Microbiology (2005), 151, 1671–1682
DOI 10.1099/mic.0.27848-0
mrpA, a gene with roles in resistance to Na+ and
adaptation to alkaline pH in the cyanobacterium
Anabaena sp. PCC7120
A. Blanco-Rivero,13 F. Leganés,1 E. Fernández-Valiente,1 P. Calle2
and F. Fernández-Piñas1
Departamento de Biologı́a, Facultad de Ciencias1 and Departamento de Quı́mica Fı́sica
Aplicada2, Universidad Autónoma de Madrid, Madrid 28049, Spain
Correspondence
F. Fernández-Piñas
[email protected]
Received 22 December 2004
Revised 16 February 2005
Accepted 17 February 2005
Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain, PHB11,
which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na+ sensitivity;
this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of
photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon
mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The
wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain
PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with
significant sequence similarity to a family of bacterial operons that are believed to code for a novel
multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to
alkaline pH. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation)
following the nomenclature of the Bacillus subtilis operon; the ORF mutated in PHB11
corresponded to mrpA. Computer analysis suggested that all seven predicted Anabaena Mrp
proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted
protein sequences encoded by mrpA, mrpB and mrpC showed significant similarity to hydrophobic
subunits of the proton pumping NADH : ubiquinone oxidoreductase. In vivo expression studies
indicated that mrpA is induced with increasing external Na+ concentrations and alkaline pH; mrpA
is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative
cyanobacterial Mrp complex is discussed.
INTRODUCTION
Cyanobacteria are the only prokaryotic organisms carrying
out an oxygen-evolving photosynthesis. They are thought to
3Present address: Umeå Plant Sciences Center, Department of Plant
Physiology, Umeå University, S-901 87 Umeå, Sweden.
Abbreviations: Chl, chlorophyll; Ci, inorganic carbon; Em, erythromycin;
Km, kanamycin sulphate; Nm, neomycin sulfate; pHex, external pH; pHin,
internal pH; Sp, spectinomycin dihydrochloride.
The GenBank/EMBL/DDBJ accession number for the sequence
reported in this paper is AF239979.
Characteristics of the putative gene products of the Anabaena mrp
locus and their predicted similarity to known protein sequences are
available in Supplementary Table S1, an alignment of MrpA from
Anabaena sp. strain PCC7120 and MrpA from other bacterial species
in Supplementary Fig. S1, a schematic diagram of the mrp region of
selected heterotrophic bacteria, Anabaena sp. strain PCC7120 and of
other cyanobacteria whose genomes have been sequenced showing
the ORF denominations in Supplementary Fig. S2, and a sequence
alignment of the presumptive LysR-type T(N11)A motif binding sites in
the promoter region of the putative Anabaena all1843–all1837 operon
in Supplementary Fig. S3 with the online version of this paper at http://
mic.sgmjournals.org.
0002-7848 G 2005 SGM
have originated during the Precambrian era (more than
36109 years ago) and as a group are known to survive a
wide spectrum of environmental stresses, such as temperature shock, photooxidation, nutrient deficiency, pH
changes, salinity and osmotic stress, and ultraviolet light.
Thus, cyanobacteria appear to be a suitable system for
analysing the active mechanism(s) developed in response to
changing environmental conditions.
Regarding pH requirements, cyanobacteria have optimum
growth between pH 7?5 and 11, being practically absent in
habitats with pH values below 4 or 5 (Brock, 1973). There
is still no satisfactory understanding of the molecular
mechanisms underlying their preference for alkaline
environments, although it should be noted that alkaline
pH favours the formation of bicarbonate and that aquatic
photosynthesizers are often limited by inorganic carbon (Ci)
availability. Studies involving a range of organisms have
identified both Na+/H+ and K+/H+ antiporters as major
means of regulating internal pH (pHin) when the external
pH is alkaline (Krulwich, 1995). However, the major Na+extruding mechanism in most bacterial cells is the Na+/H+
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1671
A. Blanco-Rivero and others
antiporter, which extrudes Na+ in exchange for H+
(Krulwich et al., 1994; Padan & Schuldiner, 1994). This
process is driven by an electrochemical gradient of protons
across the cytoplasmic membrane, which is established by
the respiratory chain or the H+-translocating ATPase. Thus,
in bacteria, the Na+/H+ antiporter plays a role in Na+
extrusion, pH homeostasis, cell-volume regulation and
establishment of an electrical potential of Na+ (Padan &
Schuldiner, 1996).
Na+ is an essential ion for most cyanobacteria, especially
when they are grown at high external pH values (Allen &
Arnon, 1955; Espie et al., 1988). Na+ is needed for the
uptake of several inorganic nutrients (inorganic carbon,
nitrate, phosphate) (Lara et al., 1993; Avendaño &
Fernández-Valiente, 1994) and for photosynthetic electron
transport at the O2-evolving complex (Zhao & Brand, 1988).
While low concentrations of Na+ are required by
cyanobacteria, higher concentrations of Na+ may be
harmful. The salt tolerance of cyanobacteria varies widely,
from low-salt-tolerance strains that tolerate a maximum of
0?5 M NaCl to strains from hypersaline environments that
can tolerate up to 3 M NaCl. For successful salt adaptation
at high salt concentrations, cyanobacteria actively extrude
Na+, accumulate K+ and maintain internal ion concentrations comparable to cells grown under low-salt concentrations (Reed & Stewart, 1985). The extrusion of Na+ by
cyanobacteria has been explained by Na+/H+ proton
exchange, since increased Na+/H+ antiporter activities
have been detected in salt-adapted cyanobacterial cells.
These transporters use the proton motive force established
by primary H+ pumps, such as H+-ATPases or respiratory
cytochrome oxidases. The main role of Na+/H+ antiporters
in ion export to achieve high salt tolerance has been clearly
shown in Escherichia coli (Padan & Schuldiner, 1993). In
contrast, from bioenergetic studies, the action of a primary
Na+-ATPase has been predicted to serve as the main source
for active Na+ extrusion in cyanobacteria (Ritchie, 1992),
since, at least under alkaline conditions, the adverse
transmembrane pH gradient prevents the generation of a
substantial proton motive force. However, although there
are several potential Na+-ATPases in the Synechocystis sp.
PCC6803 genome, no Na+-ATPase has been identified thus
far in any cyanobacterium (Ritchie, 1998). Also, bioenergetic analysis reveals that, under physiological ranges of pH,
Na+-coupled secondary ion transport across membranes
occurs in Synechococcus sp. PCC7942 (Ritchie, 1998),
implying that there is an energy-consuming Na+-efflux
mechanism in cyanobacteria. Therefore, whether or not the
predicted energy-dependent Na+-efflux protein(s) actually
exists, and the extent to which other cation ATPases may
also affect Na+ sensitivity in cyanobacteria, remain to be
established.
Most studies of salt adaptation have been performed in the
moderately halotolerant cyanobacterium Synechocystis sp.
PCC6803. Recently Wang et al. (2002) and Elanskaya et al.
(2002), independently, have mutated five genes identified as
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encoding putative Na+/H+ antiporters in the Synechocystis
genome (Kaneko et al., 1996). Elanskaya et al. (2002) found
that, with the exception of the NhaS3 mutant, which could
not be segregated, none of the mutants showed a significant
growth depression under high-salt and/or high-pH conditions. Wang et al. (2002) also found that NhaS3 may
perform essential housekeeping functions for the survival of
the organism, being critical for salt tolerance. Disruption of
nhaS2 and nhaS4 demonstrated that both genes could be
essential for the survival of cyanobacteria in freshwater
habitats characterized by large fluctuations in salt concentration and pH.
No such studies have been undertaken in the heterocystous
filamentous cyanobacterium Anabaena sp. PCC7120, a
freshwater strain with low salt tolerance. In this work, we
report the identification of a transposon-generated mutant
of Anabaena sp. PCC7120, denoted PHB11, which exhibits
pronounced Na+ sensitivity and which was initially isolated
by its inability to grow at alkaline pH.
The transposon in PHB11 inserted within ORF all1838,
which forms part of a putative seven-ORF operon (all1843–
all1837) which in turn shows significant sequence similarity
to a family of bacterial operons mainly involved in tolerance
to high salt concentrations and in adaptation to alkaline pH
(Hiramatsu et al., 1998; Putnoky et al., 1998; Ito et al., 1999;
Kosono et al., 1999). The ORF mutated in PHB11 shows the
highest similarity to ORFA of bacterial operons and has
been denoted mrpA following the nomenclature of the
Bacillus subtilis mrp (multiple resistance and pH adaptation)
operon, which is the most extensively studied (Ito et al.,
1999). The six remaining ORFs of the putative Anabaena
operon were also denoted according to their highest
similarity with the corresponding bacterial ORFs. The
evidence presented indicates that Anabaena mrpA is
involved, as are its bacterial counterparts, in Na+ tolerance,
particularly at elevated pH. Interestingly, three genes of the
putative Anabaena mrp operon, mrpA, mrpB and mrpD,
code for predicted protein sequences that also show
significant similarity to hydrophobic subunits of the
proton-pumping NAD(P)H : ubiquinone oxidoreductase
(complex I) found in mitochondria and eubacteria, which
may suggest a potential relationship between complex I and
the putative Anabaena Mrp complex.
METHODS
Strains and growth conditions. Anabaena sp. strain PCC7120
and its derivatives (Table 1) were routinely grown at 28 uC in continuous warm white light (90 mE m22 s21) on a rotary shaker in
50 ml medium AA/8 (Allen & Arnon, 1955) supplemented with
nitrate (5 mM) and buffered with 25 mM HEPES, pH 7?5, in
125 ml Erlenmeyer flasks. To buffer media at pH 9 and above,
Bistris propane (BTP) was used. Plasmid constructs (Table 1) were
introduced into cyanobacterial strains by conjugations (Elhai &
Wolk, 1988), and single and double recombinant strains were
selected as described by Cai & Wolk (1990).
The different strains were grown in the presence of appropriate
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Microbiology 151
Role of mrpA in Na+ tolerance and pH adaptation
Table 1. Anabaena strains and plasmid constructs used in this study
Strain or plasmid
PCC7120
PHB11
PHB11
(pBG2041)
SR2045-1B
DR2045-6A
PCR 2.1
TOPO
pBG2033
pBG2037
pBG2038
pBG2041
pBG2045
pDS4101
pRL623
pRL759D
pRL1075
pRL1124
pRL1342
Derivation and/or salient characteristics
Source or reference
Wild-type
NmR
NmR EmR
C. P. Wolk
This work
This work
SpR SmR EmR product of single homologous recombination of
plasmid pBG2045 with wild-type PCC7120
SpR SmR EmS product of double homologous recombination of
plasmid pBG2045 with wild-type PCC7120
Cloning vector
This work
Circularized EcoRV fragment from Anabaena sp. strain
PHB11 that bears Tn5-1063
Anabaena DNA-containing PstI–BamHI portion of pBG2033
fused to the PstI–BamHI portion of pRL759D that contains oriV,
bom, luxAB, and SmR SpR determinant
728 pb PCR fragment bearing wild-type mrpA as its only ORF
cloned in the PCR 2.1TOPO vector
Wild-type mrpA on a BamHI–XhoI fragment from pBG2038,
inserted into pRL1342 cut with BamHI and XhoI
Product of ligation of pBG2037 linearized at EcoRV, with the
FspI portion of pRL1075 that contains sacB and CmR EmR
determinants
Apr, helper plasmid based on ColK, carries mob (ColK)
Helper plasmid bearing methylase genes M.AvaI, M.Eco47II
(whose product methylates AvaII sites), and M.EcoT22I
(EcoT22I is an isoschizomer of AvaIII)
One of a family of BLOS plasmids with bom, V. fischeri luxAB,
oriV and SmR SpR determinant for in vitro replacement of
all but the termini of a transposon
Contains a sacB–oriT (RK2) CmR EmR cassette that is separated
from oriV by inverted polylinkers
Kmr derivative of pACY177 that bears the same methylase genes
as pRL623
CmR EmR RSF1010-based plasmid
This work
Invitrogen
This work
This work
This work
This work
This work
Finnegan & Sherratt (1982)
Elhai et al. (1997)
Black et al. (1993)
Black et al. (1993)
Cohen et al. (1998)
C. P. Wolk
antibiotics. Mutant PHB11 was grown in liquid AA/8 medium with
40 mg ml21 (final concentration) neomycin sulfate (Nm). Single
recombinant SR2045-1B and double recombinant DR2045-6A were
grown with 2 mg ml21 spectinomycin dihydrochloride (Sp) plus, respectively, 1 mg ml21 erythromycin (Em) or no additional antibiotic.
Mutant PHB11 bearing plasmid PBG2041 was grown with 1 mg ml21
Em and 40 mg ml21 Nm.
French press (Simoaminco FA-078) at 147 000 kPa. Cell debris was
removed by centrifugation; the supernatant was centrifuged at
100 000 g for 1 h at 4 uC. The pellet was resuspended in the same
medium containing 25 % (v/v) glycerol, 10 mM MgCl2, 10 mM
NaCl, 20 mM sodium phosphate buffer, pH 7?5, and 1 mM PMSF.
Membranes were stored at 270 uC.
Analytical methods. Culture density was determined spectrophoto-
Measurement of photosynthetic activities. Oxygen evolution
was measured at 30 uC under saturating white light (300 mmol
metrically at 750 nm. For dry-weight determinations, cells were
collected, washed and dried at 70 uC for 24 h. For chlorophyll determinations, samples were extracted in methanol at 4 uC for 24 h in
darkness. The chlorophyll content of the extract was estimated
according to the spectrophotometric method of Marker (1972).
photons m22 s21) with a Clark-type oxygen electrode (Hansatech).
Total photosynthetic flux was assayed as O2 consumption using methyl
viologen as artificial electron acceptor, as described previously (Lien,
1978).
Preparation of thylakoid membranes. Thylakoid membrane iso-
lation was performed using the method described by Mi et al.
(1995) from cell suspensions exposed to pH 7?5 or 10?5. Cultures
were harvested by centrifugation (20 000 g, 10 min). The cell pellet
was washed twice and resuspended in 25 % (v/v) glycerol, 10 mM
MgCl2, 10 mM NaCl, 20 mM sodium phosphate buffer, pH 7?5,
and 1 mM PMSF. After 1 h on ice, cells were broken in a precooled
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Photosystem II capacity in isolated membranes (20 mg Chl ml21) (Chl,
chlorophyll) was estimated as O2 evolution in the presence of 2 mM
phenyl-1,4-benzoquinone (PBQ), 10 mM CaCl2 and 0?42 mM
ferricyanide in saturating white light (300 mmol photons m22 s21).
Photosystem I activity was measured in isolated membranes (20 mg Chl
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A. Blanco-Rivero and others
ml21) as O2 consumption using sodium ascorbate (5 mM) and 2,6dichlorophenol indophenol (DCPIP) as artificial electron donors and
methyl viologen (0?13 mM) as artificial electron acceptor (Lien, 1978).
Estimation of intracellular pH. Intracellular pH was measured by
electron spin resonance (ESR), mainly as described by Belkin et al.
(1987). The nitroxide spin probes used were: 3-carbamoyl proxyl
(neutral probe); 3-aminomethyl proxyl (basic probe) and 3-carboxy
proxyl (acid probe). All assays were conducted using a Varian model
E-12 EPR spectrometer. The samples consisted of a concentrated cell
suspension (80 mg Chl ml21) to which the appropriate spin probe
was added (final concentrations of 200 mM for the neutral probe
and 100 mM for the basic and acid probes). When required, 1?75 M
nickel tetraethylenepentaamine sulphate (NiTEPA) was added to
quench the ESR signal emanating from probe molecules in the
medium and thereby visualize only the intracellular signal. Ratios of
internal to total probe concentrations were calculated from height
ratios to the midfield lines of the quenched and unquenched samples. Samples were illuminated, when required, within the spectrometer cavity with a fluence rate of 300 mmol photons m22 s21 from
a cold white light from a fibre-optic system (KL1500 Electronic,
Schott). Calculations of intracellular pH were made according to
Belkin et al. (1987).
Recovery of transposon-containing plasmids and construction of derivatives of these plasmids. Plasmid pBG2033 (Tn5-
1063 and contiguous Anabaena sp. DNA) (Table 1) was obtained by
digesting chromosomal DNA from mutant PHB11 with EcoRV and
then by recircularization of the fragments with T4 DNA ligase and
transfer to E. coli HB101 by electroporation. Colonies that grew on
LB-agar plates with 50 mg ml21 kanamycin sulphate (Km) were analysed further. Approximately 3?8 kb of Anabaena sp. strain PCC7120
genomic DNA was recovered in pBG2033.
In order to generate the PHB11 mutation in wild-type Anabaena sp.
strain PCC7120 (see Table 1), most of the transposon was removed
from pBG2032 by cutting with PstI and BamHI. The remaining 4?6 kb
piece of DNA was ligated with pRL759D (Black et al., 1993) that had
been cut with PstI and BamHI, generating pBG2037. Plasmid pRL1075,
which bears the conditionally lethal gene sacB, which in turn allows for
selection of double recombinant strains (Cai & Wolk, 1990), was cut
with FspI, and a fragment of 5?6 kb was inserted into pBG2037 that had
been cut with EcoRV, to generate pBG2045 (Table 1).
Southern analysis. Southern analysis of chromosomal DNA made
use of the Genius system (Boehringer Mannheim). DNA probes
were labelled with digoxigenin-11-dUTP from random primers
(DIG DNA Labelling Kit, Boehringer Mannheim).
Sequence analysis. Automated sequencing (ABI Prism 377 DNA
Sequencer, Perkin-Elmer) was performed on fragments that were
subcloned from pBG2033 and pBG2038. The initial sequencing from
the ends of the transposon in pGB2033 was performed from specific
primers for the left and right end of the transposon (Black et al.,
1993). Sequence analysis was performed with the UW GCG package
of the University of Wisconsin, Genetics Computer Group, version 7
(Devereux et al., 1984). Amino acid sequence analysis was performed with the DAS transmembrane prediction package (Proteomics tools) at the ExPASy Molecular Biology Server (http://www.
expasy.org/) and Pfam (Protein Search Washington University in
St Louis) (http://pfam.wustl.edu/) for conserved protein domains.
Database comparisons and alignments of the DNA and predicted
protein sequences were performed by using the default settings of the
algorithm developed by Altschul et al. (1997) at the National Center for
Biotechnology Information (NCBI) with the BLAST network service
programs (http://www.ncbi.nlm.nih.gov/blast/) and WU-BLAST from
the European Bioinformatics Institute (EMBL-EBI) (http://www.
ebi.ac.uk/blast2/index.html). Sequence alignments were performed
with the CLUSTAL X program version 1.81 (Thompson et al., 1997).
In vivo monitoring of the expression of mrpA. The luciferase
activity of aliquots from strains DR2045-6A and SR2045-1B exposed
to increasing Na+ concentrations and/or alkaline pH was measured
at specified times and used as a measure of the transcription of
mrpA. mrpA expression was also measured under Ci limitation,
which was applied by switching the aeration from 1?5 % CO2 in air
(v/v) to air alone (350 ppm, Ci-limited) (Wang et al., 2004). The
luminescence of both strains was measured with supplementation
of exogenous aldehyde (0?01 %, v/v, n-decyl aldehyde, 0?1 %, v/v,
Triton X-100). Luminescence was measured using a digital luminometer (Bio Orbit 1250 Luminometer). The luminometer was calibrated by setting the background counts to zero and the built-in
standard photon source [a sealed ampoule of the isotope 14C, activity 0?26 mCi (9?62 kBq)] to 10 mV. Calculations made according to
Hastings & Weber (1963) indicated that one unit (1 mV) corresponded to a light emission of 6?76105 quanta s21 from the vial.
Nucleotide sequence accession number. The sequence reported
Cloning of mrpA and assays of complementation of the
mutant strain. A PCR clone of wild-type Anabaena sp. strain
PCC7120 DNA bracketing the transposon in mutant PHB11 was
generated with the primers 59-TGGCCGTTGCTCATTCTAGG-39
and 59-TGCGGAATTCGGCAGGACGA-39. The resulting 728 bp
PCR fragment was firstly cloned in the vector PCR2.1TOPO
(Invitrogen) producing plasmid pBG2038 (Table 1). From pBG2038,
the same fragment was cut and inserted between the BamHI and XhoI
sites of pRL1342, a chloramphenicol- and erythromycin-resistant
RSF1010-based plasmid (obtained from C. P. Wolk), generating
plasmid pBG2041 (Table 1), which can replicate in Anabaena sp.
strain PCC7120.
in this paper has been submitted to GenBank under accession no.
AF239979.
RESULTS AND DISCUSSION
Phenotype of mutant PHB11
Plasmid pBG2041 was transferred, with pRL1342 as control, from E.
coli to cells of mutant strain PHB11, as described by Wolk et al. (1984),
using helper plasmids pDS4101 and pRL1124 (see Table 1) (Finnegan
& Sherratt, 1982). Selection was made on Petri dishes of agar-solidified
AA medium (Allen & Arnon, 1955) containing 10 mg Em ml21 and
200 mg Nm ml21.
Transposon-bearing (i.e. antibiotic resistant) exconjugant
colonies grown in AA plus nitrate medium buffered at
pH 7?5 were transferred to Petri dishes with medium
buffered at pH 10?5 (5 mM BTP). One colony that yellowed
after several days under the basic pH conditions was selected
for further study and denoted as PHB11. Southern analysis
of PHB11 showed that only one copy of the transposon had
inserted into the Anabaena genome within an EcoRV
fragment of approximately 12 kb (data not shown).
The green colonies that appeared on the filters were further restreaked
to plates of the same medium to assess their ability to grow at high pH
and/or at high salt concentration.
To determine the function of the gene affected in the
mutant, various physiological tests were carried out. First, a
time-course study of the growth of the mutant in medium
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Role of mrpA in Na+ tolerance and pH adaptation
Fig. 1. Effect of external pH on growth (a, b and c) and photosynthesis (d, e and f) of wild-type Anabaena sp. strain
PCC7120 and mutant strain PHB11. (a, d), pH 7?5 (25 mM HEPES); (b, e), pH 9 (2 mM BTP); (c, f), pH 10?5 (2 mM BTP).
Error bars show SEM, calculated from three independent experiments with duplicate samples.
buffered at three selected external pH values, 7?5, 9 and 10?5,
was undertaken (Fig. 1a–c). As shown in the figure (Fig. 1a),
at pH 7?5 the growth curve of the mutant strain was similar
to that of the wild-type, although in the long term (after
7 days of culture), the growth yield of the mutant was 10 %
less than that of the wild-type (Student’s t test, P<0?05). At
pH 9 (Fig. 1b), the mutant strain showed 8 % growth
inhibition after 24 h (Student’s t test, P<0?05); after 7 days
of culture the inhibition reached 16 %. However, at pH 10?5
(Fig. 1c), the mutant strain showed impaired growth after
only 24 h of culture (20 % less growth, Student’s t test,
P<0?05). After 10 to 11 days at an external pH of 10?5,
mutant PHB11 bleached and died. Microscopic observations showed that under these basic conditions, filaments of
the mutant strain appeared yellow, short and with distorted
cells.
Photosynthesis was also examined in the mutant strain. As
shown in Fig. 1(d–f), the photosynthetic activity was lower
in the mutant strain than in the wild-type at the three
selected external pHs, although the inhibition of photosynthesis was much more pronounced at pH 10?5 (Fig. 1f).
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The observed effect of basic pH on photosynthesis of the
mutant led us to measure the total photosynthetic flux after
rapid alkalinization of the medium by suddenly shifting
external pH from 7?5 to 9 and from 7?5 to 10?5 by addition
of 0?05 M NaOH. The whole alkalinization process was
tightly monitored with a pH electrode. After a pH shift from
7?5 to 9, the photosynthetic flux of the mutant strain was
significantly inhibited, being around 45 % that of the wildtype (39?77±9?42 versus 87?79±16?48 mmol O2 mg Chl21
h21 for the wild-type; Student’s t test, P<0?05); after a
pH shift from 7?5 to 10?5, the inhibition was around
95 % of the wild-type strain value (5?89±0?84 versus
110?27±10?99 mmol O2 mg Chl21 h21 for the wild-type;
Student’s t test, P<0?05). The rapid and significant
inhibition of the photosynthetic flux in the mutant strain
after the pH change suggests that the mutated gene may be
particularly important to cope with short-term stress.
The observed inhibition of photosynthesis may reflect an
effect on the activity of the photosystems. To test the effect of
the mutation on each photosystem, thylakoidal membranes
of both strains were isolated from cells grown at pH 7?5
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A. Blanco-Rivero and others
Table 2. Determination of intracellular pH values (pHin) of
Anabaena sp. strain PCC7120 wild-type and PHB11 mutant
strain exposed during 3 days to external pH (pHex) values
of 7?5, 9, 10?5 and 11
Standard errors were calculated from three independent experiments with duplicate samples.
pHex
7?5
9
10?5
11
Strain
pHin
Wild-type
PHB11
Wild-type
PHB11
Wild-type
PHB11
Wild-type
PHB11
6?93±0?31
7?05±0?07
7?12±0?03
7?18±0?09
7?26±0?48
7?10±0?14
7?21±0?05
7?18±0?13
and 10?5 and activities of photosystem I (PSI) and photosystem II (PSII) measured in the isolated membranes.
The most significant effect was seen at pH 10?5, at which
a 75 % inhibition of PSII activity in the mutant strain
(17?36±3?11 versus 72?96±1?53 mmol O2 mg Chl21 h21
for the wild-type), and a 20 % inhibition of PSI activity
(131?26±4?20 versus 164?27±2?15 mmol O2 mg Chl21 h21
for the wild-type) were recorded.
This initial phenotypic characterization indicated that the
mutant had serious problems of adaptation to external pH
values in the alkaline range; this may reflect difficulties of the
mutant strain in controlling its intracellular pH. To check
this, we used ESR spin-probe techniques (Belkin et al., 1987)
to measure the intracellular pH values of the two strains
exposed to increasing external pH values for 3 days.
As shown in Table 2, at alkaline external pH values, no
significant differences in the intracellular pH values were
found between the mutant strain (whose growth was already
impaired after 3 days at high pH) and the wild-type.
The experiments already described were made with cultures
grown with 3 mM NaCl in the growth medium, hereafter
referred to as the Na+ standard concentration. Sodium is
known to be involved in both the osmotic and the pH
adaptation of bacterial cells (Booth, 1985) and is essential
for many cyanobacteria, especially if they are growing at
alkaline pH (Espie et al., 1988). To further analyse the role of
the mutated gene in salt and pH tolerance, increasing concentrations of NaCl at external pHs of both 7?5 and 10?5
were tested. As shown in Table 3, at an external pH of 7?5,
the generation time of the mutant strain was significantly
higher (Student’s t test, P<0?05) than that of the wildtype at external NaCl concentrations above 25 mM. The
inhibitory effect of NaCl on the growth of PHB11 became
more pronounced at an external pH of 10?5, at which, at the
standard Na+ concentration (as also evidenced in Fig. 1c),
the generation time was 20 % higher than that of the wildtype, increasing to 42 % higher at 25 mM and to 65 % at
50 mM (Student’s t test, P<0?001). At the highest external
pH, the mutant strain did not grow above 50 mM NaCl,
while the wild-type was able to grow, albeit very slowly, even
at 150 mM NaCl. There was no comparable inhibition of
growth by added K+ (data not shown). These results
indicate that the mutant strain is sensitive to increasing Na+
concentrations and that this sensitivity is higher at alkaline
pH.
Like most living cells, at high salt concentrations,
cyanobacteria actively extrude Na+ to try to maintain
internal ion concentrations comparable to those of cells
grown under low salt concentrations. Hence, the observed
sensitivity of the mutant strain may indicate difficulties in
extruding Na+. In this regard, preliminary data suggest
higher internal sodium in the mutant, particularly at higher
pH. Since an increased level of Na+ could be cytotoxic,
particularly at alkaline pH values (Krulwich et al., 1990),
these results may explain the observed impairment of
growth and photosynthesis (Fig. 1) and suggest a role
for the mutated gene in Na+ extrusion that is particularly
Table 3. Generation times of Anabaena sp. strain PCC7120 wild-type and PHB11 mutant strain exposed during 24 h to
increasing Na+ concentrations, both at pH 7?5 and pH 10?5
Values shown are generation times (h). Standard errors were calculated from three independent experiments with duplicate samples. 2,
Growth not detected.
Na+ concentration (mM)
Strain
0?3
pHex 7?5
Wild-type
PHB11
pHex 10?5
Wild-type
PHB11
3*
25
50
75
100
150
25?03±1?08
26?67±4?38
25?32±1?40
27?16±1?51
26?50±0?84
37?24±0?63
26?72±0?00
38?05±3?55
27?03±0?03
40?25±0?35
26?98±0?93
43?87±0?13
28?20±1?65
43?08±0?12
23?41±0?62
27?09±0?58
26?70±1?49
31?98±0?39
30?94±4?10
53?21±0?28
32?78±0?31
93?14±1?17
44?51±11?74
2
48?68±8?39
2
81?46±1?02
2
*Standard Na+ concentration in the growth medium.
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Microbiology 151
Role of mrpA in Na+ tolerance and pH adaptation
important at elevated external pH. However, measurement
of internal pH (Table 2) indicates that the mutated gene
may not have a significant involvement in internal pH
regulation or, alternatively, that the activity of the single
Na+/H+ antiporters may account for these results.
The Tn5-interrupted ORF encodes a predicted protein of
193 amino acids with an expected molecular mass of
21?3 kDa and an expected pI of 5?51. Near the N-terminal
domain, three predicted transmembrane domains are
located (data not shown).
Within the full genome sequence of moderately halotolerant
Synechocystis sp. PCC6803 are six putative genes encoding
single Na+/H+ antiporters (Kaneko et al., 1996). Elanskaya
et al. (2002) and Wang et al. (2002) have independently
mutated five ORFs and have found that NhaS3 (both
groups could not get a complete segregation of the mutant)
performs housekeeping functions essential to the survival
of the organism and which are critical for salt tolerance,
especially under alkaline conditions, in this cyanobacterium.
The ORF mutated in PHB11 corresponds to ORF all1838 of
the Anabaena genome (http://www.kazusa.org.jp/cyano/).
ORF all1838 is the sixth ORF of what seems to be a large
transcriptional unit of seven ORFs. Each of the ORFs
starts with ATG, except the first, which starts with GTG.
A promoter-like sequence (235 region and 210 region;
http://www.softberry.com/berry.phtml) was found in the
upstream region (data not shown). Neither a terminatorlike nor a promoter-like sequence was found between the
seven ORFs. Therefore, it seems that the seven ORFs
comprise an operon.
Elanskaya et al. (2002) concluded that the other four
antiporters were not clearly involved in salt tolerance or in
growth at high pH, whilst Wang et al. (2002) reported that
NhaS2 y NhaS4 might be essential for survival in freshwater
habitats characterized by large fluctuations in salt concentration and pH. Unfortunately, no such mutagenesis study
has been undertaken in Anabaena sp. PCC7120, for which
analysis of the complete genome sequence also reveals the
presence of five genes encoding putative single Na+/H+
antiporters (all1303, all2113, alr0252, alr0656 and all4832;
http://www.kazusa.or.jp/cyano).
Reconstruction of the mutation in mutant
PHB11
To determine whether the phenotype of PHB11 was the
result of insertion of the transposon rather than the result
of a secondary mutation, the transposon insertion was
reconstructed (Black et al., 1993). Transposon Tn5-1063
(7?8 kb) together with approximately 3?8 kb of contiguous
Anabaena DNA was recovered from mutant PHB11 upon
excision with EcoRV and circularization and transfer to E.
coli by electroporation, producing plasmid pBG2033 (from
which plasmid pBG2045 was constructed) (see Table 1 and
Methods).
Southern blotting analysis of one strain, designated
DR2045-6A, derived from presumptive recombination of
pBG2045 with the wild-type strain Anabaena sp. PCC7120
showed that the original mutation had been reconstructed
(data not shown). The phenotype of the double recombinant strain DR2045-6A also matched that of mutant strain
PHB11 (data not shown). Strain DR2045-6A, as well as
single recombinant strain SR2045-1B, were used for
subsequent in vivo gene expression studies (see below).
Analysis of the gene interrupted by the
transposon in strain PHB11
The transposon in strain PHB11 was found to interrupt an
ORF of 579 bp (data not shown). The transposon had
inserted 443 bp 39 from the first ATG codon of the ORF,
generating a 9 bp repeat (59-CGCTATATG-39).
http://mic.sgmjournals.org
The putative protein encoded by all1838 shows significant
similarity (>50 %) with a significant stretch of amino acids
of a much larger protein (between 725 and 804 amino acids)
identified as protein A of a putative seven-protein complex
found in several bacterial species and involved in pH
adaptation and Na+ tolerance (Hiramatsu et al., 1998;
Putnoky et al., 1998; Ito et al., 1999; Kosono et al., 1999).
Supplementary Fig. S1, available with the online version
of this paper at http://mic.sgmjournals.org, shows the
sequence alignment. Besides the protein encoded by
all1838, each of the remaining six gene products encoded
by the putative cyanobacterial operon also shows significant similarity to the corresponding bacterial products.
Furthermore, the seven putative Anabaena gene products
are highly hydrophobic and show a similar number of
predicted transmembrane helices to that reported for the
homologous bacterial products (Hiramatsu et al., 1998;
Putnoky et al., 1998; Ito et al., 1999; Kosono et al., 1999).
Details of the predicted similarities of the putative Anabaena
gene products as well as several other characteristics, that
is, coding positions, number of amino acid residues and
number of predicted transmembrane helices, are available
in Supplementary Table S1 with the online version of this
paper at http://mic.sgmjournals.org.
The bacterial complex is believed to function as an unusual
multicomponent Na+,K+/H+ antiporter. This gene family
was first discovered in alkaliphilic Bacillus sp. strain C-125
(Hamamoto et al., 1994); in that organism, it is required
for pH homeostasis in an alkaline environment. A mutant
derivative of the alkaliphilic Bacillus sp. is not able to grow at
alkaline pH because of a mutation in the first of a four-ORF
operon, suggesting that this gene cluster has an important
role in pH adaptation. Soon afterwards, homologues of
the alkalophilic Bacillus operon were described in three bacteria, Bacillus subtilis, Staphylococcus aureus and Rhizobium
meliloti. In these three bacteria, the operon contains a
whole set of seven genes (ORFA–G) and is thought to
encode a multisubunit antiporter family. In Bacillus subtilis,
Ito et al. (1999) denoted the operon as mrp (multiple
resistance and pH adaptation) and demonstrated that the
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A. Blanco-Rivero and others
operon is primarily involved in Na+ resistance, particularly
at alkaline pH; simultaneously, Kosono et al. (1999) also
described the same locus in Bacillus subtilis, but denoted it as
sha (sodium/hydrogen antiporter). Hiramatsu et al. (1998)
had already described the operon in Staphylococcus aureus,
denoting it mnh (multisubunit Na+/H+ antiporter). The
mnh operon complemented a Na+/H+ antiporter-deficient
E. coli strain, thereby suggesting that it may also function as a
Na+/H+ antiporter; the authors suggested that all seven
genes (mnhA–G) were required for the antiporter activity, so
that the Mnh antiporter probably forms a ion transport
complex of substantial size. The pha (pH adaptation) locus
of Rhizobium meliloti is required for invasion of nodule
tissue to establish nitrogen-fixing symbiosis (Putnoky et al.,
1998). Pha mutants show sensitivity to K+ but not to Na+
in their growth. It seems that the pha locus may encode a
K+/H+ antiporter that is involved in pH adaptation during
the infection process.
Based on the observed similarities, we decided to denote the
gene mutated in PHB11 as mrpA and the Anabaena operon
as mrp, following the nomenclature of the Bacillus subtilis
operon, for which the most extensive study of this locus has
been performed (Ito et al., 1999, 2000, 2001; Kosono et al.,
1999, 2000).
Two significant features of the Anabaena operon are the
different gene arrangement (mrpC to mrpB) and the smaller
size of mrpA. Supplementary Fig. S2 with the online version of this paper at http://mic.sgmjournals.org shows the
schematic region of the mrp locus of selected heterotrophic
bacteria, Anabaena sp, PCC7120 and other cyanobacteria
for which the genomes have been sequenced. The same gene
arrangement found in Anabaena sp PCC7120 is also found
in five other cyanobacteria: the unicellular Synechocystis sp.
PCC6803, the unicellular thermophilic Thermosynechoccus
elongatus BP-1 (http://www.kazusa.or.jp), the unicellular
Synechococcus elongatus PCC7942, the marine filamentous
Trichodesmium erythraeum and the heterocystous Anabaena
variabilis (http://www.jgi.doe.gov). A characteristic feature
of the Synechocystis operon is the presence of two extra 59
ORFs that may have appeared as a result of gene duplication.
However, the mrp operon was absent from the genomes
of the thylakoid-less Gloeobacter violaceus (http://www.
kazusa.or.jp), the marine unicellular Synechococcus WH8102,
Prochlorococcus marinus strains MED4 and MIT9313 and
the heterocystous symbiotic Nostoc punctiforme (http://
www.jgi.doe.gov). Thus, the mrp operon does not seem to
be of universal occurrence among cyanobacteria. The absence of the operon in the marine Phrochlorococcus and
Synechococcus strains could be explained by their small
genome size and by the fact that, being marine, they
probably have an elevated requirement for Na+; however,
the filamentous T. erythraeum is marine but retains the
operon, and N. punctiforme has probably one of the largest
genomes among bacteria but apparently lacks the operon.
Another interesting feature is that three subunits of the
putative Anabaena Mrp complex, MrpA, B and D, show
1678
significant similarity (more than 50 %) with subunits 2, 4, 5
or 6 of the eukaryotic proton pumping NADH : ubiquinone
oxidoreductase (complex I); NdhB, NdhE, NdhF and NdhG
of chloroplasts; Nad2, Nad4L, Nad5 and Nad6 of plant
mitochondria NADH-dehydrogenase and NuoN, NuoK,
NuoL and NuoJ of E. coli NADH-dehydrogenase 1 (see
Supplementary Table S1).
In E. coli, complex I is assembled from 14 different subunits,
seven of which are intrinsic membrane proteins (NuoA,
NuoH, NuoJ–N) (Weidner et al., 1993). In mitochondria,
complex I may contain as many as 40 different polypeptides.
The 14 minimal subunits whose homologues make up the
bacterial complex I can be subdivided in two groups:
seven are peripheral or predominantly peripheral proteins
including all subunits predicted to bind the known redox
group; the remaining seven subunits are membrane intrinsic
proteins encoded in the mitochondrial genome (ND1–6,
ND4L; Friedrich et al., 1995). This membrane-embedded
part of the complex is involved in proton translocation
(Friedrich et al., 1995). At least 12 (NdhA–L) of the 14
minimal subunits of complex I have been found to be
coded in the genome of the unicellular cyanobacterium
Synechocystis sp. PCC6803 (Kaneko et al., 1996).
Interestingly, Prommeenate et al. (2004) have found two
additional Ndh subunits (encoded by sll1262 and slr1623) in
this cyanobacterium. Most ndh genes are present as single
copies in the Synechocystis genome. However, there are
multiple copies of ndhD (six homologues) and ndhF (three
homologues). In the Anabaena genome (Kaneko et al.,
2001) there are 11 ndh genes (ndhA–K) with five copies of
ndhD and three of ndhF, although it lacks the ndhL
homologue of Synechocystis. However, Anabaena does
contain an ORF (alr0751) that is not present in
Synechocystis but that could code for a putative catalytic
subunit homologous to NuoE of E. coli.
The fact that ndhD and ndhF are present as gene families
suggests that several types of NDH-1 complex exist in
cyanobacteria, each with different NdhD/NdhF subunits
and with each potential complex having different functions (Klughammer et al., 1999; Ohkawa et al., 2000). The
NdhD1.NdhD2 type of NDH-1 complex would be involved
in the PSI-dependent cyclic electron transport pathway as
well as in cellular respiration, while the NdhD3.NdhD4
NDH-1 complexes are essential for CO2 uptake (Ohkawa
et al., 2000). Prommeenate et al. (2004) have found two large
NDH-1 complexes, termed A (460 kDa) and B (330 kDa),
with similar protein profiles by SDS-PAGE, by which they
have identified hydrophilic as well as hydrophobic modules.
However, NdhF3 was never found in the NDH-1 complexes.
The authors argue that some of the annotated NdhD and
NdhF subunits may actually have roles unrelated to NDH-1
function. More recently, Zhang et al. (2004) have also
analysed the subunit composition and functional roles of
the NDH-1 complexes of Synechocystis, and have found that
the two larger NDH-1 complexes also lack the NdhD3 and
NdhF3 subunits. In this regard, Putnoky et al. (1998) and
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Role of mrpA in Na+ tolerance and pH adaptation
Hiramatsu et al. (1998) have suggested that the observed
similarities between subunits of the bacterial mrp operons
and hydrophobic subunits of Complex I point towards a
common origin and function for both systems. The authors
speculate that the membrane-embedded part of complex I
evolved from an ancestral cation/proton antiporter and
became specialized for concerted actions with the cytoplasmic subunits involved in electron transfer. Recently,
Mathiesen & Hägerhäll (2003) have hypothesized that a
multisubunit antiporter complex formed by MrpA (homologous to NuoL), MrpD (homologous to NuoM/N) and
MrpC (which the authors have found homologous to
NuoK) may have been recruited to the ancestral complex I,
which would contain a NuoKLMN subunit module.
Cloning of the wild-type version of mrpA and
complementation of the mutation
A 728 bp PCR fragment of Anabaena sp. PCC7120 DNA was
shown by sequencing to contain the mrpA gene as the only
ORF (data not shown). The sequence of the cloned gene was
identical to that obtained from the transposon-mutagenized
form of the fragment recovered on pBG2033 (Table 1; see
Methods). The 728 bp fragment bearing the PCR-amplified
wild-type mrpA was cloned into the RSF1010-based plasmid
pRl1342, generating plasmid pBG2041, which can replicate in Anabaena. Plasmid pBG2041 was transferred by
conjugation to mutant strain PHB11, and colonies resistant
to erythromycin were selected. The complemented strain,
denoted PHB11 : pBG2041-6, behaved in the same way as
the wild-type strain at both an external pH of 10?5 and an
external pH of 10?5 supplemented with 50 mM Na+ (data
not shown).
In vivo expression of mrpA
Transposon Tn5-1063 generates transcriptional fusions
between the Vibrio fischeri luxA and luxB genes, which
encode luciferase, and genes into which the transposon
becomes inserted (Wolk et al., 1991), thus permitting the
monitoring of gene expression in vivo, provided that the
transposon is correctly oriented. However, the transposon in mutant PHB11 placed luxAB antiparallel to the
gene mrpA (not shown). Plasmid pBG2037 (Table 1; see
Methods) was constructed in order to reconstruct the
mutation, placing luxAB parallel to the direction of
transcription of mrpA. Single recombinant [Emr Spr Smr
(sucrose sensitive)] and double recombinant [Ems Spr Smr
Fig. 2. Luciferase activity of cell suspensions from the double recombinant strain DR2045-6A (a, c) and single recombinant
strain SR2045-1B (b, d). Cells were exposed to increasing concentrations of NaCl at pH 7?5 (a, b) or increasing
concentrations of NaCl at pH 10?5 (c, d). Samples were taken and their luminescence was measured at the times indicated.
Error bars show SEM, calculated from three independent experiments with duplicate samples.
http://mic.sgmjournals.org
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1679
A. Blanco-Rivero and others
(sucrose resistant)] strains were obtained (see above). Single
recombinant SR2045-1B and double recombinant DR20456A were selected for the in vivo expression studies.
The in vivo expression of mrpA from cell suspensions of the
single and double recombinant strains was first monitored
as a function of time under increasing external Na+
concentrations at pH 7?5. As shown in Fig. 2(a, b), the
pattern of expression of the gene in the double (a) as well as
the single recombinant strain (b) is very similar. There is
a clear induction of mrpA in the presence of increasing
concentrations of Na+ in the medium; depending on the
external Na+ concentration, the induction is maximal after
4–8 h incubation, with the highest level of induction at
100 mM Na+.
The second environmental condition tested was the
combined effect of alkaline pH (pH 10?5) and increased
Na+. In this case, there was a remarkable difference between
the double recombinant (Fig. 2c) and the single recombinant strain (Fig. 2d). In the double recombinant, there is a
clear induction of the expression of mrpA at relatively
low external Na+ concentrations (3–25 mM). However, at
higher Na+ concentrations there is no induction of
expression, indicating that alkaline conditions combined
with high Na+ concentrations affect the viability of the
double recombinant strain, as already found with the
original mutant strain PHB11. However, the pattern of
induction of mrpA in the single recombinant strain, which
retains a wild-type copy of the gene, is clearly different, with
maximum levels of induction at the highest Na+ concentrations tested. The induction is maximal after around
1 h of exposure to basic pH, reaching luminescence
values significantly higher than those found at pH 7?5.
Therefore, expression of mrpA is upregulated in response to
increased Na+ concentration in the culture medium. The
level of induction is higher at alkaline pH values. Both the
phenotypic results and the expression studies imply a role
for mrpA, and probably for the whole operon, in resistance
to Na+, particularly at alkaline pH, in this cyanobacterium.
Wang et al. (2004) reported that the transcription of two
genes of Synechocystis sp. PCC6803 whose products resemble subunits 5 and 6 of NDH-1, subsequently named
as ndhD5 and ndhD6, were upregulated in response to a
CO2 downshift. These genes were located in a large transcriptional unit that we have identified here as the Synechocystis mrp operon. We checked whether transcription
of the Anabaena mrpA gene was also upregulated in response
to a Ci downshift. Fig. 3 shows the pattern of mrpA
expression in both the double (Fig. 3a) and single (Fig. 3b)
recombinant strains after switching the aeration from 1?5 %
CO2 in air (v/v) to air alone (350 ppm CO2, Ci limited). As
can be seen in the figure, the transcription of mrpA is
induced twofold under Ci-limiting conditions in the double
recombinant and threefold in the single recombinant strain.
The results reported by Wang et al. (2004) together with
ours suggest an involvement of the cyanobacterial mrp
operon in low-Ci acclimation.
1680
Fig. 3. Luciferase activity of cell suspensions from the double
recombinant strain DR2045-6A (a) and single recombinant
strain SR2045-1B (b). Cultures were bubbled with air supplemented with 1?5 % CO2 and then switched to air alone. The
luciferase activity of cell suspensions supplemented with 1?5 %
CO2 is shown as a control. Samples were taken and their luminescence measured at the times indicated. Error bars show
SEM, calculated from three independent experiments with duplicate samples.
It is noticeable that the induction of mrpA under alkaline/
Na+ stress and low Ci is very quick (Figs 2 and 3); this again
suggests a relevant role for mrpA and probably the whole
mrp operon in the response to short-term stress, whereas the
growth data shown in Fig. 1 may indicate that, in the long
term, compensatory mechanisms are induced that allow
some growth of the mutant until it finally bleaches and dies.
Wang et al. (2004) also found that disruption of ndhR (Figge
et al., 2001) activated the transcription of Synechocystis mrp
genes. NdhR is a LysR-family regulator of Ci uptake, initially
described as a transcriptional regulator for ndh genes in
Synechocystis sp. PCC6803 (Figge et al., 2001). The T(N11)A
sequence is often present in the promoter regions of genes
controlled by LysR-type regulators in proteobacteria. Figge
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Microbiology 151
Role of mrpA in Na+ tolerance and pH adaptation
et al. (2001) already proposed the TCAATG–(N10)–
ATCAAT sequence as the consensus motif in Synechocystis
sp. PCC6803. Three presumptive NdhR-binding sites have
been identified in the promoter region of the mrp operon in
Synechocystis (Wang et al., 2004). We checked the promoter of the mrp operon of Anabaena and found eight
presumptive NdhR-binding sites (shown in Supplementary
Fig. S3 with the online version of this paper at http://
mic.sgmjournals.org). We searched for a homologue of
ndhR in the Anabaena genome and found two possible
candidates: all4986, which shows 69 % similarity to ndhR,
and all3953, which shows 49 % similarity. all4986 is next
to all4985, which putatively encodes sucrose synthase.
all3953 is close to, although divergently transcribed from,
genes ndhF (alr3956) and ndhD (alr3957), which are within
a large transcript unit of nine ORFs (alr3954–alr3959,
asr3959–asr3961).
Finally, with all this evidence, a major question arises
regarding which is/are the specific role(s) of a putative
Mrp complex in cyanobacteria. As has been proposed in
heterotrophic bacteria (Hiramatsu et al., 1998), it may be a
multicomponent Na+/H+ antiporter that is energized by
electron transport through the subunits resembling hydrophobic components of complex I. However, additional
experiments are required to clarify whether or not this
system truly has a Na+/H+ antiporter activity in cyanobacteria and to determine which protein(s) in the complex
function as the Na+/H+ antiporter, and the relationship, if
any, between the subunits resembling the hydrophobic core
of NDH-1 and NDH-1 itself. Also, if the Mrp complex is a
true Na+/H+ antiporter, the Na+ gradient created may
drive nutrient uptake, in other words, HCO32, as suggested
for Synechocystis (Wang et al., 2004). Also unresolved is the
relationship, if any, between this multisubunit complex
and the single Na+/H+ antiporters in Anabaena strain sp.
PCC7120. A more complete mutational and biochemical
analysis of this complicated and unexplored cyanobacterial
locus is needed to fully resolve these issues
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ACKNOWLEDGEMENTS
This work was supported by MCYT grants BMC2000-0128 and
BMC2003-03583.
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