved in nitrogen metabolism consists of two parts: a unique sigma
factor, σ54, and the proteins that activate it, bacterial enhancer binding proteins (bEBPs)
(reviewed in Bush and Dixon 2012, see also Buck et al. 2000; Cannon et al. 2000; Joly et
al. 2007).
Sigma54 is the rpoN gene product and differs from the common sigma factor, σ70,
the rpoD gene product, in two distinct ways. Though both promoters have consensus
binding sequences, the variability of the σ70 -35 and -10 boxes is much greater than the
45
well conserved -24 and -12 binding sites of σ54 (Domenzain et al. 2012; Francke et al.
2011). Also, the σ70-RNA polymerase complex is sufficient to melt the bases in the -10
box to initiate transcription whereas the σ54-RNA polymerase complex requires ATP
hydrolysis to mediate the melting of the -12 bases, which is catalyzed by bEBPs
(Friedman and Gelles 2012; Saecker et al. 2011).
NifA is a bEBP and contains the three conserved bEBP domains: an N-terminal
domain to regulate oligomer formation, a central domain for ATP hydrolysis, and a Cterminal domain for DNA binding (Bush and Dixon 2012; Masepohl and Kranz 2009).
Typically bEBPs exists as dimers incapable of interacting with σ54 and hydrolyzing ATP,
but upon activation a hexameric ring forms (Figure 2.1). The transition between the
dimeric and hexameric states is controlled by the N-terminal regulatory domain, known
as the GAF domain for NifA, which is post-translationally regulated through at least one
of the following: metabolite binding, protein interaction, or covalent modification
(Aravind and Ponting 1997; Kern et al. 1999; Little and Dixon 2003; Meyer et al. 2001;
Pawlowski et al. 2003; Zou et al. 2008). Once a signal modifies the regulatory domain to
induce hexamer formation, the central domain can then interact with σ54 to hydrolyze
ATP (Wigneshweraraj et al. 2008). The central domain belongs to the AAA+ functional
group that consists of two subdomains. The α/β subdomain contains the Walker A and B
sequences and Arg finger that is required for ATP binding and hydrolysis; the α-helical
subdomain contains the sensor 2 region that also coordinates the ATP (Hanson and
Whiteheart 2005). The active site for ATP hydrolysis is only formed when the bEBPs are
46
Figure 2.1 Oligomeric state of bEBPs. bEBPs naturally exist as dimers but upon
activation reform as hexamers. (A) Crystal structure of NtrC1 from Aquifex aeolicus
lacking the DNA binding domain (pdb accession 1NY5). As a dimer, the AAA+
domains, composed of the α/β and α-helical subdomains, face each other and are
stabilized by the linker helix to the regulatory domain. (B) The crystal structure of ZraR,
a bEBP involved with Zn metabolism, as a hexamer from Salmonella enterica sv.
Typhimurium (pdb accession 1OJL), from which the regulatory domain has been
truncated. When bEBPs form a hexamer they are then capable of hydrolyzing ATP. The
inset shows the ATP binding pocket with side chains from critical residues of the Walker
A, Walker B, Sensor 2, and Arg finger indicated.
47
in the hexameric state because the active site is composed of residues from adjacent
peptides (Rappas et al. 2007). The central domain from bEBPs also contains a unique
loop that is not found in other AAA+ containing proteins. This loop has a conserved
sequence, GAFTGA, and relays the conformational changes from ATP hydrolysis to σ54
resulting in promoter melting (Francke et al. 2011). Among the bEBPs, the AAA+
domain of NifA from some bacteria, including the NSP bacteria and the bacteria that
form symbiotic rhizomes, is unique because it contains a Cys motif, C-X11-C-X19-C-X4C, in which the individual Cys are critical for activity through an unknown mechanism
but is hypothesized to require a metal cofactor (Fischer et al. 1988; Fischer 1994;
Oliveira et al. 2009). The C-terminal domain is the DNA binding domain that typically
forms a helix-turn-helix motif (Bush and Dixon 2012). This domain directs the bEBPs to
palindromic sequences hundreds of bases upstream of the σ54 binding sites. To facilitate
bEBP- σ54 interaction, most promoter sequences also contain an integration host factor
(IHF) binding site to bend the DNA (Masepohl and Kranz 2009).
NifA is a bEBP that is required to interact with σ54 to initiate nitrogenase gene
transcription (Masepohl and Kranz 2009).
In this chapter, the post-translational
regulation of NifA from Rb. sphaeroides was explored through targeted deletions and
chimeric exchanges. Attempts were made to determine the effect of these modifications
on the oligomerization state through recombinant expression and purification in order to
gain further insight into the role of NifA in gene expression.
48
MATERIALS AND METHODS
Growth Conditions. E. coli and Rb. sphaeroides were grown as described in
Chapter 1.
Bacterial strains and plasmids. The bacterial strains and plasmids used in this
chapter are listed in Table 2.1. Standard molecular biology techniques, unless otherwise
stated, were used for gene cloning and construction; oligonucleotides used are listed in
Table 2.2.
E. coli strain JM109 was used for maintenance and construction of all
plasmids, and strains S17-1 and SM10 were used to conjugate the plasmids into Rb.
sphaeroides. Conjugations were performed as described in Chapter 1.
Strain 145. To construct a strain harboring a nonpolar deletion of nifA derived
from strain 2.4.1, a nifA deletion fragment was constructed by ligating approximately 400
bases upstream of a putative ribosome binding site of nifA to approximately 400 bases of
the 3’ coding region into plasmid pCR-Blunt II-TOPO. This construct was then cloned
into the suicide vector pJQ200mp18Km, transformed into strain S17-1, and conjugated
into strain 2.4.1. Double recombinants were screened as in Chapter 1 to isolate strain
145.
49
Table 2.1 Strains and plasmids used in Chapter 2.
Strain or Plasmid
E. coli
JM109
S17-1
SM10
ER2566
Rb. sphaeroides
2.4.1
145
Plasmids
pCR-Blunt IITOPO
pJQ200mp18Km
pGtf2
pET-28a
pET-nifA
pBBRsm2MCS5
pBBR-nifAwt
pBBR-nifAdG1
pBBR-nifAdG2
pBBR-nifAhis
pBBR-nifAIDL
pBBR-nifAasub
Description
Source or Reference
Cloning strain
Conjugation strain, Smr
Conjugation strain, Kmr
IPTG inducible T7 polymerase
expression strain
Yanisch-Perron et al. (1985)
Simon, Priefer, Pühler (1983)
Simon, Priefer, Pühler (1983)
NEB
Type strain
ΔnifA derivative of strain 2.4.1
van Niel (1944)
This study
Cloning vector
Invitrogen
Allelic exchange vector harboring sacB; Kmr
Chaperon expression plasmid
His-tag expression vector
pET-28a vector containing the nifA sequence
from strain 2.4.1
Broad host range vector, Smr
Laguna (2010)
Takara
Invitrogen
This study
pBBRsm2MCS5 containing a nifA WT cassette
pBBRsm2MCS5 containing a nifA cassette that
lacks amino acid residues 1-179
pBBRsm2MCS5 containing a nifA cassette that
lacks amino acid residues 1-255
pBBRsm2MCS5 containing a nifA cassette
derived from plasmid pET-nifA
pBBRsm2MCS5 containing a chimeric nifAntrC cassette in which the regions from the
α-subdomain to the interdomain linker were
exchanged
pBBRsm2MCS5 containing a chimeric nifAntrC cassette in which the α-subdomains
were exchanged
50
Schneider et al.
(2012)
This study
This study
This study
This study
This study
This study
Table 2.2 Oligonucleotides used in Chapter 2. Each oligonucleotide pair was used to
clone the indicated construct.
5’-3’ sequence
Name
F Rs 548 T speI
R nifA pro ncoI
Construct
GTACTAGTGCAAGGTCCTGCAGGAG
GTCCATGGCCAGACCTCCGT
Upstream HRSa for
nifA deletionb
F nifA T sacI
R nifa T xbaI
GCGAGCTCGAGAACTGCATC
GGCTCTAGATTTGTCGCACCCC
Downstream HRS
for nifA deletion
F nifA IDLc ds
R nifA IDLc us
ACCGTCCCCTCCGCGC
CGGCAGCACGATGGGCAC
Inverse primers for
nifAIDL
F ntrC IDL us
R ntrC IDL ds
CCCATCGTGCTGCCGTCGCTGCGCGAACGGG
CGCGGAGGGGACGGTCGAGGAGGACAGCTTCTCGCC
ntrC region for
nifAIDL
F nifA aSub ds
R nifA IDLc us
TCGGCCGATCTCTGGCG
CGGCAGCACGATGGGCAC
Inverse primers for
nifAasub
F ntrC IDL us
ntrC region for
CCCATCGTGCTGCCGTCGCTGCGCGAACGGG
nifAasub
R ntrC aSub ds
CCAGAGATCGGCCGACAGCACAGCCTCGACCTCG
a
HRS, homologous recombination site
b
an incomplete amplification product was ultimately used in which the 3’ primer
sequence plus an additional 3 base pairs were missing
51
Plasmids. The promoter region, cloned from approximately 400 bases upstream of
the nifA ATG start codon, was ligated to the start codon of all nifA alleles via an
engineered NcoI site and cloned into plasmid pBBR-SM2-MCS5 to construct
complementation vectors designated pBBR-nifA.
WT nifA, corresponding to the
sequence from strain 2.4.1, was used to construct plasmid pBBR-nifAwt and also was
cloned into the His-tag expression vector pET28a via the BamHI and EcoRI restriction
sites to construct plasmid pET-nifA. This His-tagged NifA encoded gene was cloned
from plasmid pET-nifA to construct plasmid pBBR-nifAhis.
To construct plasmids
pBBR-nifAdG1 and pBBR-nifAdG2 the first 537 and 675 bases of nifA were not
included resulting in a truncation of the first 179 and 225 residues of NifA respectively.
To construct the chimeric nifA-ntrC constructs, inverse amplification was performed on a
nifA containing vector and was joined to an ntrC amplification product using the strain
2.4.1 genome as a template via the infusion reaction (Clontech, Mountain View, CA).
For plasmid pBBR-nifAIDL, the ntrC sequence from position 846 to 1134 was
exchanged for that of nifA from position 1203 to 1575. For plasmid pBBR-nifAasub, the
ntrC sequence from position 846 to 1071 was exchanged for that of nifA from position
1203 to 1440.
Enzyme assays and biochemical procedures. Acetylene reduction assays and
Western immunoblot determinations were performed as described in Chapter 1
52
Models and protein structures. SWISS MODEL (Arnold et al. 2006; Kiefer et
al. 2009; Peitsch 1995) and I-TASSER (Roy, Kucukural, Zhang 2010; Roy, Yang, Zhang
2012; Zhang 2008) were used to generate structural models of NifA.
Pymol
(http://pymol.org/educational/) was used to visualize all structural diagrams.
Protein Purification
Recombinant protein synthesis.
E. coli strain ER2566 (NEB, Ipswich, MA)
harboring the tetracycline inducible chaperon expression plasmid pGtf2 (Takara) and the
IPTG inducible His-tag nifA expression plasmid pET-nifA was grown in LB media
supplemented with 1% glucose, 75 mM MOPS, pH 7.5 with NaOH, kanamycin, and
chloramphenicol. Overnight cultures were grown at 37°C in sealed but vented bottles
with 100 ml media and used to inoculate 2 L of media equilibrated with nitrogen gas and
containing 10 ng/ml tetracycline. After 4-5 h of growth at 37°C and shaking at 90 rpm in
a sealed jar equipped with tubes for sampling and venting, when the OD600 of the culture
was between 0.4-0.5, ferrous ammonium sulfate, cysteine, and IPTG were added to the
culture for a final concentration of 0.1 mM, 2 mM, and 0.3 mM respectively. The culture
was then grown at room temperature overnight and then harvested anaerobically at 6000
rpm for 15 min at 4°C with a Beckman JA-10 rotor.
Recombinant protein purification.
All manipulations were performed under
anaerobic conditions in an anaerobic chamber (Coy Laboratory Products, Grass Lake,
MI) or using sealed bottles. Cell pellets were suspended in 20 ml of LW buffer (50 mM
sodium phosphate pH 8, 300 mM NaCl, 10 mM imidazole) and lysed in a French Press at
53
1000 psi, then clarified by centrifugation at 16000 rpm for 15 min at 4°C with a Beckman
JA-20 rotor. The supernatant was added to a column containing Ni-NTA resin (Qiagen,
Valencia, CA) equilibrated in LW buffer. Washes and elution was performed with
increasing concentrations of imidazole up to 250 mM in LW buffer.
RESULTS
Modeling of Rb. sphaeroides NifA. NifA from Rb. sphaeroides was modeled
against various proteins as indicated below using the programs SWISS-MODEL and ITASSER (Arnold et al. 2006; Kiefer et al. 2009; Peitsch 1995; Roy et al. 2010; Roy et
al. 2012; Zhang 2008). Both programs produced similar models, so only those from
SWISS-MODEL are shown (Figure 2.2). The central region through the C-terminus was
modeled against the bEBP involved in Zn metabolism, ZraR, from Salmonella enterica
sv. Typhimurium (pdb accession 1OJL), and the GAF domain was modeled against the
GAF domain containing enzyme, methionine-R-sulfoxide reductase, from Neisseria
meningitides (pdb accession 3MMH). Two regions of the GAF domain exhibited low
modeling accuracy as determined by ANOLEA predictions; they were contained within
the second helix and the second sheet. For the central and C-terminal domains, only the
two loops that protrude from the AAA+ domain into the center of the helix, one of which
54
Figure 2.2 SWISS-MODEL of NifA from Rb. sphaeroides. (A) ANOLEA evaluation
of the regulatory GAF domain; darker residues indicate poor evaluation. (B) The AAA+
domain through the C-terminus. Predicted ATP binding residues of the Walker A,
Walker B, and Sensor 2 region are indicated as side chains in shades of blue; the Cys of
the unique motif are indicated as black space-filled side chains in the α-helical
subdomain.
55
contains the GAFTGA motif, exhibited low modeling accuracy. The residues that are
predicted to be the Walker A, Walker B, and Sensor 2 motifs reside in the same position
as those for ZraR, and the residues of the unique Cys motif are located around the sensor
2 region in the α-helical subdomain (Figure 2.2).
Complementation of strain 145. The nonpolar nifA deletion strain, strain 145,
was constructed from the WT strain 2.4.1. Cultures of strain 145 were incapable of
growth under nitrogen-fixing conditions, but when complemented with the WT NifA
encoded by a gene on plasmid pBBR-nifAwt or a GAF domain truncation variant
encoded by a gene on plasmid pBBR-nifAdG2, growth rate was restored to WT levels
(Table 2.3). Strain 145 complemented with an empty vector or plasmid pBBR-nifAdG1,
which contains a gene encoding for a partial GAF domain truncation variant of NifA
were incapable of growth under nitrogen-fixing conditions.
Cultures of strain 145
containing plasmid pBBR-nifAdG2 displayed moderate nitrogenase derepressive
phenotypes as shown by Western immunoblots directed against NifH and nitrogenase
activity assays of cultures grown with ammonia (Figure 2.3, Table 2.4). With these
cultures, nitrogenase was not fully derepressed as compared to cultures of strains 2.4.1,
145 (pBBR-nifAwt), or 145 (pBBR-nifAdG2) grown with alternative nitrogen sources.
To determine if the Cys motif of NifA can be functionally replaced with a
homologous region from a bEBP that does not contain a Cys motif, the α-helical
subdomain from the AAA+ domain of Rb. sphaeroides NtrC was exchanged for that of
NifA. Two chimeras were constructed and expressed from the nifA promoter: plasmid
56
Table 2.3 Nitrogen-fixing growth of nifA complementation strains. Doubling times,
reported in hours, were calculated for strains that contain different nifA alleles grown
under nitrogen-fixing conditions.
Strain
Description
Doubling time
2.4.1
WT
8.8 ± 0.8
145
ΔnifA
NGa
145 (pBBR-Sm2-MCS5) Empty vector
NG
145 (pBBR-nifAwt)
WT nifA
8.0 ± 1.2
145 (pBBR-nifAdG1)
Shortened GAF domain truncation
NG
145 (pBBR-nifAdG2)
Full GAF domain truncation
9.7 ± 0.4
145 (pBBR-nifAIDL)
α-helical to DNA binding NtrC chimera
NG
145 (pBBR-nifAasub)
α-helical NtrC chimera
NG
a
NG, no growth
57
Figure 2.3 Western immunoblot of crude extracts from cultures of nifA
complementation strains using antisera to NifH. Cell extracts from strain 2.4.1 (lane
1), strain 145 (lanes 2 and 3), and strain 145 containing plasmids pBBR-nifAwt (lanes 4
and 5), pBBR-nifAdG1 (lanes 6 and 7), and pBBR-nifAdG2 (lanes 8 and 9) were blotted
with antibodies directed against NifH to visualize nitrogenase. All samples contained
NifH when the cells were cultured with glutamate (lanes 1, 3, 5, 7, and 9) as the nitrogen
source except for samples from strain 145, but only strains containing plasmid pBBRnifAdG2 contained detectable levels of NifH when cultures were grown with ammonia
(lanes 2, 4, 6, and 8).
58
Table 2.4 Nitrogenase activities of nifA complementation strains. Each strain was
cultured with the indicated nitrogen source and assayed for acetylene reduction activity.
Results are reported in nmol of ethylene generated/min/mg of dry cell weight.
Strain
2.4.1
145 (pBBR-nifAwt)
145 (pBBR-nifAdG1)
145 (pBBR-nifAdG2)
a
ND, not detected
Nitrogen Source
Ammonia Glutamate
NDa
70 ± 5
ND
111 ± 6
ND
14 ± 1
3±3
144 ± 9
59
pBBR-nifAIDL contains an exchange of the α-helical subdomain to the DNA binding
domain and plasmid pBBR-nifAasub contains an exchange of just the α-helical
subdomain (Figure 2.4). Both of these plasmids failed to complement strain 145 for
nitrogen-fixing growth (Table 2.3).
Purification of recombinant His-tagged NifA. N-terminal His-tagged NifA
cloned from a pET28a vector behaved as WT NifA in complementation studies (data not
shown); therefore the purification of His-tagged NifA was attempted with Ni-affinity
resin. Soluble, His-tagged, full length NifA was synthesized in an E. coli recombinant
host containing additional chaperones in anaerobic conditions with additional Fe and Cys.
Though much of the 66 kD His-tagged NifA separated from the lysate with the pelleted
fraction, that which remained in the supernatant was adsorbed to the Ni-NTA resin as
indicated by its absence in the flow through fraction (Figure 2.5). Subsequent washes
with increasing concentrations of imidazole resulted in the elution of the His-tagged NifA
(Figure 2.5).
Unfortunately, a buffer was not discovered that would maintain the
solubility of the eluate for an extended period of time. In testing various buffers, a 40
mM Tris, 10 mM NaCl buffer was found to maintain His-tagged NifA solubility upon
lysis, but it prevented the protein from binding the Ni-column (Figure 2.6).
60
NifA
NtrC
TLRVDVRLVTATNKDLERAVANGTFRADLYFRICVVPIVLPPLRDRKEDIGLLAQGLLER 420
----APRIMSTSQVDLASRLESGAFRQDLYYRLGGVTLHVPSLRERVDDIPLLADHFLAR 301
*:::::: **
: .*:** ***:*: *.: :*.**:* :** ***: :* *
NifA
NtrC
FNKRNGMKKKLHPSAVAALAQCNFPGNVRELENCIARVAALSPETVIHADDLACHHDHCL 480
GERDLGATRRLSNEARDLVRAYSWPGNVRQLENTLRRLMVTSAEAEITRAEVEAVLGN-- 359
:: * .::* .*
:
.:*****:*** : *: . *.*: *
:: . .:
NifA
NtrC
SADLWRLQTGSASPVGGLAQGPLELPVLGSRPPAAAPSAPPPPPPTVPSAPLDGEAAERE 540
-------QPAMEPLKGGGEGEKLSSSVARHLRRYFDLHGGALPPPGVYQRILR--EVEAP 410
*.. . **
*. .*
. . *** * . *
.*
NifA
NtrC
ALIEAMERAGWVQAKAARLRGMTPRQIGYALKKYNIRVEKF---- 581
LIEIALDATAGNQAKCADLLGINRNTLRKKITDLDIRVTRRRKLM 455
: *:: :. ***.* * *:. . :
:.. :*** :
Figure 2.4 Homology between NifA and NtrC. A partial Clustal-W2 sequence
alignment between NifA and NtrC of Rb. sphaeroides is shown. The yellow highlighted
residues indicate the α-helical subdomain; red highlighted residues indicate the Cys of the
motif; gray highlighted residues indicate the DNA binding domain. The vertical bar
represents the start of the chimeric exchanges. For plasmid pBBR-nifAIDL, NtrC
sequence from position 281 to 378 was exchanged for that of NifA from position 401 to
535 to switch the regions from the α-helical subdomain to the DNA binding domain. For
plasmid pBBR-nifAasub, NtrC sequence from position 281 to 357 was exchanged for
that of NifA from position 401 to 485 to switch only the α-helical subdomains.
61
Figure 2.5 Purification of recombinant His-tagged NifA. A Coomassie-stained SDSPAGE shows the purification of the 66 kD His-tagged NifA, indicated by arrow, from
strain ER2566 containing plasmid pGtf2. Pre-induced whole-cell lysate, lane 1; Postinduced whole-cell lysate, lane 2 and 3; centrifuge-clarified lysate: pellet lane 4,
supernatant lane 5; column flow-through, lane 6; 40, 80, 120 mM imidazole wash lanes
7-9 respectively; 250 mM imidazole elution, lanes 10-15. Sizes of the Markers (M) are
250, 150, 100, 75, 50, 37, 25, and 20 kD; approximate sizes of the chaperones are 10, 56,
and 60 kD.
62
Figure 2.6 Solubility of recombinant His-tagged NifA. A Coomassie-stained SDSPAGE shows the solubility of the 66 kD His-tagged NifA, indicated by arrow, from strain
ER2566 containing plasmid pGtf2 lysed in buffer containing 40 mM Tris and 10 mM
NaCl. (A) Post-induced whole-cell lysate, lane 1; centrifuge-clarified lysate, lane 2. (B)
Centrifuge-clarified lysate, lane 1; column flow-through, lane 2. Sizes of the Markers
(M) are 250, 150, 100, 75, 50, 37, 25, and 20 kD; approximate sizes of the chaperones are
10, 56, and 60 kD.
63
DISCUSSION
Nitrogenase is an oxygen-sensitive enzyme that catalyzes the fixation of nitrogen
gas into the biologically relevant nitrogen source ammonia; therefore it is only expressed
when fixed nitrogen is needed and when oxygen tensions are low, via microaerobic
growth conditions or increased cellular respiration rate (Masepohl and Kranz 2009).
These two parameters are also manifested in the post-translational regulation of NifA, the
transcriptional activator for the nitrogenase regulon, because it is inactivated by high
nitrogen and oxygen tensions.
The N-terminal GAF domain was responsible for
repressing NifA activity when the cell had abundant nitrogen because its full deletion
resulted in a NifA variant that could no longer completely repress nitrogenase synthesis
upon growth with ammonia. This conclusion is supported by studies of nitrogenase
derepression due to changes in the GAF domain of NifA from other species (Laguna
2010; Paschen et al. 2001; Rey et al. 2007; Zou et al. 2008).
Interestingly, complementation of strain 145 with plasmid pBBR-nifAdG2, which
encodes for the full GAF deletion NifA protein, could not fully derepress nitrogenase.
This phenotype is unusual when Rb. sphaeroides is compared to other organisms,
because observations in other species have shown that upon nifA-induced derepression,
nitrogenase was expressed at full activity (Heiniger et al. 2012). Additionally, it was
shown that just increasing the copy number of nifA through plasmid based expression of
64
the WT gene in other organisms, was sufficient to induce a moderate derepressive
phenotype, which was not observed in Rb. sphaeroides (Paschen et al. 2001). Rb.
sphaeroides is unique among nitrogen-fixing organisms because it does not contain the
DraT/G system that post-translationally regulates the activity of nitrogenase through
adenylation of the NifH subunit of the nitrogenase complex; therefore nitrogen-fixation is
only known to be regulated at the level of transcription (Masepohl and Kranz 2009). This
indicates that other transcription factors or signals may be necessary for complete
derepression in Rb. sphaeroides, which is consistent with the observation that the
nitrogenase-derepressed strain 16PHC contained mutations in genes encoding nitrogen
regulatory proteins in addition to the nifA gene (Chapter 1; Laguna 2010). Additionally,
the unique sigma factor, σ54, is required to express genes involved in alternative nitrogen
metabolism.
In most species, σ54 is constitutively expressed, including the NSP
bacterium Rps. palustris, but in the nitrogenase-derepressed strain 16PHC of Rb.
sphaeroides, the transcript for σ54 was up regulated 7 fold (Laguna 2010; Rey et al.
2007). Therefore this sigma factor might need to be deregulated for optimal nitrogenase
gene expression.
In other species, there has been only limited success in being able to complement
ΔnifA strains with a GAF deletion variant of NifA for nitrogen fixation, as also was
observed for strain 145 (pBBR-nifAdG1) (Arsene, Kaminski, Elmerich 1996; Souza et al.
1999; Zou et al. 2008). The difference between plasmids pBBR-nifAdG1and pBBRnifAdG2 is that the GAF domain truncation variant of NifA encoded on plasmid pBBRnifAdG1 still contained the helix linking the GAF domain to the AAA+ domain. This
65
helix has been hypothesized to stabilize the inactive dimer oligomerization state based on
recent crystal structures of a NifA homologue (Batchelor et al. 2013). It was proposed
that upon co-inducer binding, the globular portion of the GAF domain restructures to
destabilize the linker helix, thus promoting the destabilization of the dimer and
rearrangement to the active hexamer of NifA. The regions of the GAF domain that
change conformation correspond to the strands of the β-sheet and α-helix that were
constrained in the NifA model. Unfortunately, the structures from this study have not
been released, so the hypothesis that the ANOLEA values of these regions will improve
upon modeling to the ligand bound structure cannot yet be tested.
The current structure-function studies of Batchelor et al. (2013) can explain the
different phenotypes of strains 145 (pBBR-nifAdG1) and 145 (pBBR-nifAdG2). Since
strain 145 (pBBR-nifAdG1) synthesized a NifA that does not contain the globular region
of the GAF domain, the linker helix cannot be destabilized through the normal structural
rearrangements; therefore these NifA variants would be constrained to the inactive dimer
configuration until thermodynamics can overcome the activation energy to switch
configurations to the hexamer. This would explain the decreased abundance of NifH and
activity of nitrogenase in strain 145 (pBBR-nifAdG1) grown with glutamate compared to
strain 145 (pBBR-nifAwt). In contrast, strain 145 (pBBR-nifAdG2) synthesized a NifA
in which the linker helix was also truncated; therefore these NifA variants would not be
able to maintain a dimer so they would be constrained to the active hexameric state
leading to nitrogenase derepression when grown with ammonia and also overexpression
when grown with glutamate as was observed in this study.
66
To determine if the oligomerization states of these GAF domain truncated
proteins occur as predicted, they have to be purified. Unfortunately, purified WT NifA
containing an N-terminal His tag was not able to be stably maintained after enrichment
through a Ni-affinity column even when its potential oxygen sensitivity was
accommodated through anaerobic synthesis with additional Fe and Cys.
Therefore
further attempts to compare the stability of NifA variants could not be performed. An
interesting observation of the eluate of NifA was its amber coloration, which would
suggest that NifA is a metalloprotein.
An Fe-S cluster has been proposed to be coordinated to the Cys residues of the
unique motif, C-X11-C-X19-C-X4-C, to regulate NifA activity in regards to oxygen
tensions (Fischer 1994). Since each Cys residue in the motif is necessary for activity
(Fischer et al. 1988; Oliveira et al. 2009), attempts were made to construct an active and
oxygen insensitive NifA through an exchange of the entire region that contains the motif.
Two different exchanges with the bEBP NtrC were constructed: one that started at the αsubdomain of the AAA+ domain and ended at the HTH motif, encoded on plasmid
pBBR-nifAIDL, and another that was just the α-subdomain, encoded on plasmid pBBRnifAasub.
Neither was capable of complementation of strain 145 for growth with
nitrogen gas.
This is probably the result of NtrC from Rb. sphaeroides and Rb.
capsulatus not being typical bEBPs. These proteins lack the GAFTGA motif that is
required for interaction with σ54, and it has been shown for the Rb. capsulatus NtrC that it
actually activates the σ70 bound RNA polymerase (Bowman and Kranz 1998). This
promoter activation still required NtrC to bind ATP but the Rb. capsulatus NtrC did not
67
display any ATPase activity (Bowman and Kranz 1998). Therefore future studies of
NifA chimeras should be constructed from bona fide bEBPs that can hydrolyze ATP.
In conclusion, the GAF domain of NifA from Rb. sphaeroides has been shown to
be a regulatory domain that repressed nitrogenase gene expression upon growth with
ammonia because strains containing NifA in which the GAF domain was completely
truncated displayed aberrant regulation. In addition, support for the role of the linker
helix in stabilizing the inactive dimer has been shown through comparisons of
nitrogenase synthesis in strains that synthesized NifA proteins that lacked the GAF
domain with those that lacked the globular subdomain but still included the linker helix.
Finally, evidence from studies that compared nitrogenase expression levels show that in
Rb. sphaeroides other factors might be necessary for nifA induced nitrogenase
derepression, like the deregulation of the alternative sigma factor.
68
Chapter 3: Nitrogenase-Mediated Repression of cbb Transcription
INTRODUCTION
Homeostatic control elements regulate all aspects of organisms’ complex network
of metabolism to coordinate and maintain balanced growth. Though metabolic pathways
need to be studied in isolation so the individual reaction steps can be identified, the
function of the pathway needs to be studied in the context of the entire cell so all of its
regulatory mechanisms can be determined. For example, the CBB pathway has been
historically studied to determine the reactions that contribute to the reduction of CO2 such
that this carbon can be incorporated into cell material. This information is important to
establish and identify the role of the CBB catalysts in autotrophic growth; however, it
was also observed that the levels of these enzymes were decreased when cells were
cultured with poor nitrogen sources (Selao, Nordlund, Noren 2008; VerBerkmoes et al.
2006). A proposed reason for this repression is that carbon assimilation needs to be
decreased in order for the cell to maintain its carbon-nitrogen balance when the
organisms is grown with alternative nitrogen sources, which are harder to metabolize.
Indeed, few testable hypotheses have been put forth that have led to a mechanistic
understanding of this regulation.
A primary alternative nitrogen metabolic process that has been hypothesized to be
responsible for CBB repression is the nitrogenase complex and its enzymatic activity
69
(McKinlay and Harwood 2010; Tichi and Tabita 2000; Wang et al. 2010). Nitrogenase is
a complex of NifH, NifD, and NifK peptides that is responsible for catalyzing the
conversion of nitrogen gas into an easily metabolized form of nitrogen, ammonia.
Typically nitrogenase synthesis is repressed when ammonia is the nitrogen source for
growth, but the genes may be expressed under nitrogen-fixing conditions and also when
amino acids, specifically glutamate, are used as the nitrogen source for growth, as has
been extensively studied in the NSP bacterium Rb. capsulatus (Hillmer and Gest 1977;
Masepohl and Kranz 2009). When glutamate is the nitrogen source, the nitrogenase
complex is not necessary for growth, so (i) nitrogenase mutants that would normally be
lethal under nitrogen-fixing conditions can be studied and (ii) all nitrogen gas can be
removed from such cultures, resulting in nitrogenase catalyzing only hydrogen
production, thus not affecting cellular nitrogen levels (Hillmer and Gest 1977; Hoffman
et al. 2013; Tao et al. 2012).
To maintain cellular carbon levels, the CBB cycle is required for autotrophic
growth when CO2 is employed as the sole carbon source and also under
photoheterotrophic growth conditions in the presence of organic carbon substrates,
though the expression level of the cbb genes is lower under photoheterotrophic growth
conditions (Gibson and Tabita 1993; Gibson et al. 2002). The cbb genes are arranged on
two distinct operons in Rb. sphaeroides; upstream promoter sequences of both operons
contain binding sites for transcriptional regulators RegA and CbbR (Dangel et al. 2005;
Dubbs et al. 2000; Dubbs and Tabita 2003). RegA, a response regulator protein, is part
of a two component system with its kinase RegB, which senses the redox state of the
70
quinone pool (Dubbs and Tabita 2004; Wu and Bauer 2008; Wu and Bauer 2010). Upon
RegB binding reduced quinols, it is activated to catalyze phosphorylation of RegA (Wu
and Bauer 2010). RegA is a global regulator that activates transcription of many genes
that are induced upon microaerobic and anaerobic growth conditions, including the cbb
operons and the nif operon, which encodes for nitrogenase (Joshi and Tabita 1996). Each
of the operons under the control of RegA also have additional regulators to maintain an
additional level control (Dubbs and Tabita 2004). The other known regulator of the cbb
operons is CbbR, which is a member of the LysR-type transcriptional regulators (Dubbs
et al. 2000; Dangel and Tabita 2009). The activity of this family of regulators is often
controlled by interactions with small molecules that are usually metabolites in the
regulated pathway (Schell 1993). CbbR is activated by the unique metabolite of the CBB
pathway, RuBP, which is the product of phosphoribulokinase (PRK) catalysis (Dangel et
al. 2005). PRK enzymes are classified into two groups. Those that typically occur in
anoxygenic photosynthetic organisms are inhibited by AMP and allosterically activated
by NADH (Gibson and Tabita 1987; Novak and Tabita 1999; Rindt and Ohmann 1969).
Conversely, oxygenic phototrophs contain PRK enzymes that are not known to respond
to NADH levels; instead, those of eukaryotes are regulated by redox sensitive disulfide
bonds and inhibitory complex formation with a small protein, CP12, while the PRK from
the cyanobacterium Synechococcus elongatus PCC 7942 is only regulated in vivo by
inhibitory complex formation with CP12 (Kobayashi et al. 2003; Miziorko 2000; Tamoi
et al. 1998; Tamoi et al. 2005). Since the RegAB system is activated by reduced quinols
71
and CbbR-mediated transcription is regulated by RuBP, the level of which is controlled
by NADH-activated PRK, then both CBB regulators ultimately respond to redox.
In this chapter, a hypothesis that links nitrogen metabolism to CBB regulation,
based on a recent proposal for Rps. palustris, was tested (McKinlay and Harwood 2010).
Perturbations to the nitrogen metabolism of Rb. sphaeroides were accomplished through
changes to the composition of the growth media and to the genomes of different strains.
Regulation of cbb expression was controlled through genetic manipulations and
complementations. The resulting phenotypic changes were observed through protein
abundances, enzyme activities, and metabolite levels. Thus, in vivo methods were used
to establish a link between nitrogen and carbon metabolism in Rb. sphaeroides.
MATERIALS AND METHODS
Growth Conditions. E. coli and Rb. sphaeroides were grown as described in
Chapter 1.
Bacterial strains and plasmids. The bacterial strains and plasmids used in this
chapter are listed in Table 3.1. Standard molecular biology techniques, unless otherwise
stated, were used for gene cloning and construction; oligonucleotides used are listed in
Table 3.2.
E. coli strain JM109 was used for maintenance and construction of all
72
Table 3.1 Strains and plasmids used in Chapter 3.
Strain or Plasmid
E. coli
JM109
S17-1
SM10
Rb. sphaeroides
2.4.1
2.4.1C1
B214
15165
15165C1
NK10
145
Plasmids
pCR-Blunt IITOPO
pJQ200mp18Km
pJQdsac
pVKCI
pVKCII
pBBRsm2MCS5
pBBR-nifAwt
pET-S7PRK
pBBR-F2B
pBBR-F2C
Description
Source or Reference
Cloning strain
Conjugation strain, Smr
Conjugation strain, Kmr
Yanisch-Perron et al. (1985)
Simon, Priefer, Pühler (1983)
Simon, Priefer, Pühler (1983)
Type strain
Strain 2.4.1 with a chromosomal cbbI-lacZ
fusion
ΔcbbPII derivative of strain 2.4.1
ΔcbbPI derivative of strain B214
Strain 15165 with a chromosomal cbbI-lacZ
fusion
ΔnifK strain derived from strain 2.4.1
ΔnifA derivative of strain 2.4.1
van Niel (1944)
This study
Chapter 1
Chapter 1
This study
Chapter 1
Chapter 2
Cloning vector
Invitrogen
Allelic exchange vector harboring sacB;
Kmr
ΔsacB vector derived from plasmid
pJQ200mp18Km
cbbI-lacZ containing vector
cbbII-lacZ containing vector
Broad host range vector, Smr
pBBRsm2MCS5 containing a nifA WT
cassette
His-tag expression vector containing
Synpcc7942_0977
pBBRsm2MCS5 containing a cbbII
promoter fusion to cbbPII
pBBRsm2MCS5 containing a cbbII
promoter fusion to Synpcc7942_0977
Laguna (2010)
73
Chapter 1
Dubbs and Tabita (1998)
Dubbs and Tabita (2003)
Schneider et al. (2012)
Chapter 2
Kobayashi et al. (2003)
This study
This study
Table 3.2 Oligonucleotides used in Chapter 3. Each oligonucleotide pair was used to
clone the indicated construct.
5’-3’ sequence
Name
F FII pro xba fus
R FII pro nco fus
AATTGGGCCCTCTAGACAACGGTCCGCCGACAAG
TCTTCGCCATGGCTCCTCCTGCCTCTG
F PRKb nco fus
R PRKb hind fus
GAGCCATGGCGAAGAAATATCCCATCATTTCCGTGG
GCTATGCATCAAGCTTGCCTCAGGCCC
R PRK 6301 nco GAAGGTCGTCCCATGGGCAAG
F PRK 6301 hind GGATGAAAGCTTGAGCAACCTAGACGC
74
Construct
cbbII promoter
cbbPII coding
region
Synpcc7942_0977
coding region
plasmids, and strains S17-1 and SM10 were used to conjugate the plasmids into Rb.
sphaeroides. Conjugations were performed as described in Chapter 1.
Strains 2.4.1C1 and 15165C1.
Strains harboring a chromosomal cbbI-lacZ
promoter fusion were constructed by excising the cbbI-lacZ promoter fusion from
plasmid pVKCI with EcoRI and ligating it into plasmid pJQdsac. Then this vector was
transformed into strain S17-1, and conjugated into strains 2.4.1 and 15165. Kanamycin
resistant colonies were screened for plasmid integration by sequencing the cbbI promoter
regions.
Plasmids. For PRK complementation vectors, the entire cbbII promoter region up
to the start codon from plasmid pVKCII was cloned upstream of the start codon of genes
that encode for PRK and inserted into plasmid pBBR-Sm2-MCS5. The cbbPII gene
encoding form-II PRK from Rb. sphaeroides was amplified from the genome of strain
2.4.1 and used to construct plasmid pBBR-F2B.
The gene encoding PRK from
Synechococcus elongatus PCC 7942 (His-tag free Synpcc7942_0977), was obtained from
a vector derived from plasmid pET-S7PRK and used to construct plasmid pBBR-F2C.
Enzyme assays and biochemical procedures. Acetylene reduction assays and
Western immunoblot determinations were performed as described in Chapter 1.
β-galactosidase activity.
Cultures were harvested and cell pellets lysed as
described in Chapter 1. Cell extract was added to Z-buffer (50 mM sodium phosphate,
75
pH 7; 10 mM KCl; 1 mM MgSO4; 5 mM β-mercaptoethanol) containing 0.8 mg/ml onitrophenyl-β-galactoside in a total volume of 1 ml. A molar extinction coefficient of
3.1*103 cm-1M-1 was used to calculate the rate of production of o-nitrophenol from the
continuously measured absorbance at 405 nm and was normalized against the amount of
protein used in the assay, as described in Chapter 1, to calculate specific activity. Unless
otherwise stated, triplicate cultures were assayed in duplicate and the values are reported
as averages of the cultures ± standard deviations.
NADH quantification. Cultures were harvested as described in Chapter 1. NADH
was determined using the EnzyChrom NAD+/NADH Assay Kit (BioAssay Systems,
Hayward, CA) as described by manufacturer’s directions, except cell pellets were
sonicated in 200 μl extraction buffer. NADH levels were normalized to the amount of
protein contained in the assay as described in Chapter 1.
Unless otherwise stated,
triplicate cultures were assayed in duplicate and the values are reported as averages of the
cultures ± standard deviations.
RESULTS
Influence of nitrogen metabolism on cbb gene regulation. To determine if
nitrogenase catalysis in general, rather than nitrogen fixation specifically, is sufficient to
induce CBB repression, lysates were analyzed from cultures of strain 2.4.1 grown with
76
ammonia or nitrogen gas as the sole nitrogen source and compared to cultures grown with
glutamate as the nitrogen source in which the headspace was sparged with argon; in this
growth mode nitrogenase just catalyzes proton reduction since the substrate for nitrogen
fixation has been removed.. The abundances of the CBB cycle protein PRK were higher
in lysates from cultures of strain 2.4.1 grown with ammonia as the nitrogen source than
with nitrogen gas or with glutamate (Figure 3.1); therefore CBB repression occurred
regardless of whether nitrogenase catalyzed nitrogen fixation or just hydrogen
production. Because these Western immunoblots cannot distinguish between form-I
PRK synthesized from the cbbI operon gene cbbPI from form-II PRK synthesized from
the cbbII operon gene cbbPII, β-galactosidase activities from strains containing lacZ
fusions to either the cbbI or cbbII promoter were determined (Figure 3.2). Both cbb
promoters displayed decreased expression when β-galactosidase activities from extracts
from glutamate grown cultures of strain 2.4.1 were compared to ammonia grown
cultures. Additionally, when both cbb promoter fusion activities were measured from
nitrogenase null strain NK10, which contains a deletion of nifK, grown with glutamate,
not only were they no longer decreased, but β-galactosidase levels were instead increased
over those of ammonia grown cultures (Figure 3.2). Also, the cbbI promoter activities
from strain 2.4.1 and strain NK10 from ammonia grown cultures were similar, as were
the cbbII promoter activities. Western immunoblots of lysates from strains 2.4.1 and an
additional nitrogenase null strain 145, which contains a nonpolar deletion of nifA that
encodes for the nif operon transcriptional activator, using antisera to PRK supported this
77
Figure 3.1 Western immunoblot of crude extracts from cultures of strain 2.4.1
grown with different nitrogen sources. Cell extracts from strain 2.4.1 grown with
ammonia (lane 1), glutamate (lane 2), or nitrogen gas (lane 3) as the nitrogen source were
blotted with antibodies directed against PRK (A) to visualize the co-migrating CBB
proteins form-I and form-II PRK or NifH (B) to visualize nitrogenase. The headspace of
the cultures was sparged with argon, for growth with ammonia or glutamate, or nitrogen,
for growth with nitrogen gas. Only samples grown with alternative nitrogen sources
(lanes 2, 3) contained detectable levels of NifH; these samples also contained less PRK.
78
Figure 3.2 Plasmid based cbb promoter fusion activities from lysates of strains 2.4.1
and NK10. β-galactosidase activities were measured from lysates of strains 2.4.1 or
NK10 complemented with plasmid pVKCI or pVKCII containing the cbbI- or cbbII-lacZ
promoter fusion, respectively. Lysates from glutamate grown cultures (dark bars)
contained lower activities than ammonia grown cultures (light bars) for strain 2.4.1, but
the opposite was observed for lysates from strain NK10.
79
increase in cbb expression in nitrogenase null strains grown with glutamate, and also
showed that upon complementation with the WT nifA gene both PRK and NifH protein
levels returned to that of strain 2.4.1 (Figure 3.3). Finally, when the nitrogenase inhibitor
tungsten replaced molybdenum in the growth media of strain 2.4.1 cultures, PRK levels
were no longer repressed (Figure 3.4). These data indicate that nitrogenase activity can
repress cbb gene expression.
Involvement of PRK in CBB regulation. Strain 15165 is a CBB null strain
derived from strain 2.4.1 and contains nonpolar deletions of the cbbP genes from both
cbb operons. For unknown reasons, this strain exhibited a high rate of recombination as
observed after sequencing plasmids that have been conjugated into this strain, but isolates
were obtained that could stably maintain a cbbI-lacZ fusion integrated into the
chromosome, thereby creating strain 15165C1. Strains 15165 and 15165C1 were also
able to stably maintain pBBR-F2B or pBBR-F2C as plasmids containing the genes for
form-II PRK from Rb. sphaeroides or PRK from Synechococcus elongatus PCC 7942
respectively.
Strains 15165 and 15165 (pBBRsm2MCS5) required DMSO for
photoheterotrophic growth with ammonia whereas strain 15165 complemented with
plasmids pBBR-F2B or pBBR-F2C did not; these results are consistent with the theory
that a functional CBB cycle is needed for photoheterotrophic growth with malate.
Additionally, strains 15165 (pBBR-F2B) and 15165 (pBBR-F2C) grew with similar
doubling times (Table 3.3), indicating that the PRK isozyme from the cyanobacterium
Synechococcus elongatus PCC 7942 complemented strain 15165 as well as the Rb.
sphaeroides PRK.
80
Figure 3.3 Western immunoblot of crude extracts from cultures of strain 145. Cell
extracts from ammonia grown cultures (lanes 1, 3, and 5) or glutamate grown cultures
(lanes 2, 4, and 6) were blotted with antibodies directed against PRK (A) to visualize the
co-migrating CBB proteins form-I and form-II PRK or NifH (B) to visualize nitrogenase.
Samples were collected from strains 2.4.1 (lanes 1 and 2), 145 (lanes 3 and 4), 145
(pBBR-nifAwt) (lanes 5 and 6). All samples grown on ammonia have similar levels of
PRK. Strain 145 grown with glutamate had increased levels of PRK while its WT
complemented strain grown with glutamate displayed decreased levels of PRK. All
strains grown with glutamate, except for strain 145, have detectable levels of NifH.
81
Figure 3.4 Western immunoblot of crude extracts from cultures of strain 2.4.1
grown with tungsten. Cell extracts of strain 2.4.1 grown with ammonia (lanes 1 and 2)
or glutamate (lanes 3 and 4) were blotted with antibodies directed against PRK (A) to
visualize the co-migrating CBB proteins form-I and form-II PRK or NifH (B) to visualize
nitrogenase. Lanes 2 and 4 contained media without the normal complement of
molybdenum and was supplemented with 5 μM sodium tungstate. The substitution of
Mo for W did not affect NifH levels nor PRK levels from cultures grown with ammonia;
it did influence PRK levels from cultures grown with glutamate.
82
Table 3.3 Growth of PRK complementation strains. Strains were grown with the
indicated nitrogen source in media that contained spectinomycin at 25μg/ml, except for
strain 15165, which was grown without antibiotics. Generation times are reported in
hours.
Nitrogen source
Strain
Ammonia
Glutamate
a
15165
NG
9.7 ± 0.5
15165 (pBBRsm2MCS5)
NG
11.0 ± 0.6b
15165 (pBBR-F2B)
7.1 ± 0.1
12 ± 1
15165 (pBBR-F2C)
7.5 ± 1.3
12 ± 2
a
NG, no growth without the addition of dimethylsulfoxide
b
average ± range for two samples, all other values were derived
from at least triplicate samples
83
Western immunoblots were used to detect the presence of the additional CBB
proteins form-I and form-II RubisCO, because strain 15165 does not contain the genes
that encode PRK. Form-I RubisCO is encoded by the cbbLS genes of the cbbI operon
and the form-II RubisCO is encoded by the cbbM gene of the cbbII operon; therefore the
protein abundances of each isozyme can be used to distinguish regulation of each operon.
Western immunoblots and cbbI-lacZ promoter activities showed that extracts from strains
15165 or 15165C1 complemented with cbbPII expressed from the cbbII promoter had cbb
expression levels similar to those of strain 2.4.1 (Figure 3.5, Figure 3.6). Interestingly,
upon complementation with plasmid pBBR-F2C, encoding for the cyanobacterial PRK,
strains 15165 and 15165C1 exhibited altered cbb expression when grown with glutamate
(Figure 3.5, Figure 3.6). The form-I RubisCO protein levels and cbbI promoter activity
levels from strains 15165 (pBBR-F2C) and 15165C1 (pBBR-F2C) cultured with
ammonia were similar to each other and also to all the other strains cultured with
ammonia, but in addition the levels from lysates of these strains cultured on glutamate
were also similar to the lysates from cultures grown with ammonia. For the cbbII operon
regulation, the form-II RubisCO levels from extracts of cultures grown with glutamate
were slightly elevated compared to the other glutamate grown strains but were less than
those grown with ammonia.
This indicated that the cyanobacterial PRK isozyme
abrogated the nitrogenase-induced repression of cbb expression for the cbbI operon and
partially for the cbbII operon.
84
Figure 3.5 Western immunoblot of crude extracts from cultures of strain 15165.
Cell extracts were blotted with antibodies directed against form-I RubisCO (A) or form-II
RubisCO (B). Lanes 14 and 15 contained extracts from strain 2.4.1 grown with ammonia
or glutamate respectively, all other lanes contained extracts from strain 15165. Lanes 1-3
contained extracts from three different cultures of strain 15165 (pBBR-F2B) grown with
ammonia and lanes 4-6 were grown with glutamate. Lanes 7 and 8 contained of extracts
from strain 15165 (pBBR-F2C) grown with ammonia and lanes 9-11 were grown with
glutamate. Lane 12 contained extracts from strain 15165 grown with ammonia and
dimethylsulfoxide, and lane 13 contained extracts from strain 15165 grown with
glutamate.
85
Figure 3.6 Chromosomal based cbbI promoter fusion activities from lysates of
strains 2.4.1C1 and 15165C1. β-galactosidase activities were measured from lysates of
strains 2.4.1C1 and 15165C1 grown with ammonia (light bars) or glutamate (dark bars).
Similar levels of activity were observed from lysates of all cultures that could grow with
ammonia and also the glutamate grown culture of strain 15165C1 (pBBR-F2C). All
other lysates from glutamate grown cultures had similar levels of activity. Strain
15165C1 was only able to grow with ammonia if it also contained a gene encoding for
PRK; NG, no growth.
86
Metabolite analysis. To determine if the intracellular levels of NADH changed
upon nitrogenase expression, NADH pools were measured with the BioAssay Systems
EnzyChrom kit (Table 3.4). Both strains 2.4.1 and NK10 had similar levels when grown
with ammonia but strain NK10 had higher levels when grown with glutamate.
In
performing the assay, for unknown reasons, it was noticed that samples that were spiked
with known amounts of standards were inconsistent when comparisons of samples from
cultures grown with different nitrogen sources were made; therefore comparisons can
only be made among samples from cultures grown with the same nitrogen source.
DISCUSSION
Though many metabolic pathways have been determined, in many instances how
these pathways are regulated remain to be elucidated. This is because regulation is
complex and often arises in unexpected circumstances. Studies of NSP bacterial cultures
grown photoheterotrophically with various nitrogen sources led to observations that the
nitrogen source affects carbon metabolism, specifically the CBB cycle (Edgren and
Nordlund 2004; Selao et al. 2008; VerBerkmoes et al. 2006). Many studies have shown
the interaction between carbon and nitrogen metabolism, but very few of them have
described how carbon metabolism responds to nitrogen metabolism; most describe
87
Table 3.4 NADH levels. Strains were grown with the indicated nitrogen source. NADH
levels were quantified using the EnzyChrom assay kit and are reported as nmol of
NADH/mg of protein.
Nitrogen Source
Strain Ammonia Glutamate
2.4.1 0.3 ± 0.1 0.7 ± 0.2
NK10 0.3 ± 0.05 2.1 ± 0.1
88
the reciprocal regulation in which nitrogen metabolism responds to carbon metabolism
(Doucette et al. 2011; Ninfa 2007).
In Rb. sphaeroides, nitrogenase activity leads to repression of cbb gene
expression. This repression occurred regardless of whether nitrogenase was actively
fixing nitrogen or whether it just catalyzed the reduction of protons since repression is
observed even when no nitrogen gas was present in the culture. Similar findings have
been reported for other NSP bacteria as well (McKinlay and Harwood 2010; Smith and
Tabita 2002; Tichi and Tabita 2001), however in the current study it was clear that
nitrogenase activity and not merely the presence of nitrogenase protein was important for
cbb repression. This conclusion was supported by the phenotype of nitrogenase null
mutants which no longer repressed cbb gene expression, whether the null phenotype was
induced through the deletion of genes required for nitrogenase activity or through the
addition of nitrogenase inhibitors. Studies of strains where nitrogenase was specifically
induced upon growth with ammonia through nifA mutations have also shown cbb
transcript repression (McKinlay and Harwood 2010).
PRK was hypothesized to be required for the expression of the cbb genes because
its catalytic product, RuBP, activated CbbR, the master regulator required for cbb
transcription (Dangel et al. 2005; Gibson and Tabita 1993; Smith and Tabita 2002; Tichi
and Tabita 2001). Therefore complementation experiments of PRK deletion strains,
strains 15165 and 15165C1, were conducted to determine if PRK activity links
nitrogenase activity with the observed cbb gene repression. Even though bacterial and
cyanobacterial PRK have low sequence conservation and are arranged in different
89
oligomeric states, they still catalyze the same reaction (Tabita 1988). Therefore it was
not surprising that a comparison of the growth rates of strain 15165 complemented with
plasmid pBBR-F2B, containing cbbPII from Rb. sphaeroides, or plasmid pBBR-F2C,
containing the PRK gene Synpcc7942_0977 from a cyanobacterium, indicated that each
plasmid restored growth to similar rates. Additionally, analyzing the phenotypes of these
complemented strains allowed conclusions to be made about the impact of PRK activity
and regulation on cbb expression because cyanobacterial PRK activity is regulated
differently than PRK from Rb. sphaeroides (Kobayashi et al. 2003; Miziorko 2000;
Tamoi et al. 1998; Tamoi et al. 2005, Tabita 1988).
Synthesis of a functional
cyanobacterial PRK abolished nitrogenase-induced repression of the cbbI operon in PRK
deletion strains but not did not completely revert cbbII operon repression. These results
indicated that PRK was a link between nitrogenase activity and cbb gene expression and
also that additional repressive effects may influence the cbbII operon.
Differential
regulation of the two cbb operons has been observed in different contexts in Rb.
sphaeroides (Gibson and Tabita 1993; Gibson et al. 2002).
Further determination of the molecular mechanism between nitrogenase activity
and cbb gene repression requires establishing a link between nitrogenase activity and
PRK regulation as hypothesized by McKinley and Harwood (2010).
Nitrogenase
catalysis indirectly results in the consumption of NADH via the Rhodobacter nitrogen
fixation (RNF) complex. The RNF complex coordinates the oxidation of NADH and the
reduction of ferredoxin using reverse electron flow coupled to the proton motive force
(Biegel et al. 2011). Ferredoxin then donates the electrons to nitrogenase, catalyzed by
90
the NifH subunit of the nitrogenase complex (Ludden 1991). Therefore nitrogenase
catalysis could decrease the pool size of NADH, which is known to be required for
activation of PRK from Rb. sphaeroides (Gibson and Tabita 1987; Novak and Tabita
1999; Rindt and Ohmann 1969).
The hypothesis of NADH cellular pool sizes
coordinating nitrogenase activity with cbb repression was supported by the
complementation studies using different PRK isozymes because the activity of
cyanobacterial PRK is not known to be regulated by NADH. In further support for the
NADH hypothesis, the nitrogenase null strain NK10 had higher levels of cbb expression
and also possessed higher levels of NADH upon growth with glutamate. Presumably the
increased levels of NADH might then stimulate PRK activity to catalyze the production
of more RuBP, thus causing the observed increase in cbb expression. Whether this is true
will depend on future experiments as the method of “fixing” the metabolite levels in the
cell might need to be optimized, and other methods that allows for comparisons among
growth with different nitrogen sources will need to be developed.
Other studies
attempted to measure changes in NADH levels upon nitrogenase induction, but these
studies were inconsistent with each other (Haaker et al. 1974; Nordlund and Hoglund
1986; Noren and Nordlund 1994). Therefore more scrupulous data must be generated
before any strong conclusions can be made.
In conclusion, through gene deletion and tungsten inhibition studies, the activity
of the nitrogenase complex was determined to be responsible for initiating repression of
the cbb genes. Also through gene deletion and complementation studies, Rb. sphaeroides
PRK appeared to be part of a regulatory link between nitrogenase activity and cbb gene
91
expression. These studies provide additional information for differential control of the
cbb operons in Rb. sphaeroides. Finally, additional studies are required to determine
what other factor(s) cause the repressive effect on the cbbII operon and whether NADH is
also part of this regulatory chain.
92
Conclusions and Future Directions
Studies of perturbing carbon metabolism through deletions of cbb genes led to the
isolation of mutants in nitrogen metabolism. Through studies of strain 16PHC, it was
observed that (i) the GlnA T255A enzyme was sufficient to derepress nitrogenase
synthesis, (ii) the K16R substitution in NifK was a neutral substitution, and (iii) the NifK
H466R protein could decrease nitrogenase activity. Studies of the PHC mutant strains
that alter nitrogen metabolism led to an interest in NifA regulation. The deletion of the
GAF domain of NifA was sufficient to derepress nitrogenase synthesis, but other factors
are still needed for full derepression in Rb. sphaeroides. Finally, studies of nitrogen
metabolism led back to studies of carbon metabolism. These investigations showed that
nitrogenase activity was sufficient to cause cbb repression, with PRK being the enzyme
that relayed the signal; however other proteins may also be involved.
To further characterize the GlnA T255A enzyme, studies investigating the
oligomerization state of this enzyme should be conducted to determine if this substitution
does indeed destabilize the dodecamer; additionally, studies of the C-terminal helix,
especially of the conserved Tyr, hypothesized to be the bonding partner for T255, will
supplement the data already generated. The derepressive effect of the GlnA T255A
enzyme was hypothesized to be due to its decreased enzymatic activity, which would
lower the cellular glutamine pools; therefore to confirm this hypothesis, amino acid
93
quantifications of lysates from strains HR, 321, and 16PHC should be performed. While
the GlnA T255A enzyme imparts a nitrogenase-derepressive phenotype to the cell, it was
insufficient to induce full derepression. Therefore, investigations into the effects of other
mutations and combinations of mutations found within the genome of strain 16PHC
should be conducted to determine what mutation(s) fully derepress nitrogenase synthesis.
The reciprocal experiments of repairing the mutations in strain 16PHC should also be
conducted to determine what mutations are necessary to induce the PHC phenotype.
Once the genotype that is necessary and sufficient to induce a full nitrogenasederepressive phenotype has been determined, the mutation resulting in the NifK H466R
substitution should be repaired to construct a strain with a nitrogenase-derepressed and
active phenotype in Rb. sphaeroides from known mutations.
Nitrogenase regulation is unique in Rb. sphaeroides compared to other NSP
bacteria because neither spontaneous nor targeted mutations in nifA have resulted in full
nitrogenase gene expression when strains were cultured with ammonia. To gain a further
understanding of the role of NifA, a comparative analysis of NifA variants, including the
completely truncated GAF domain variant, should be conducted by expressing the genes
in other NSP organisms. Additionally, rpoN should be constitutively expressed in strain
145 (pBBR-nifAdG2) to determine if the deregulation of this additional transcription
factor coupled with the presence of the truncated GAF domain variant of NifA can induce
complete nitrogenase derepression in Rb. sphaeroides. Progressing towards the complete
characterization of NifA requires in vitro experimentation; therefore different buffer
compositions need to be tested in order to isolate and stabilize NifA. When this has been
94
achieved, oligomerization data of the GAF truncation variants should be generated, as
well as determining if NifA contains a redox active Fe-S cluster. Additionally, in vitro
experimentation should be conducted to determine what metabolites or proteins interact
with the GAF domain to regulate the nitrogen response of NifA.
If a strain of Rb. sphaeroides can be constructed through NifA and σ54 variants
that can derepress nitrogenase specifically, then this strain will help to elucidate the
regulatory pathway between nitrogenase activity and cbb repression. Current studies of
this regulatory pathway compared strains grown with rich or poor nitrogen sources, but if
a nitrogenase derepressed strain can be constructed, then variants of this strain can be
compared when grown solely with ammonia. This would eliminate all other variables that
change upon nitrogen deprivation; therefore all changes would result specifically from
nitrogenase synthesis. While the current PHC strain of Rb. sphaeroides, 16PHC, can
derepress nitrogenase synthesis, it does so presumably by artificially inducing poornitrogen conditions through the inefficient catalysis of GlnA T255A; thereby effectively
converting ammonia into a poor nitrogen source.
Additionally more studies of the current strains can be completed.
If the
increased cbb expression of strain NK10 is actually due to increased NADH levels,
resulting in increased PRK activity, then studies of a ΔPRK strain derived from strain
NK10 that has been complemented with plasmid pBBR-F2C should negate the increased
cbb expression. Also the differential expression of the cbb operons should be further
characterized by finding isolates of strain 15165 that can incorporate a cbbII-lacZ
promoter fusion into its chromosome while also maintaining pBBR-F2B and pBBR-F2C
95
as plasmids. In regards to other factors that could contribute to the differential regulation,
studies of the other known cbb regulator should be conducted. This regulator is RegA of
the RegAB two component system. Substitutions have been characterized in RegA that
mimic constitutive phosphorylation (Du et al. 1998); thereby studies of a ΔRegAB strain
complemented with this constitutive RegA would allow conclusions to be made of its
role relaying additional regulatory signals induced upon nitrogenase activity. Finally
since PRK has been determined to be a link in the regulatory chain, the product of its
catalysis, RuBP, should be quantified.
Lastly, experiments to study the CBB cycle’s connection to other metabolic
pathways should be conducted. It has been well documented that CBB null strains
derepress nitrogenase for redox homeostasis, but only one study has shown redox
balancing mechanisms with another metabolic pathway: one that produces H2S (Rizk et
al . 2011). The CBB null strain 193 is unlikely to derepress nitrogenase because nifA has
been deleted; after a long lag phase when this strain was cultured with ammonia, cultures
developed that produced H2S (data not shown). These adaptive strains could then be
characterized to discover additional connections to whole cell metabolism.
Life depends on balance and experiments of homeostasis revealed unexpected
connections. In this work, experiments explored the unexpected balance among carbon
metabolism, redox maintenance, and nitrogen metabolism. Knowledge of metabolism is
useful not just for basic science but can also be applied towards the production of highvalue products. For example, the redox balancing mechanisms of strain 193 could be
used to produce reduced bioproducts from oxidized metabolites. Synthetic biology seeks
96
to construct novel pathways for these products, but regulation of the pathway and its
niche in the context of the whole cell need to be understood or unexpected and unwanted
results will occur. Successful metabolic engineering depends on perturbing a balance and
understanding the consequences to control the production of a desired product. From a
simple chemical reaction to an organism’s complex metabolism, balances are disrupted
not just for product development but also to understand life.
97
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