FEMS Microbiology Reviews 28 (2004) 353–376
www.fems-microbiology.org
Regulators of nonsulfur purple phototrophic bacteria and
the interactive control of CO2 assimilation, nitrogen fixation,
hydrogen metabolism and energy generation
James M. Dubbs a, F. Robert Tabita
b,*
a
b
Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand
Department of Microbiology and Plant Biotechnology Center, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA
Received 28 July 2003; received in revised form 29 October 2003; accepted 23 December 2003
First published online 20 February 2004
Abstract
For the metabolically diverse nonsulfur purple phototrophic bacteria, maintaining redox homeostasis requires balancing the
activities of energy supplying and energy-utilizing pathways, often in the face of drastic changes in environmental conditions. These
organisms, members of the class Alphaproteobacteria, primarily use CO2 as an electron sink to achieve redox homeostasis. After
noting the consequences of inactivating the capacity for CO2 reduction through the Calvin–Benson–Bassham (CBB) pathway, it was
shown that the molecular control of many additional important biological processes catalyzed by nonsulfur purple bacteria is linked
to expression of the CBB genes. Several regulator proteins are involved, with the two component Reg/Prr regulatory system playing
a major role in maintaining redox poise in these organisms. Reg/Prr was shown to be a global regulator involved in the coordinate
control of a number of metabolic processes including CO2 assimilation, nitrogen fixation, hydrogen metabolism and energy-generation pathways. Accumulating evidence suggests that the Reg/Prr system senses the oxidation/reduction state of the cell by
monitoring a signal associated with electron transport. The response regulator RegA/PrrA activates or represses gene expression
through direct interaction with target gene promoters where it often works in concert with other regulators that can be either global
or specific. For the key CO2 reduction pathway, which clearly triggers whether other redox balancing mechanisms are employed, the
ability to activate or inactivate the specific regulator CbbR is of paramount importance. From these studies, it is apparent that a
detailed understanding of how diverse regulatory elements integrate and control metabolism will eventually be achieved.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: CBB cycle; RegA; PrrA; CbbR; Rhodobacter; Rhodopseudomonas; Gene regulation; Redox balance
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Reg/Prr system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Structure and biochemistry of RegB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Structure and biochemistry of RegA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Reg/Prr gene organization and a regulation . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Involvement of cytochrome cbb3 oxidase in Reg/Prr system signal transduction . . .
2.5. Possible involvement of CcoQ in the Reg/Prr system signal. . . . . . . . . . . . . . . . .
2.6. Possible involvement of RdxB in the Reg/Prr system signal. . . . . . . . . . . . . . . . .
*
Corresponding author. Tel.: +1-614-292-4297;
fax: +1-614-292-6337.
E-mail address: [email protected] (F. Robert Tabita).
0168-6445/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsre.2004.01.002
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2.7. Possible involvement of PrrC/SenC in the Reg/Prr system signal . . . . . . . . . . . . .
2.8. Reg/Prr regulation of CBB cycle gene expression. . . . . . . . . . . . . . . . . . . . . . . .
2.9. Reg/Prr regulation of photosystem gene expression . . . . . . . . . . . . . . . . . . . . . .
2.10. Reg/Prr regulation of electron transport components . . . . . . . . . . . . . . . . . . . . .
2.11. Reg/Prr regulation of nitrogen fixation genes. . . . . . . . . . . . . . . . . . . . . . . . . . .
2.12. Reg/Prr regulation of other pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.13. RegA homologs in other bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.14. Reg/Prr-regulated promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.15. Model for Reg/Prr-mediated gene regulation . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.16. Is the Reg/Prr system always required?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HvrA and Spb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. HvrA regulation of photosystem gene expression. . . . . . . . . . . . . . . . . . . . . . . . .
3.2. HvrA regulation of nitrogen fixation gene expression . . . . . . . . . . . . . . . . . . . . . .
3.3. HvrA regulation of electron transfer components . . . . . . . . . . . . . . . . . . . . . . . .
3.4. HvrA regulation of CBB cycle gene expression . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Spb regulation of photosynthesis gene expression . . . . . . . . . . . . . . . . . . . . . . . .
3.6. Mechanism of HvrA and Spb function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CbbR-mediated regulation of cbb gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other potential regulators of cbb gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A final word on integrative control of gene expression . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
4.
5.
6.
1. Introduction
All organisms are faced with the problem of how to
efficiently balance the activity of pathways that generate
reducing power with those that consume reductant in
order to maintain redox homeostasis. For prokaryotes
like the nonsulfur purple bacteria (NSP), solving this
fundamental problem is made even more complex since
they have evolved the ability to make a living under a
diverse variety of growth modes. This group of facultative anoxygenic phototrophs belongs to the class Alphaproteobacteria and includes a number of genera
within the orders Rhodospirillales, Rhodobacterales and
Rhizobiales. The extreme metabolic versatility of these
organisms allows them to grow by anoxygenic photosynthesis, aerobic or anaerobic respiration and fermentation. During photoautotrophic and chemoautotrophic
growth, NSP bacteria can grow using H2 as an electron
donor while obtaining fixed carbon through the reduction CO2 using the CBB–Benson–Bassham (CBB) cycle
[1,2]. Phototrophic growth can also occur at the expense
of fixed carbon compounds that serve as both carbon
source and electron donor [3]. In all cases reducing
equivalents produced either through photosynthesis or
the oxidation of organic compounds are funneled to a
host of electron carriers where they can be used to
perform energy intensive tasks such as ATP synthesis,
CO2 fixation and N2 fixation, to name a few. Excess
reducing power is then transferred to a variety of terminal electron acceptors such as O2 , or to CO2 or N2
through the processes of CO2 and N2 fixation. A num-
ber of other electron acceptors can also be used as dimethyl sulfoxide (DMSO), trimethylamine oxide
(TMAO) and NO3 [4]. In order to maintain redox homeostasis cells possess regulatory mechanisms that balance the expression of energy supplying systems, such as
photosynthesis, with energy-utilization pathways such
as CO2 and N2 fixation. Physiological studies in NSP
bacteria have long indicated that there are regulatory
links between photosynthesis and the assimilation of
CO2 and N2 [5,6], and in recent years specific regulatory
systems controlling two or more of these processes have
begun to be characterized. This review deals primarily
with the current state of knowledge concerning regulator
molecules found in these organisms, with special reference to cbb gene regulation in the NSP bacteria; included is a global Reg/Prr two-component regulatory
system and a specific ‘‘one-component’’ regulator protein, CbbR, plus other potential regulators. In addition,
the influence that the CBB system has on the regulation
of other redox balancing systems in the cell will be
considered, including nitrogen fixation and hydrogen
metabolism.
2. The Reg/Prr system
The genes of the Reg/Prr regulatory system were
originally identified as regulators of photosystem gene
expression. Screens of a genomic library for the ability
to complement Rhodobacter capsulatus mutants that
displayed decreased photopigment synthesis during an-
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
aerobic growth [7] led to the isolation of the regA gene,
encoding a 184 amino acid protein that was similar in
sequence to the response regulators of two-component
regulatory systems, and regB, encoding a 462 amino
acid histidine sensor kinase [8]. In the related bacterium
Rhodobacter sphaeroides, a regA analog of identical
length was independently isolated using two different
methods. Eraso and Kaplan [9] isolated prrA (photosynthesis response regulator) from a mutant strain that
was unable to form photosynthetic complexes during
anaerobic growth. Phillips-Jones and Hunter [10] identified the analogous gene, from a R. sphaeroides genomic
library of a different strain, by its ability to complement
a R. capsulatus regA mutant. The regB analog prrB was
subsequently identified when an insertion mutant in the
region upstream of prrA showed reduced photopigment
synthesis [11]. The phenotypes of mutants in the reg/prr
genes follow the same general pattern in both organisms
and indicate that the system positively regulates photopigment gene expression. Inactivation of regB/prrB results in a decrease in photopigment synthesis, however,
the mutants are still able to synthesize enough photopigment for phototrophic growth at high light intensity
[7,8,11]. The same is true for a R. capsulatus regA mutant, however, in a R. sphaeroides prrA mutant, photopigment synthesis is decreased to the point that the
organism is incapable of phototrophic growth at any
light intensity [9].
355
signal transduction since mutations within the transmembrane region generally tend to result in O2 insensitive expression of the photosystem [11,17]. In vitro
phosphorylation experiments using truncated RegB/
PrrB proteins, lacking some or all of the transmembrane
domain, were capable of autophosphorylation and
phosphotransfer to RegA [13,18,19]. Similar studies
using full length RegB/PrrB from R. sphaeroides showed
autophosphorylation and phosphotransfer kinetics that
were similar to those of truncated RegB/PrrB [20]. A
possible regulatory role for the transmembrane domain
was also indicated since its presence dramatically reduced the half-life of RegB/PrrBP compared to the
truncated form [18,20]. The presence or absence of O2
had no affect on the kinetics of either autophosphorylation or phosphotransfer indicating that the default
state of RegB is in the kinase dominant form, as had
been previously suggested [17], and that an additional
regulator is involved in controlling the rate of RegB
autophosphorylation [20]. Both the full length and
truncated forms of RegB/PrrB were able to actively
dephosphorylate RegAP [18,20]. This phosphatase
activity is likely to be physiologically relevant since it
has been shown that overexpression of RegB/PrrB in
both a wild-type and a RegB/PrrB constitutive mutant
background reduces expression of the photosystem in
R. sphaeroides [11,21,22].
2.2. Structure and biochemistry of RegA
2.1. Structure and biochemistry of RegB
The RegB sequences that have been determined so far
in NSP bacteria show a high degree of sequence conservation with sequence identities of approximately 56–
62% [12]. The majority of sequence conservation occurs
in the carboxy terminal portion beyond approximately
amino acids 166 and 182 in R. capsulatus and R. sphaeroides, respectively [12]. This region contains a conserved H box that spans a histidine, at amino acids 195
and 211 in R. capsulatus and R. sphaeroides RegB/PrrB,
respectively, that is the site of autophosphorylation [13].
Conserved regions corresponding to the N, Gl and G2
boxes known to be involved in autophosphorylation
activity are also present [12,14,15]. Hydrophobicity plots
predict that RegB/PrrB contains an N-terminal transmembrane domain that contains six membrane spanning regions and five exposed loops. Experimental
evidence is consistent with the predictions. Structural
studies that localized the positions, either cytoplasmic or
periplasmic, of the alkaline phosphatase or b-galactosidase moieties of RegB fusion proteins indicate that the
carboxy and amino-terminal portions of RegB/PrrB are
on the cytoplasmic side of the membrane with three of
the exposed loops facing the periplasm and two facing
the cytoplasm [16]. It has been suggested that the
transmembrane domain plays a role in O2 sensing and
Sequence analyses show that the RegA/PrrA homologs from purple bacteria and others harbor a carboxy
terminal Helix–Turn–Helix DNA-binding motif that is
100% conserved and has been shown to be necessary for
DNA binding [12,23]. The carboxy terminal DNAbinding motif is separated from an amino terminal receiver domain by a stretch of four conserved proline
residues [12,24] and contains a conserved aspartate
residue at position 63 that serves as the site of phosphorylation [12,13,19,25]. It is clear that both phosphorylation and DNA binding are necessary for RegA/
PrrA function in vivo since mutations in either the Helix–Turn–Helix DNA-binding motif or the phosphorylation site at position 63 result in a phenotype similar to
a regA/prrA mutant [19,25]. However, the exact role
phosphorylation plays in RegA/PrrA-mediated gene
regulation has yet to be elucidated. One line of investigation indicates that phosphorylation of RegA/PrrA
increases its binding affinity for its DNA target site. A
constitutively active mutant of RegA (RegA*), containing an alanine to serine substitution at position 95,
conferred increased aerobic and anaerobic expression of
the photosystem in a R. capsulatus regB mutant background [24]. It was found that RegA* bound to the same
site within the puc operon promoter as wild-type
RegA, but with an increased affinity compared to that of
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wild-type RegA [18,24] and RegA*P was found to be
much more stable than wild-type RegAP [18]. Additionally, studies by Bird et al. [18] indicated that phosphorylation increased the affinity of R. capsulatus
RegA* for its binding site by about 2-fold, and that of
wild-type RegA by about 16-fold. A similar effect on
binding affinity was observed for the close RegA homolog RegR in Bradyrhizobium japonicum [26]. Since
RegA* was able to activate photosystem gene expression in the absence of the histidine kinase RegB, it was
suggested that the conformation of RegA* mimics that
of RegAP, resulting in a constitutively active protein
due to increased DNA-binding affinity. Alternatively, it
was proposed that RegA* activation may arise from the
increased stability of RegA*P that is the result of
RegA* phosphorylation by alternate histidine kinases
[18]. Other evidence indicates a different role for phosphorylation. Hemschemeier et al. [23] found that phosphorylation of RegA had only a small positive affect on
DNA-binding affinity. Changing the phosphorylation
site aspartate at position 63 to a lysine did not appear to
affect the DNA-binding behavior of the protein [25].
They also found that modified RegA proteins, lacking
the carboxy terminal DNA-binding domain, were still
able to mediate activation of photosynthesis gene expression (puc and puf operons) in response to reductions
in O2 concentration [25]. They suggested that phosphorylation of RegA was not required for DNA binding
but rather increased the efficiency of transcription initiation. There is also evidence that the aspartate at position 63 is important for RegA function in the absence
of phosphorylation. Comolli et al. [19] have shown in
vitro that unphosphorylated PrrA is still able to activate
transcription of the cycA P2 promoter albeit less efficiently than PrrAP. Nonphosphorylated PrrA, in
which the aspartate at position 63 was changed to an
alanine, was no longer able to activate transcription
from cyc A P2 [19]. Thus, phosphorylation of RegA/
PrrA may induce changes in the structure of the protein
that may affect not only DNA-binding affinity but also
alter its ability to interact with regulatory partners and
stimulate transcription initiation.
from the nonsulfur purple bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans [12] and show the
same pattern of gene organization with the exception
that no ORFs similar to either hvrA or spb are found
downstream of regA in these organisms. The transcription patterns of both the R. capsulatus and R. sphaeroides genes appear to be somewhat similar. In
R. capsulatus, a transcript has been identified derived
from senC–regA and hvrA that originates from a promoter within the regB–senC intergenic region [28,31].
Two smaller transcripts have also been identified that
are specific to regA–hvrA and hvrA [28]. It has not been
determined if the smaller transcripts are the result of
RNA processing or secondary promoters downstream
of senC. regB is encoded on a monocistronic message
originating from a promoter within the regB–senC intergenic region that overlaps the promoter for senC [31].
Analysis of senC and regB promoter fusions to lacZ in
R. capsulatus has shown that both promoters are negatively regulated by RegAP during anaerobic growth
[31]. Moreover, negative regulation was due to RegA
binding in a region that overlaps the RNA polymerasebinding sites in both promoters [31]. In R. sphaeroides,
prrB is also transcribed on a single monocistronic message [32]. A prrC–prrA transcript most likely originating
from a point within the prrB–prrC intergenic region has
been identified. An additional promoter within the 30
portion of prrC gives rise to a large prrA–spb transcript
[32]. Two additional smaller monocistronic messages
encoding prrA and spb have been identified that may be
the result of either RNA processing of the larger prrA–
spb message or partial transcriptional read through of a
putative termination site downstream of prrA [32]. It
should be noted that earlier Northern analyses performed by Mizoguchi et al. [33] were only able to
identify a single 400 nucleotide monocistronic message
encoding spb and not the larger prrC–spb message. This
may reflect differences in the quality of the RNA preparations. Western blot analyses, examining changes in
the levels of PrrA during aerobic versus anaerobic
growth indicate that the same negative autoregulation of
PrrA expression occurs in R. sphaeroides [19].
2.3. Reg/Prr gene organization and a regulation
2.4. Involvement of cytochrome cbb3 oxidase in Reg/Prr
system signal transduction
The genetic organization of the R. capsulatus and R.
sphaeroides reg/prr operons is conserved. The gene order
is <regB senC > regA > hvrA> in R. capsulatus and
<prrB prrC > prrA > spb > in R. sphaeroides with regB
and prrB transcribed divergently from the other three
genes. The function of the products of senC and prrC is
unknown, however, both display sequence similarity to
the putative cytochrome oxidase assembly factors SCO1
and SCO2 in yeast [27]. hvrA and spb encode light-responding transcriptional regulators of photosynthesis
genes [28–30]. The reg/prr genes have also been isolated
Little is known about the exact nature of the primary
signal that activates the Reg/Prr system or the additional
components, if any, that may be involved in conducting
that signal to RegB. A body of work in R. sphaeroides
indicates that the signal that activates/derepresses the
Reg/Prr system requires functional cbb3 cytochrome
oxidase. R. sphaeroides is known to contain two terminal cytochrome oxidases that accept electrons from the
cytochrome bc1 complex, via a soluble cytochrome c2 or
a membrane-bound cytochrome cy, and reduce O2 to
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
form H2 O during aerobic respiratory growth [34–36].
The low affinity aa3 type oxidase is the predominant
cytochrome oxidase during aerobic growth, while the
high affinity cbb3 type oxidase is expressed under conditions where O2 levels are low or completely absent
[34,37]. Inactivation of any of the genes in the R. sphaeroides ccoNOQP operon, encoding the four subunits
of the cbb3 oxidase [38], results in the aerobic expression
of genes encoding photosynthetic complexes (puf and
puc) [39–42] as well as a number of other Reg/Prr-regulated processes such as CO2 fixation (cbb) [43] and
DMSO reduction [44,45]. Studies focusing on the effect
of mutations in R. sphaeroides ccoNOPQ, and other
electron transport components on photopigment expression, were able to show that the relaxation of Reg/
Prr control was specific to the cbb3 oxidase since inactivation of the aa3 oxidase had no affect [46]. Moreover,
inactivation of genes encoding electron transport components upstream of the cbb3 oxidase such as the cytochromes c2 and cy , and the cytochrome bc1 complex,
resulted in the same aerobic expression of photosynthetic complexes [46]. Also, the magnitude of photosystem gene derepression appeared to be proportional to
the electron flow through the cbb3 -oxidase since inactivation of the genes encoding either cytochrome cy or c2
resulted in aerobic derepression of the photosystem to
levels that were less than those of either a cytochrome
cbb3 (ccoN) null mutant or a cytochrome cy (cycY),
cytochrome cy (cycA) double mutant [46]. A link between cbb3 and the Reg/Prr system was unambiguously
demonstrated by the fact that inactivation of either prrA
or prrB in a cbb3 -deficient ccoP mutant halts the aberrant expression of photosynthetic complexes during
aerobic growth and severely reduced expression during
dark anaerobic growth in the presence of DMSO [41].
These observations have lead to the hypothesis that
electron flow through the terminal cbb3 type cytochrome
oxidase generates a signal that prevents the phosphorylation of RegA/PrrA by membrane-bound RegB/PrrB.
While much attention has been placed on the role of
oxygen in the control of the Reg/Prr system, there is
some evidence to suggest that electron flow to something
other than O2 may be sensed by the system. Cytochrome-cbb3 oxidase-Reg/Prr-dependent gene regulation also occurs in the absence of oxygen since
inactivation of cytochrome cbb3 -oxidase has been shown
to cause a Reg/Prr-dependent increase in photopigment
synthesis during anaerobic growth, while overexpression
of cytochrome-cbb3 oxidase leads to reduced expression
of the photosystem during anaerobic photosynthetic
growth [41,42]. This evidence has lead to the suggestion
that there may be an additional anaerobic acceptor for
electrons from the cytochrome-cbb3 oxidase [42]. The
Reg/Prr system has also been shown to regulate a
number of genes in the presence of high levels of O2 . In
R. capsulatus, the Reg/Prr system is able to both activate
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nifA2 and repress hupSLC expression to similar degrees
in both the presence and absence of CO2 [47]. In addition, Reg/Prr-dependent activation of the R. sphaeroides
cbbI I operon occurs during chemoautotrophic growth at
O2 concentrations as high as 10% [43,48]. While these
Reg/Prr-dependent effects may be due to O2 consumption rates that result in low intracellular O2 concentrations, it is also possible that a second O2 independent
signal is involved.
2.5. Possible involvement of CcoQ in the Reg/Prr system
signal
Identification of the component(s) involved in the
communication of the inhibitory signal from cbb3 -oxidase to RegB/PrrB has proved difficult. Several loci have
been implicated as encoding part of the signal transduction chain from cbb3 to Reg/Prr. One of these is ccoQ
that encodes the smallest subunit of the cyt cbb3 -oxidase
[49]. Inactivation of ccoQ in R. sphaeroides resulted in
the aberrant aerobic expression of photosynthetic complexes, however, the mutant expressed an active seemingly normal cyt cbb3 -oxidase during dark anaerobic
growth in the presence of DMSO as well as during
anaerobic photoheterotrophic growth [42]. It was suggested from this that ccoQ is involved in the transduction of the inhibitory signal from cyt cbb3 -oxidase to
RegB/PrrB [42]. Subsequent investigation of the role of
CcoQ in maintaining cyt cbb3 -oxidase activity revealed
that CcoQ functions to protect the cyt cbb3 -oxidase
from O2 -mediated damage under aerobic conditions.
A lack of CcoQ under aerobic conditions results in instability of the oxidase subunits CcoP and CcoO [50].
Thus, the aerobic derepression of photosynthesis gene
expression in the ccoQ mutant was a result of the
absence of active cyt cbb3 -oxidase.
2.6. Possible involvement of RdxB in the Reg/Prr system
signal
The rdxBHIS operon, located 234 bp downstream of
R. sphaeroides ccoNOQP, has also been theorized to be
involved in the signal transduction chain from cbb3 oxidase to Reg/Prr. This idea was based primarily on the
observations that insertional inactivation of rdxB, or inframe deletion of the individual genes within rdxBHIS,
results in aberrant aerobic expression of the photosynthetic complex [40–42,51]. Furthermore, mutants in
rdxB still produced functional cbb3 -oxidase under anaerobic conditions [51]. Analysis of the homologous
operon ccoGHIS in R. capsulatus indicates that CcoH,
CcoI, CcoS and possibly CcoG are involved in assembly
and maturation of the cbb3 -oxidase [52]. An R. capsulatus mutant in ccoG, an analog of rdxB, was capable of
producing active cyt cbb3 -oxidase, under semi-aerobic
conditions, with activity levels that were slightly reduced
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(10%) relative to wild-type. The ccoG mutant also
showed impaired cyt oxidase activity when assayed using a whole colony colorimetric method [52]. The exact
function of ccoG/rdxB is unknown, however, it has been
suggested that the product of these genes may play a role
in making copper available to the cyt cbb3 -oxidase
[52,53]. While ccoG/rdxB is not essential for the synthesis of active cyt cbb3 -oxidase, its absence does appear
to have a slight but measurable effect on cyt cbb3 -oxidase activity raising the possibility that the Rdx-dependent aerobic derepression of photosystem gene
expression is due to an affect on cyt cbb3 -oxidase assembly. The recent demonstration that the cco and rdx
operons are transcriptionally linked is consistent with
their proposed role in cbb3 -oxidase assembly [54].
2.7. Possible involvement of PrrC/SenC in the Reg/Prr
system signal
The final candidate suspected as an intermediary in
the cyt cbb3 -oxidase to RegB/PrrB signal transduction
pathway is the product of the senC gene of R. capsulatus, otherwise known as the product of prrC in R. sphaeroides. This protein is an attractive candidate since the
gene encoding it is located immediately upstream of
regA/prrA in the reg/prr operons that have been sequenced to date and is cotranscribed with regA/prrA in
both R. capsulatus and R. sphaeroides [11,27,28,32].
SenC/PrrC shows significant sequence identity with
SCO1 and SCO2 in yeast. While the function of SCO2 is
unknown SCO1 has been shown to be necessary for the
assembly of the CuA center of the terminal cytochrome c
oxidase in Saccharomyces cerevisiae mitochondria
[55,56]. SenC/PrrC contains a putative N-terminal
transmembrane domain and localization studies using a
PrrC::PhoA fusion indicate that PrrC is membrane
bound [32]. Evidence indicates that SenC/PrrC may be a
redox active protein. Both SenC and PrrC contain a
conserved –CXXXCP– metal-binding motif and PrrC of
R. sphaeroides has been demonstrated to bind Cu
[27,32,57]. It has been shown that the four cysteines of
PrrC form two intramolecular disulfide bonds under
reducing conditions and that Cu binding only occurs in
this reduced state, suggesting that PrrC may have a
thiol-disulfide oxidoreductase activity in addition to
binding copper [57]. Results of mutagenesis studies of
senC/prrC in R. capsulatus and R. sphaeroides are
sometimes contradictory and difficult to interpret since
senC/prrC and regA/prrA are cotranscribed. Initial insertional inactivation studies of the senC/prrC genes in
both organisms resulted in similar phenotypes, characterized by reduced photosynthetic gene expression during dark semiaerobic growth as well as during anaerobic
growth either photoheterotrophically for R. capsulatus
[27] or in the dark in the presence of DMSO for
R. sphaeroides [11]. In R. sphaeroides, the prrC
phenotype was substantially, but not fully, relieved by
complementation with plasmid born regA while complementation with prrC had no affect, indicating that the
prrC mutation was polar with respect to the expression
of regA [11]. Similar results were obtained using a
slightly different approach in a R. capsulatus senC insertion mutant. Complementation of insertion mutants
in either senC or regA was performed with plasmids
containing regA combined with either an inactivated
senC carrying an X-insertion or an inactive senC carrying a small N-terminal deletion resulting in a nonsense
mutation [27]. A plasmid containing regA and an insertionally inactivated senC was unable to complement
either the senC or regA mutant strains with respect to
photosynthesis gene expression indicating that the senC
insertion was polar with respect to regA expression [27].
However, a plasmid containing regA and the senC
nonsense mutation was able to complement a regA
mutant, but not a senC insertion mutant, suggesting that
the senC mutant phenotype was due to the mutation in
senC and not disruption of regA expression [27]. In both
cases the results were taken as an indication that SenC/
PrrC is directly involved in the regulation of photosynthesis gene expression [11,27]. A more recent study of R.
sphaeroides prrC indicates that an in-frame deletion
mutant of prrC, that still expresses prrA from a promoter within the prrC N-terminal region, results in aberrant aerobic expression of photosynthesis genes, as
well as the product of the Reg/Prr-regulated dorA gene
encoding DMSO reductase [32,45]. Complementation
with plasmid borne prrC returned aerobic expression of
the photosystem to normal levels [32]. Once again the
results were taken to indicate that SenC/PrrC was involved in the transduction of an inhibitory signal from
cyt cbb3 -oxidase to the Reg/Prr system [32]. As with all
the other signal transduction candidates, SenC/PrrC
also may play a role in maintaining cyt cbb3 -oxidase
activity under aerobic conditions in R. capsulatus since
assays of membranes from aerobically grown cultures of
a senC insertion mutant revealed cyt cbb3 -oxidase levels
that were 3.4-fold lower than those of the wild-type
strain [27]. In R. sphaeroides, a similar affect of a prrC
mutation on aerobic cyt oxidase activity has been hinted
at, however, since R. sphaeroides expresses two cytochrome oxidases, a cbb3 -type and an aa3 -type, under
aerobic conditions, the measurement of cbb3 -oxidase
activity is difficult [32]. It is possible that the effect of
senC/prrC on photosynthesis gene expression is indirectly due to a reduction in cyt cbb3 -oxidase activity.
The goal of unambiguous identification of signal transduction components leading to RegB from cyt cbb3 oxidase remains elusive. Given the data to this point it
seems reasonable to expect that any signal transduction
components having close association to cbb3 -oxidase in
the membrane might also have a structural function. If
this is true, the participation of RdxB, CcoQ and PrrC
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
in signal transduction cannot be completely ruled out.
However, individual amino acid changes generated either randomly or through site directed means will
probably be necessary in order to identify regions
within a given protein that are involved in the signaling
function.
2.8. Reg/Prr regulation of CBB cycle gene expression
In the nonsulfur purple bacteria, the CBB cycle is the
major pathway involved in the assimilation of CO2
during the phototrophic and chemoautotrophic growth
modes. In recent years the majority of studies examining
the regulation of CBB gene expression in the nonsulfur
purple bacteria have focused on two related organisms,
R. capsulatus and R. sphaeroides. Both organisms differentially regulate the expression of two distinct forms
of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) denoted form I (L8 S8 ) ðcbbLI cbbSI Þ
and form II RubisCO ðcbbMII Þ. In each case, the
structural genes for the two forms of RubisCO are organized within two operons (cbbI and cbbII Þ (Fig. 1)
along with genes that encode other CBB pathway enzymes [58,59]. Despite these similarities there are differences between the two organisms with respect to both
gene organization and regulation summarized below.
Rhodopseudomonas palustris, whose genome was recently sequenced (http://genome.jgi-psf.org/finishedmicrobes/rhopa/rhopa.home.html), also contains two CBB
operons. Of note are three interesting open reading
frames juxtaposed between the cbbR and cbbLS genes of
R. palustris (Fig. 1). These genes encode (50 from cbbL
and 30 from cbbR, in order) two putative response
regulators (RRl and RR2) and a large protein (Pas) that
contains several interesting motifs including 2 PAS domains, one PAC domain, and both phospho-receiver
and phosphor-donor domains. Recent studies indicate
that the Pas protein regulates autotrophic CO2 -depen-
Fig. 1. The two major cbb operons from R. sphaeroides, R. capsulatus
and R. palustris. The divergently transcribed cbbR gene encodes for an
LTTR protein that specifically binds to cbb promoters and activates
transcription under certain growth conditions.
359
dent growth and the transcription of both the cbbI and
cbbII operons of R. palustris (C.-S. Oh and F.R. Tabita,
manuscript in preparation). This unique gene arrangement and unprecedented involvement of the large Pas
protein has sparked additional studies in our laboratory
of its involvement, along with RR1 and RR2, in
mediating CO2 -dependent growth as well as CbbRdependent control.
The regulation of cbb gene expression in R. sphaeroides is quite complex [60]. Expression of the genes in
both the cbbI and cbbII operons is highly induced during
anaerobic photoautotrophic growth and moderately
induced during aerobic chemoautotrophic growth [61].
During growth under CO2 fixing conditions, expression
of each operon is modulated independently in response
to a number of environmental parameters such as the
level of CO2 and the reduction state of organic carbon
compounds supplied for growth [62–66]. This independent regulation results in shifts in the relative abundance
of proteins encoded within each operon. In general,
growth under photoheterotrophic conditions, with a
fixed (organic) carbon source, results in about a 2-fold
excess of cbbII expression over cbbI . Interestingly, during
photoheterotrophic growth CO2 fixation functions to
maintain cellular redox balance by acting as an electron
sink. Photoheterotrophic growth can occur in the absence of the CBB cycle if an alternate electron acceptor,
such as DMSO, is supplied [62]. Maximal expression
from both operons is observed under photoautotrophic
and chemoautotrophic conditions; i.e., when CO2 is
used as the sole carbon source, with the expression of
the cbbI operon exceeding that for the cbbII operon by
about 2-fold [66]. A mechanism for interdependent cbb
regulation is also present that results in a compensatory
increase in the expression of one operon when the other
is inactivated [58,62,64,65]. This compensatory response is under the control of the product of the cbbR
gene that is located immediately upstream and divergently transcribed from cbbFI [67]. CbbR is a LysR-type
transcriptional regulator (LTTR) and positively regulates the expression of both the cbbI and cbbII operons
[67,68].
Reg/Prr system involvement in the regulation of CBB
cycle gene expression was first demonstrated in R. sphaeroides when it was shown that a regB insertion mutant
not only displays reduced cbbI and cbbII promoter activity
during phototrophic and chemoautotrophic growth, but
is also unable to induce high level RubisCO expression
when incubated under anaerobic conditions in a carbon
free medium [69]. An R. sphaeroides regA insertion mutant is incapable of phototrophic growth, presumably due
to inadequate expression of photosystem genes, a fact
that has complicated efforts to determine the effect of
RegA on cbb expression [70,71]. However, it has been
demonstrated that RegA is necessary for the induction of
both form I and form II RubisCO during incubation
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under phototrophic conditions [71]. DNase I footprinting
studies, using the constitutively active mutant RegA*
from R. capsulatus, have shown that RegA binds to
multiple sites within both the cbbI and cbbII promoter
operator regions. Five RegA-binding sites have been
identified within the cbbI promoter centered at approximately: )1 bp (site 1), )70 bp (site 2), )100 bp (site 3),
)316 bp (site 4) and )407 bp (site 5) upstream of the cbbI
transcription start [[71], J.M. Dubbs and F.R. Tabita,
unpublished results] (Fig. 2). Sites 1 and 2 bracket, and
partially overlap, a CbbR-binding site [71]. While the
position of RegA sites 1 and 2 suggests a possible physical
interaction between RegA and CbbR, it seems equally
possible that they may function to regulate cbbR expression. Promoter deletion analyses indicate that RegA sites
2, 3, 4 and 5 do play a role in cbbI activation. Site 4, which
binds RegA at a higher affinity than the other sites, contributes the most to cbb activation during photosynthetic
growth [68,71]. Analysis of the expression of cbb1 :: lacZ
promoter fusions in an R. sphaeroides regA insertion
mutant showed that chemoautotrophic expression of cbbI
is regA independent and that different regions of the
promoter are important for chemoautotrophic cbbI expression versus phototrophic expression [43]. RegA binds
the R.sphaeroides cbbII promoter-operator at six site
centered at approximately; )294 bp (site 1), )592 bp (site
2), )739 bp (site 3), )773 bp (site 4), )842 bp (site 5) and
)870 bp (site 6) [48] (Fig. 2), A region of the cbbII promoter containing RegA site 1 contributed the majority of
the cbbII activation observed during photoheterotrophic,
photoautotrophic and chemoautotrophic growth. RegA
site 1 of the cbbII promoter is found in approximately the
same position and is similar in sequence to RegA site 3 of
the cbbI promoter, suggesting that both of these sites
function similarly during cbb activation. The region
containing sites 4, 5, 6 and 7 has a positive effect on cbbII
expression during photoautotrophic and chemoheterotrophic growth and a negative affect on chemoautotrophic cbbII expression. Surprisingly, it was found that,
unlike cbbI , chemoautotrophic expression of cbbII is
RegA-dependent. The reason for the retention of RegAmediated regulation during chemoautotrophic growth
may be related to the fact that form II RubisCO and the
enzymes encoded by the cbbII operon allow the CBB
pathway to play the somewhat specialized role of terminal electron acceptor, thus giving the organism an enhanced ability to regulate redox poise under this growth
condition.
As previously alluded to, the regulation of the cbbI
and cbbII operons in R. capsulatus is somewhat distinct
from that of R. sphaeroides. The cbbI operon is only
expressed during photoautotrophic growth at low CO2
concentrations (i.e., 1.5% CO2 ), while the cbbII operon is
expressed during phototrophic and chemoautotrophic
growth with maximal expression occurring during photoautotrophic growth where it is the predominant form
of the enzyme [72–74]. In contrast to R. sphaeroides,
each of the R. capsulatus cbb operons contains a divergently transcribed cbbR gene [72]. CbbRI and CbbRII of
R. capsulatus have been shown to preferentially regulate
their cognate cbb operons [73,75]. A regA insertion
mutant of R. capsulatus differs from the R. sphaeroides
prrA strain in that it is able to grow under both
phototrophic and chemoautotrophic conditions. However, photoautotrophic growth occurs at a markedly
reduced rate due to the inability of the mutant to induce
cbbI and cbbII transcription. Inactivation of regB results
in a similar phenotype, however, the affect on cbb gene
expression was less severe [75]. Footprinting studies
using R. capsulatus RegA* indicate that RegA binds to
the cbbI promoter at a minimum of two sites centered at
approximately )11 bp (site 1) and )110 bp (site 2)
(Fig. 2). Site 1 of the R. capsulatus cbbI promoter is
similar to site 1 of the R. sphaeroides cbbI promoter in
that it overlaps part of the CbbR-binding site as well as
the )10 region and the cbbI transcription start. The R.
capsulatus cbbII promoter contains at least two RegAbinding sites centered at approximately )108 bp (site 1)
and )146 bp (site 2) [75].
2.9. Reg/Prr regulation of photosystem gene expression
Fig. 2. Locations of the mapped RegA/PrrA-binding sites in R. capsulatus and R. sphaeroides. R. sphaeroides promoters are within the
dashed box. The binding sites for other known regulators that have
either been mapped or inferred from the presence of conserved binding
motifs are also indicated. Negatively regulated promoters are shown in
the inset. References are listed in the text.
In R. capsulatus and R. sphaeroides, structural genes
that encode components for the photosynthetic apparatus are organized into three operons denoted puf, puc
and puh. The puf and puh operons encode structural
proteins of the photosynthetic reaction center and the
light harvesting I complex (LHI), while the puc operon
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
encodes the structural proteins of light harvesting II
complex (LHII) (for a review see [76]). The regulation of
photosystem gene expression in both organisms is extremely complex. In addition to the Reg/Prr system, the
R. capsulatus puf, puc and puh operons have been shown
to be under the control of one or more of the following:
the aerobic repressors AerR and CrtJ [77,78], the low
light activator HvrA [28] and Integration Host Factor
(IHF) [79]. In R. sphaeroides, the list is equally long and
includes the CrtJ homolog PpsR [80], the transcriptional
activator PpaA, an AerR homolog [81], the anaerobic
regulator FnrL [82] and the high light repressor Spb, an
HvrA homolog [83].
In R. sphaeroides and R. capsulatus, the Reg/Prr
system has been shown to positively regulate the puf, puc
and puh operons in response to anaerobiosis [7–9,11]. In
R. sphaeroides, inactivation of prrB results in a strain
that is unable to grow photoautotrophically [69] and has
a high light requirement for photoheterotrophic growth
[11], while regA/prrA mutants are unable to grow phototrophically under any light intensity [9]. Both the prrA
and prrB mutants showed reduced levels of RC and LH
complexes along with reduced levels of puf, puc and puh
transcripts during anaerobic growth, although the effect
of the prrA mutation was more severe [9,11]. The Reg/
Prr system was also implicated in the regulation of carotenoid and bacteriochlorophyll synthesis when it was
observed that both of these accumulated under aerobic
conditions in a constitutive prrB mutant (prrB78)
background [11].
In R. capsulatus, mutants in regA and regB showed
similar phenotypes and were unable to grow photoheterotrophically at low light intensities, grew at reduced
rates under intermediate light intensities and grew at
nearly wild-type rates under high intensity illumination
[7,8]. Unlike R. sphaeroides, R. capsulatus reg mutants
were capable of photoautotrophic growth [75]. Both
mutants showed reduced levels of RC, LH I and LHII
complexes during anaerobic growth [7,8]. Promoter fusion studies have shown that a R. capsulatus regA mutant is unable to anaerobically induce puf or puh operon
expression, while puc operon expression was severely
reduced in response to anaerobiosis [7]. regB mutants
retained some ability to regulate puf, puc and puh expression, but at drastically reduced levels [8]. Footprinting studies in R. capsulatus have shown that RegA
binds to the puc promoter strongly, at a site centered at
approximately )60 bp that was demonstrated to be involved in transcriptional activation in vitro [24,84]. A
weak binding site, centered at approximately )76 bp,
was also detected, however, it was not necessary for
activation in vitro [24,84]. The puc operon has also been
shown to be under the control of other transcriptional
regulators. R. sphaeroides PpsR and its homolog CrtJ in
R. capsulatus repress transcription of the puc operon, as
well as several operons involved in bacteriochlorophyll
361
and carotenoid biosynthesis, under aerobic conditions
[78,80,85–88]. In the presence of oxygen, both PpsR and
CrtJ form an intramolecular disulfide bond that enables
the protein to bind to its DNA target sequence [89]. In
R. sphaeroides, the action of PpsR is antagonized by the
flavin-binding antirepressor AppA [90]. In vitro, AppA
has been shown to break the intramolecular disulfide
bond in oxidized PpsR and form a stable AppA–PpsR2
complex [91]. Excitation of the bound flavin by blue
light renders AppA incapable of forming the complex,
effectively resulting in blue light repression of target gene
transcription observed during semiaerobic growth in R.
sphaeroides [91,92]. CrtJ binds to the puc promoter at
two widely spaced sites centered at )49 and )301 bp
[87]. The proximal CrtJ-binding site ()49 bp) overlaps
the puc promoter consensus )35 sequence as well as the
strong RegA-binding site where it has been shown to
compete for binding with RegA [84]. A second recently
discovered repressor, AerR, is also involved in aerobic
repression of the R. capsulatus puc promoter in concert
with CrtJ [77]. Genetic evidence indicates that the nonspecific transcriptional regulator IHF functions to enhance the rate of puc mRNA accumulation in response
to anaerobiosis in both R. sphaeroides and R. capsulatus
and DNaseI footprint and gel mobility shift analyses
show that IHF binds to the puc promoter at multiples
sites in both organisms [79,93,94]. Genetic evidence in
R. sphaeroides suggests that FnrL, a homolog of the
anaerobic activator FNR of Escherichia coli [82], is involved in puc expression since a FnrL mutant was unable to fully induce puc transcription upon a shift from
high (30%) to low (2%) oxygen. A region of the puc
promoter that was found to be responsible for a portion
of the FnrL-dependent activation contained overlapping
consensus binding sequences for IHF and FNR. To
date, less is known concerning the regulation of the puf
and puh promoters. Both are under the positive control
of the Reg/Prr system in R. sphaeroides and R.capsulatus
in response to anaerobiosis [8,9], In R. capsulatus, AerR
has been shown to repress the puf operon under aerobic
conditions [77], while both the puf and puh operons are
subject to low-light activation by the product of hvrA
[28], which has been reported to bind the puh promoter
at approximately 90–100 bp upstream of the transcription start [76]. In R. sphaeroides, the R. capsulatus HvrA
homolog Spb is encoded in a similar position in the
regB–senC–regA–hvrA gene cluster. Interestingly, spb
encodes a high-light repressor of puf and puh expression
in R. sphaeroides. In vitro footprinting studies have
shown RegA to bind to the R. capsulatus puf promoter
at two sites centered at approximately )37 and )74 bp
[24,84]. No published data for RegA binding to the
puh operon is available. The puf promoter also contains a single IHF-binding site centered at )57 bp
that partially overlaps the proximal RegA-binding site
[79].
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2.10. Reg/Prr regulation of electron transport components
Indications of Reg/Prr involvement in the regulation
of electron transport components came with the demonstration in R. sphaeroides that PrrA was a positive
effector of cytochrome c2 (cycA) transcription during
both aerobic and anaerobic dark growth in the presence
of DMSO [9]. The direct involvement of PrrA was
confirmed when it was shown that RegA* bound to a
region centered at approximately )50 bp relative to the
cycA P2 transcription start where it activated transcription in vitro and in vivo [95,96]. Promoter deletion
analysis indicated that the PrrA/RegA-binding site was
also functional during anaerobic phototrophic growth
and suggested that upstream promoter sequences may
be targets for additional regulators [96].
Analyses of promoter-lacZ fusions in a R. capsulatus
regA background showed RegA to be involved in the
regulation of expression of operons encoding components of the cyt bc1 complex (petABC), cbb3 cytochrome
oxidase (ccoNOQP), quinol oxidase (cydAB), cytochrome cy (cycY) and cytochrome c2 (cycA) [97].
Moreover, RegA was found to have both positive and
negative affects on the expression of some of these
promoters that varied according to growth conditions.
Assays of promoter activity under anaerobic phototrophic, semi-aerobic (slow shaking) and aerobic (fast
shaking) growth conditions showed that, to a greater or
lesser degree, RegA positively regulated petABC, cycA
and cydAB expression under all growth conditions.
RegA was found to be a positive effector of cycY under
semi-aerobic conditions but had only slight to no positive effect during anaerobic phototrophic and aerobic
growth, respectively. Both cbb3 and quinol oxidase expression were highest under semi-aerobic conditions and
were RegA-dependent. RegA had a large positive affect
on the aerobic and semi-aerobic expression of both the
cbb3 and quinol oxidase and a large negative affect on
cbb3 oxidase expression during anaerobic phototrophic
growth [97]. This suggested that unphosphorylated
RegA could actually function as an activator under
aerobic and semi-aerobic conditions and phosphorylated RegA could act as a negative regulator during
anaerobic phototrophic growth conditions. Analysis of
cbb3 promoter expression in a regB mutant seems to
support this idea since inactivation of regB returned
aerobic and semi-aerobic expression levels to those of
wild-type. As would be predicted, anaerobic expression
of cbb3 oxidase was increased in the regB mutant,
however, the expression level was 2-fold higher than that
of the wild-type [98]. This discrepancy was explained
when it was shown that FnrL positively regulate the
ccoNOQP operon under anaerobic photosynthetic and
semiaerobic conditions in both R. capsulatus and
R. sphaeroides, possibly acting at a consensus FNRbinding sequence that is centered approximately 100 bp
upstream of the ccoN transcription start in R. capsulatus
[98,99] and 73.5 and 41.5 bp upstream of the FnrL-dependent P2N transcription start in the R. sphaeroides cco
promoter [37]. In an interesting twist, FnrL was shown
to negatively regulate the R. capsulatus cydAB operon
under semi-aerobic conditions leading to the speculation
that in R. capsulatus, cbb3 oxidase has a higher affinity
for O2 than quinol oxidase and is therefore, preferentially expressed under low O2 conditions [98].
2.11. Reg/Prr regulation of nitrogen fixation genes
A detailed description of the current state of knowledge concerning the regulation of nitrogen fixation in
nonsulfur purple bacteria is outlined in a number of
recent reviews [100,101] and will not be dealt with here.
However, a brief description of the general mechanism
of nif regulation in the nonsulfur purple bacteria is
necessary. In R. capsulatus, expression of the nifHDK
operon is primarily regulated in response to the availability of a fixed nitrogen source such as ammonia and
the oxygen concentration [100,101]. Nitrogen control is
mediated through a regulatory cascade involving GlnB,
NtrB, NtrC and NifA. Expression of the nitrogenase
structural genes nifHDK is under the direct control of
the transcriptional activator NifA that is in turn under
the positive control of phosphorylated NtrC. The levels
of NtrCP are controlled through the interaction of
GlnB with the protein kinase NtrB. Briefly, during
growth in the presence of fixed nitrogen, GlnB interacts
with the NtrB kinase in a manner that inhibits NtrBdependent phosphorylation of NtrC. Consequently, the
synthesis of NifA and ultimately nifHDK is not induced.
During growth in the absence of fixed nitrogen, uridylation of GlnB disrupts its interaction with NtrB, allowing it to phosphorylate NtrC. NtrCP-dependent
expression of NifA leads to the expression of nifHDK.
The mechanism of oxygen control or nitrogenase expression is at least in part due to an inherent sensitivity
of NifA to oxygen [100,102].
Involvement of the Reg/Prr system in the regulation
of nitrogen fixation was first observed in mutants of
CBB cycle-deficient strains of R. sphaeroides (strain
16PHC) and Rhodospirillum rubrum (I-19) that derepress
nitrogenase synthesis in the presence of ammonia and
use the nitrogenase enzyme complex as an alternative
method of dissipating excess reducing power during
photoheterotrophic growth [103]. Inactivation of regB
in R. sphaeroides strain 16PHC reversed the derepression of nitrogenase expression. Moreover, inactivation
of regB in wild-type R. sphaeroides results in an inability
to grow at the expense of N2 , confirming that a functional Reg/Prr system is necessary for normal regulation
of nif [103]. Expression studies using a R. sphaeroides
nifHDK promoter fusion to lacZ confirmed that the
Reg/Prr system functions as a positive regulator of nif-
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
HDK expression in addition to the nif-specific regulator
NifA [70]. The positive regulation of nitrogenase expression in R. sphaeroides was found to be a direct result
of RegA/PrrA binding to the nifHDK promoter at two
sites. The first site, centered at position +12 relative to
the nifHDK transcription start, overlaps it, while the
second site, centered at position )28 partially overlaps
the 50 side of a r54 -consensus promoter sequence (J.M.
Dubbs and F.R. Tabita, unpublished results). Regulation of nif expression in R. capsulatus is also under Reg/
Prr control, however, the mechanism of regulation is
different from that in R. sphaeroides. RegA/PrrA does
not bind to the nifHDK promoter but rather exerts its
positive affect on nif expression indirectly through the
activation of expression of one of the two copies of the
nif-specific positive activator, nifA2 [47]. RegA-dependent positive regulation occurred both in the presence
and absence of O2 and was the result of RegA binding to
a single site within the nifA2 promoter at a site midway
between the transcription start and two upstream NtrCbinding sites [47,104]. It is not known if RegA mediates
regulation of nifA in R. sphaeroides.
2.12. Reg/Prr regulation of other pathways
The RegA/PrrA system is known to be involved in the
regulation of a number of other cellular processes that
illustrate the truly global nature of this regulatory system. In addition to those already discussed, the Reg/Prr
system contributes to the regulation of hydrogenase
synthesis [47], DMSO and nitrate reduction [45,105],
formaldehyde assimilation [106] and chemotaxis [107].
In R. capsulatus, expression of [Ni/Fe] hydrogenase,
encoded by the hupSLC operon, is positively regulated
in the presence of hydrogen through the concerted action of the hydrogen responsive HupT/HupR twocomponent regulatory system and IHF [108,109]. The
Reg/Prr system negatively regulates hupSLC expression
directly through the binding of RegA to the hupSLC
promoter at two sites centered at )39 and )88 bp, relative to the hupS transcription start [47]. RegA-dependent negative regulation occurred under both aerobic
and anaerobic photoheterotrophic conditions [47]. The
upstream RegA-binding site overlaps the binding site for
IHF [109] and it is thought that RegA-mediated negative regulation results from competition for binding
between RegA and IHF [47].
Rhodobacter capsulatus and R. sphaeroides have the
ability to grow anaerobically both in the dark and
photoheterotrophically using DMSO as a terminal
electron acceptor [4]. DMSO reductase, the enzyme responsible for the reduction of DMSO, is encoded by the
dorBCD operon. DMSO-dependent induction of this
operon is under the control of a two-component regulatory system composed of the sensor kinase DorS and
the response regulator DorR [44,110]. The dorBCD
363
operon is expressed at very low levels under aerobic
conditions and this aerobic versus anaerobic control is
indirectly due to the regulation of DorS by FNR
[111,112]. Analysis of the anaerobic photoheterotrophic
expression of the dorBCD operon in R. capsulatus has
recently shown that RegA/PrrA negatively regulates
dorBCD under this condition [45]. It is known that inactivation of cyt cbb3 -oxidase in both R. capsulatus and
R. sphaeroides results in increases in the expression of
dorBCD in the presence and absence of DMSO suggesting that RegA/PrrA plays a similar regulatory role
in both organisms [44].
The Reg/Prr system also regulates an additional
electron-accepting pathway in R. sphaeroides involving
respiration via the reduction of nitrite to nitric oxide
(NO) by the enzyme nitrite reductase [105]. The expression of the nirKV operon that encodes nitrite reductase (nirK), and a protein of unknown function
(nirV), is expressed under low oxygen conditions in the
presence of nitrate and is under the control the NO
sensing positive regulator NnrR [113,114]. The Reg/Prr
system was found to positively regulate nirK under microaerobic conditions in the presence and absence of
nitrate [105]. Interestingly, inactivation of cyt cbb3 -oxidase resulted in an increase in nirK expression under
microaerobic conditions. It is not known whether the
increase in the microaerobic expression of nirK in the cyt
cbb3 -oxidase mutant is mediated by the Reg/Prr system
[105]. The Reg/Prr system also positively regulates the
ppaZ gene under low O2 concentrations. The ppaZ gene,
encoding a pseudoazurin, is located immediately
downstream of and divergently transcribed from nirKV
[115]. Expression of ppaZ occurs only under low O2
concentrations and is positively regulated by the Reg/Prr
system in concert with NnrR and FnrL [115].
The Reg/Prr system also links the redox state of the
cell with the aerotactic response in R. sphaeroides.
Inactivation of regB/prrB diminished the ability of
R. sphaeroides to move away from atmospheric concentrations of oxygen [107]. The exact mechanism by
which the Reg/Prr system affects aerotaxis is not known,
however, the Reg/Prr system has been shown to negatively regulate the expression of the cheOP2 promoter
through the binding of RegA/PrrA [107].
Rhodobacter sphaeroides has the ability to derive energy in the form of NADH through the oxidation of
formaldehyde using a glutathione-dependent formaldehyde dehydrogenase, encoded by adhI [116]. RegA/PrrA
is a positive effector of adhI promoter expression during
aerobic growth. However, it has not been determined if
this positive affect was due to the direct action of RegA/
PrrA. A RegA/PrrA-independent activation of the adhI
promoter during growth in the presence of formaldehyde, or methanol suggested the presence of an
additional formaldehyde-specific regulatory system
[106].
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2.13. RegA homologs in other bacteria
The two-component regulatory systems RegSR of
B. japonicum and ActSR of Sinorhizobium meliloti have
been shown to be quite similar to the Reg/Prr regulatory
system in nonsulfur purple bacteria [26,117,118]. Both
RegSR and ActSR share similarities with the Reg/Prr
system with respect to the pathways they regulate.
RegSR positively regulates the expression of the fixR–
ifA operon in B. japonicum under both aerobic and anaerobic conditions through the binding of RegR to an
UAS within the fixR promoter [119]. A regR mutant
showed an impaired ability to grow in the free-living
state anaerobically and is unable to fix nitrogen symbiotically [119]. ActSR appears to play a global regulatory
role in S. meliloti where it is involved in the regulation of
expression of proteins involved in CO2 fixation as well
as formate dehydrogenase, formaldehyde dehydrogenase and methanol dehydrogenase [120]. ActRS also
confers acid tolerance in R. meliloti [121]. Functional
similarity between RegSR, ActSR and the Reg/Prr system is evident since the soluble carboxy-terminal portion
of RegS was able to phosphorylate both RegR and
R. capsulatus RegA/PrrA in vitro [117]. Both regA/prrA
and actR were also able to complement a B. japonicum
regR mutant with respect to fixR–nifA expression and
nitrogen fixation [117]. Furthermore, RegR restored
wild-type level expression of a puc promoter-lacZ fusion
in an R. capsulatus regA mutant [117].
In Pseudomonas aeruginosa, an analog of the Reg/Prr
system composed of the response regulator RoxR and
the sensor kinase RoxS positively regulates the expression of a cyanide insensitive quinol oxidase (CIO) in
response to exposure to cyanide through the binding of
RoxR to the cioAB promoter [19]. The deduced amino
acid sequences of RoxR and R. sphaeroides PrrA are
similar (50.7% identity) and either gene was able to
complement a R. sphaeroides prrA or P. aeruginosa roxR
mutant [19]. The sequence identity between RoxS and
PrrB was not as great (22.6%) and was localized primarily within the carboxy terminal kinase domains of
the two proteins [19]. Functional similarity was further
demonstrated by the fact that PrrB was able to phosphorylate RoxR in vitro and phosphorylated RoxR was
also able to activate the R. sphaeroides cycA P2 promoter in vitro [19]. However, roxS was unable to complement a R. sphaeroides prrB mutant, suggesting
differences in the signal transduction chains of the two
systems [19].
2.14. Reg/Prr-regulated promoters
Identification of a consensus Reg/Prr-binding sequence based on sequence comparisons of RegA/PrrAbinding sites was initially hampered by the fact that the
genomes of the organisms in which binding sites have
been mapped are extremely GC-rich. This problem was
overcome by Emmerich et al. [118] who identified the
consensus core-binding sequence, 50 -GNGRCRTTNNGNCGC-30 , for the RegA/PrrA homolog RegR of
B. japonicum using in vitro binding site selection coupled
with site directed mutagenesis of a known RegR-binding
site. Using the RegR consensus sequence as a reference,
similar sequences have been found in mapped RegA/
PrrA-binding sites in R. capsulatus and R. sphaeraides,
and a similar core-binding site for R. capsulatus RegA of
50 -G C/T G G/C G/C G/A NN T/A T/A NNC G/A C-30
has been proposed [97,118]. The number and placement
of RegA/PrrA-binding sites within Reg/Prr-regulated
promoters varies greatly (Fig. 2). Promoters examined
so far contain anywhere from 1 to 6 sites that can be
located from the transcription start to nearly 900 bp
upstream. However, in most promoters RegA/PrrAbinding sites are found within 100 bp of the transcription start. This variability in binding site position,
combined with the fact that RegA/PrrA often functions
in concert with other regulators, indicates that RegA/
PrrA-mediated regulation probably occurs through
more than one mechanism.
Almost nothing is known about RegA/PrrA interactions with RNA polymerase. It has been inferred, based
upon the binding site position, that Reg/Prr system autoregulation occurs when bound RegA blocks RNA
polymerase access to the divergent senC and regB promoters [31]. Information concerning interactions during
gene activation is scant. Current evidence indicates that
RegA/PrrA regulates both r54 and r70 responsive promoters and is able to activate transcription in vitro in
the presence of either E. coli or Rhodobacter r70 -RNA
polymerases [[84,96,119], J.M. Dubbs and F.R. Tabita,
unpublished results]. In vitros experiments using a defined transcription system suggest that RegA/PrrA activation of the R. capsulatus puf and puc promoters
involve multiple modes of interaction with RNA
polymerase [84]. Both promoters displayed RegAPdependent activation in the presence of R. capsulatus
r70 -RNA polymerase. However, the RegAP-binding
site in the puf promoter, centered at )37, overlaps a
region that has no similarity to the E. coli r70 -RNA
polymerase consensus )35 sequence, while in the puc
promoter RegAP binds a region centered at )60, 14 bp
upstream of a good E. coli r70 -RNA polymerase consensus )35 element [84]. This difference in binding site
position has lead to the suggestion that, in the case of
the puf promoter, RegAP interacts with the RNA
polymerase r70 subunit, while activation of puc may
result from interactions with the a-subunit of RNA
polymerase [84]. The majority of the Reg/Prr activated
promoters (see Fig. 2) appear to be similar to the puc
promoter in that they contain a binding site immediately
upstream of, but not overlapping, the )35 region. The
only promoter thus far examined that appears to be
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
similar to the R. capsulatus puf promoter is the R. sphaeroides nifH promoter in which one of its RegA/PrrAbinding sites overlaps the 50 portion of the consensus r54
promoter sequence (J.M. Dubbs and F.R. Tabita, unpublished results). The function of RegA binding at
other sites is even less clear. A number of RegA/PrrA
activated promoters contain binding sites that overlap
(R. capsulatus petA, cycA and cycY; R. sphaeroides cbbII ,
nifH) or are immediately adjacent to (R. capsulatus
cbbII ) the transcription start [71,75,97] suggesting that
RegA/PrrA may interact with RNA polymerase on the
30 side of the promoter. The R. sphaeroides cbbI , cbbII
and the R. capsulatus cycA promoters contain one or
more RegA/PrrA-binding sites occurring at distances
greater than 200 bp upstream of the transcription start
[48,71,97] indicating that RegA/PrrA can activate transcription from a distance. This is best illustrated in the
case of the R. sphaeroides cbbI and cbbII promoters
where two RegA/PrrA-binding sites, centered at )315
bp in cbbI and )295 bp in cbbII , are responsible for a 40and 15-fold enhancement of expression during photo-
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autotrophic growth, respectively [48,68,71]. As for the
cbbI promoter, the function of this region is helical
phase-dependent, suggesting that RegA/PrrA bound at
these sites may interact with the CBB cycle-specific activator CbbR, RNA polymerase and additional RegA
molecules bound near the transcription initiation site
through the formation of a DNA loop [71].
RegA/PrrA commonly functions in concert with
other regulatory proteins. The only instance where the
nature of the interaction between RegA/PrrA and
another regulator has been clearly demonstrated is
between RegA and the aerobic repressor CrtJ. The
RegA-binding site within the R. capsulatus puc promoter
overlaps one of the two CrtJ-binding sites and competition for binding between RegAP and CrtJ has been
demonstrated in vitro [84]. In the case of several other
promoters, interaction between RegA/PrrA and other
regulatory proteins has been inferred based on the
relative positions of mapped binding sites. In the
R. capsulatus hupSLC operon, one of the two RegA/
PrrA-binding sites overlaps that of the positive regulator
Fig. 3. Prevailing model for Reg/Prr gene regulation under phototrophic growth conditions. Asterisks indicate operons that also display positive Reg/
Prr-dependent regulation at reduced levels during aerobic growth; Question marks indicate operons that show nearly identical patterns of regulation
in the presence and absence of O2 .
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IHF and it is thought that RegAP negatively regulates
hupSLC by blocking IHF binding at this site [47]. A
possible interaction between RegA/PrrA and CbbR is
suggested by overlapping binding sites in the cbbI
promoters of both R. sphaeroides and R. capsulatus
[71,75].
2.15. Model for Reg/Prr-mediated gene regulation
A proposed model for Reg/Prr-mediated gene regulation that is based on previous models for Reg/Prr
function in R. sphaeroides and R. capsulatus [46,97,122] is
outlined in Fig. 3. The system is a redox sensor that responds to an as yet unidentified inhibitory signal that is
thought to be dependent on electron flow through the
cbb3 -type cytochrome oxidase. Electron flow through
cytochrome cbb3 oxidase generates an inhibitory signal
affecting RegB kinase activity. This signal may be conducted by the membrane-bound polypeptide SenC/PrrB.
Under conditions where the redox state of the cell is in
‘‘normal’’ balance, the Reg/Prr system is inactive and
phosphorylation of RegA/PrrA by RegB/PrrB does not
occur. Under conditions that generate excess reducing
potential, caused by either increases in the input of reductant into the system or a shortage of the acceptor
molecule, the signal is disrupted, leading to activation of
the RegB kinase and phosphorylation of RegA. RegAP
would then activate expression of pathways that consume reductant such as the CBB cycle (cbb genes), nitrogen fixation (nif genes) and increase electron transport
capacity (cydAB, petABC, cycA), while at the same time
down-regulating processes that produce reductant such
as hydrogenase (hupSLC). Pathways that determine the
aerotactic responses of the cell would also be affected to
position the cell in an environment that is optimal for the
particular growth mode [107]. The unnecessary expression of genes is avoided since RegAP almost always
acts in concert with one or more regulators responding to
a variety of other regulatory signals that can be either
global or specific in nature. For example, RegA-mediated activation of the CBB cycle or nitrogen fixation
genes requires positive, regulatory input from the cbbspecific activator CbbR, and the nif-specific activators
NtrC and NifA [67,100] while simultaneously subject to
the regulatory affect of HvrA [123–125].
2.16. Is the Reg/Prr system always required?
PrrA mutants of R. sphaeroides do not grow phototrophically, so determining the direct involvement of
Fig. 4. Proposed model of cbb gene regulation in R. sphaeroides during chemoautotrophic growth (from Gibson et al., 2002, with permission). The
cbb3 cytochrome (Cyt) oxidase transmits an inhibitory signal to PrrB resulting in a shift in the equilibrium towards unphosphorylated PrrA and
subsequent lack of activation of the cbbII promoter. Also shown is a signal derived from cbb3 cytochrome oxidase activity going to an alternate
transcriptional regulator that in turn activates the cbbI promoter. The model depicts transcriptional activation of both cbb operons by CbbR in
response to a signal that presumably reflects the carbon status of the cell.
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
the Prr system on cbb gene expression could not be
addressed under these growth conditions. However, this
organism can grow under dark aerobic chemoautotrophic conditions where CO2 is the sole carbon source and
under this growth mode cbb gene expression is enhanced
[74]. This provides a convenient system to assess the role
of the Reg/Prr system for CO2 -dependent growth because Reg/Prr mutant strains can grow under these
conditions. Thus, in a recent study, it was found that
RegA/PrrA may not be an obligatory part of the proposed pathway for redox signal transduction in a ccoP
prrA double mutant of R. sphaeroides [43]. The basic
finding was that expression of the cbbI promoter is either
unaffected, or in some cases, enhanced by a prrA. mutation, while expression from the cbbII promoter is severely reduced during chemoautotrophic growth. Thus,
in a ccoP prrA double mutant, the positive effect for the
ccoP mutation on cbbII expression was almost completely negated in the prrA background, as might be
expected by the prevailing model (Fig. 3), but exactly the
opposite effect was observed for the cbbI promoter [43].
There are several potential explanations for these these
results, and certainly the idea of differential regulation of
the two cbb operons [66] is reinforced by these observations (also considered in [48]). However, the lack of a
negative effect of the prrA mutation on cbbI transcription makes it difficult to reconcile with the proposed
pathway for redox signal transduction via PrrA (Fig. 3)
in the ccoP prrA double mutant. Thus, if PrrA is an
obligatory component in the signal transduction pathway leading from CcoP [41], then cbbI and cbbII promoter activity in the ccoP prrA double mutant would be
expected to be identical to that observed in the single
prrA mutant unless PrrA was acting to repress activity.
That the Reg/Prr system is not always linked to cbb3
cytochrome oxidize activity and these results suggest the
existence of additional transcriptional activator(s), also
implied from studies that indicate that different parts of
the cbbI promoter region are important during chemoautotrophic versus photoautotrophic growth. Taken
together, these results have led to the development of a
revised model for cbb gene regulation and signal transduction involving the Prr/Reg system, CbbR, and other
potential regulators independent of Reg/Prr (Fig. 4) [43].
3. HvrA and Spb
Accumulating evidence in R. capsulatus indicates that
the product of the hvrA gene is a global regulator linking
photosynthesis with nitrogen fixation and CO2 fixation.
As has been mentioned previously, hvrA is part of the
<regB-senC > regA > hvrA operon where it is cotranscribed with senC and regA. HvrA is a 102 amino acid
residue protein with a predicted molecular mass of 11.52
kDa [28]. It is positively charged and contains a slightly
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N-terminal helix–turn–helix DNA-binding motif [28].
HvrA is approximately 30% identical to the E. coli
global regulatory protein H-NS and has been shown to
partially complement an E. coli H-NS mutant as well as
bind to an E. coli bla promoter fragment containing an
H-NS-binding site [126]. The positional counterpart to
hvrA in R. sphaeroides, denoted spb, encodes a 105
amino acid protein of a predicted molecular mass of
11.5 kDa that is 53% identical to R. capsulatus HvrA
[83]. Like HvrA, Spb contains an amino terminal helix–
turn–helix DNA-binding domain and a predicted
leucine zipper motif that has been proposed to be involved in dimerization [83]. Spb belongs to the H-NS
family of regulatory proteins and like hvrA has been
shown to partially complement an E. coli H-NS mutant
[126].
3.1. HvrA regulation of photosystem gene expression
Initial investigation into the role of hvrA showed it to
be a low light activator of photosynthetic reaction center
and light harvesting complex I genes since an R. capsulatus hvrA mutant was unable to increase expression
of the puf and puh promoters in response to growth
under low light conditions [28]. No effect was seen on
the expression of either the LHII encoding puc operon
or the bacteriochlorophyll synthesis gene promoters,
bchC and bchH. Inactivation of hvrA also resulted in
reduced phototrophic growth rates under low and medium light intensities [28].
3.2. HvrA regulation of nitrogen fixation gene expression
HvrA involvement in the regulation of nitrogen fixation genes was initially discovered during a screen for
R. capsulatus transposon mutants that no longer exhibited ammonium control of nif expression [124].
During anaerobic photoheterotrophic growth in the
presence of ammonia, inactivation of hvrA resulted in
nifH promoter expression levels that were comparable to
the wild-type grown in the absence of ammonia. Growth
of the hvrA mutant in the absence of ammonia resulted
in a further 2-fold increase in nifH expression [124] indicating that some level of ammonia control of nifH
expression was retained. Oxygen control of nifH expression was completely abolished in the hvrA mutant
since, in the absence of ammonia, comparable abnormally high levels of nifH expression were detected under
both aerobic and microaerobic conditions [124]. Further
work, using strains containing lacZ fusions to promoters
of other nif genes grown phototropically in the presence
and absence of ammonia, showed that in addition to
nifH, HvrA negatively regulates the expression of nifB1,
but not nifA1 or nifA2 [125]. In the presence of ammonia, HvrA was also shown to negatively affect rpoN
expression to a relatively small degree [124]. Since all of
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the experiments were performed at saturating light intensities [124], it remains to be seen how alterations in
light intensity affect HvrA-mediated regulation of nif. In
addition, R. capsulatus contains the anf-encoded alternative nitrogenase system; what effect HvrA has on this
system remains to be determined.
3.3. HvrA regulation of electron transfer components
Expression studies using promoter fusions to lacZ
found that HvrA negatively affects the expression of the
ccoNOQRP operon during dark semi-aerobic growth
[98]. Conversely, HvrA is a positive effector of cydAB
expression under anaerobic phototrophic conditions
[98]. It has been proposed that HvrA works in concert
with RegA to insure high expression of the putative
higher affinity quinol oxidase under low oxygen concentrations, while maximizing expression of what is
thought to be the putative lower affinity cytochrome
cbb3 oxidase when O2 levels are higher [98].
3.4. HvrA regulation of CBB cycle gene expression
There is some evidence to indicate that HvrA may
function as a positive regulator of cbb gene expression in
R. capsulatus. An hvrA mutant showed an approximately 3-fold reduction in RubisCO activity during
photoautotrophic growth [123]. No effect on RubisCO
gene expression was seen during growth under either
photoheterotrophic or chemoautotrophic conditions.
The light intensity was not varied during these experiments; however, the fact that the growth rates of the
wild-type and hvrA mutants were similar indicates that
they were growing under light saturating conditions. It
is not known if the positive effect on RubisCO gene
expression is a direct or indirect effect of HvrA nor is
it known how alterations in light intensity affect cbb
expression in the hvrA mutant.
3.5. Spb regulation of photosynthesis gene expression
In spite of its similarities to HvrA, the regulatory role
played by Spb is very different. Spb has been shown to
be a light responsive repressor of both puf and puc
transcription in R. sphaeroides [29]. Inactivation of spb
results in significantly increased puf and puc transcription during phototrophic growth under low light with
more modest increases under high light, but had no effect on the oxygen-dependent regulation of puf and puc
transcription [29]. Regulation of the puf operon is due to
the direct binding of Spb to a site between 709 and 722
bp upstream of the pufB translation start. This region of
the puf promoter has been shown to be involved in oxygen-dependent regulation in both R. capsulatus and
R. sphaeroides [127,128]. The binding of Spb to this region of the puf promoter was detected during aerobic
dark growth and semiaerobic growth in the light, conditions under which the puf operon is expressed at low
levels [29,30]. No binding was detected during semiaerobic growth in the dark, a condition under which puf is
more highly expressed [29,30]. Binding of Spb in crude
extracts was enhanced by illumination with broadspectrum blue light (360–600 nm) and phosphatase
treatment [30]. The mechanism of Spb-mediated repression of puf and puc expression is not known. However, in the puf operon, the Sbp-binding site partially
overlaps the imperfect repeat sequence 50 -GCGGCGATCCGGCGC-30 that is a close match to the
consensus RegR-binding sequence 50 -GCGRCRTTNNGNCGC-30 (J.M. Dubbs and F.R. Tabita, personal
observations). Spb may compete for binding with RegAP or some other regulator at this site. In any case,
Sbp and HvrA have quite different functions with regard
to the regulation of puf and puc operons. The effect, if
any, of Spb on nif and cbb gene expression has yet to be
investigated.
It is interesting to note that an spb mutant retains the
ability to increase expression of the puf and puc operons
in response to low light during semi-aerobic growth [29],
suggesting the presence of an additional light responsive
activation pathway(s) in R. sphaeroides. Blast searches
of the R. sphaeroides genome using both the R. capsulatus HvrA and R. sphaeroides Spb protein sequences
reveals the presence of an additional HvrA homolog.
The putative HvrA-like protein (NCBI Accession No.
ZP 00007461) is 105 amino acids in length and is 50%
identical to HvrA and 67% identical to Spb.
3.6. Mechanism of HvrA and Spb function
What little is known about the regulatory effects of
HvrA seems to indicate that it too is involved in balancing energy supply and energy utilization in the cell. A
lowering of light intensity would result in a reduction in
the energy supply due to a decrease in the photosynthetic rate. The action of HvrA under these conditions
would increase the photosynthetic rate through the
stimulation of light harvesting antennae expression
while simultaneously repressing the energetically expensive process of N2 fixation [124,125]. This is seemingly at odds with the fact that HvrA is a positive
regulator of the energy expensive process of CO2 fixation during phototrophic growth [123]. The fact that the
effect on nif expression was measured in the presence of
a fixed carbon source (photoheterotrophic conditions)
while the effect on cbb expression was measured under
conditions where H2 served as an electron donor and
CO2 was the sole carbon source (photoautotrophic
conditions) may explain this.
The nature of the regulatory signal controlling HvrA
and Spb is unclear. HvrA has been reported to contain a
nucleotide-binding domain leading to speculation that it
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
might monitor light intensity directly through the use of
a bound chromophore such as flavin or sense the intracellular ATP/ADP ratio [129]. The observation that
HvrA relieves the O2 control of nif expression as well as
controlling the expression of both cco and cyd operons
in response to O2 in the dark indicates that HvrA may
not be sensing light per se. However, to date there is no
direct evidence in support of any of these hypotheses.
Based on amino acid sequence similarity it might be
expected that Spb and HvrA respond to similar signals.
The opposite regulatory effects mediated by the two with
regard to photosynthesis gene expression is not unique
and is a reflection of the very different strategies employed by R. capsulatus and R. sphaeroides for the regulation of photosystem genes. A similar difference in the
function of homologous regulatory proteins has already
been observed between R. capsulatus and R. sphaeroides.
In R. capsulatus, AerR is an aerobic repressor of photosynthesis gene expression while its homolog in
R. sphaeroides, PpaA, is an activator of photosynthesis
gene expression that is under the control of the aerobic
repressor PpsR [77,81].
4. CbbR-mediated regulation of cbb gene expression
As previously described, the CBB cycle performs two
different and essential functions, acting as both a supplier of fixed carbon during autotrophic growth and as
an electron sink involved in maintaining redox balance
during photoheterotrophic growth. These two separate
functions require that cbb expression be under the simultaneous control of regulatory systems responding to
the carbon status as well as the redox state of the cell.
Thus, an understanding of CBB cycle (cbb) gene regulation can serve as a model for understanding how
pathway-specific regulatory signals are integrated with
more global redox-responding regulatory systems like
Reg/Prr. In the purple nonsulfur bacteria R. sphaeroides
and R. capsulatus, CBB cycle-specific regulation of cbb
gene expression is under the control of the LysR-type
transcriptional (LTTR) activator CbbR [67,68,74,
75,130]. In R. sphaeroides, expression of both the cbbI
and cbbII operons requires the product of a single cbbR
gene that is located immediately upstream and divergently transcribed from the cbbI operon [67,68,130]. In
R. capsulatus, a divergently transcribed cbbR gene is
located immediately upstream of each of the two cbb
operons in this organism [72,73]. Each of these CbbRs,
designated CbbRI and CbbRII , has been shown to specifically regulate its cognate cbb operon [74,75]. A similar linkage of cbbR genes with CBB cycle operons has
been observed in a large number of other autotrophic
bacteria [131,132 and references therein]. The binding
sites of R. sphaeroides CbbR and R. capsulatus CbbRI
and CbbRII within the promoter operator regions of the
369
cbb operons they regulate have been mapped by DNaseI
nuclease protection [48,71,75]. All of the binding sites
occur within approximately 80 bp from the transcription
start and are characterized by a region of protection
bisected by one or more DNaseI hypersensitive sites that
are probably the result of CbbR induced DNA bending
[133].
One common characteristic of many LTTR regulators is that they generally activate transcription in response to an inducer molecule that is often an
intermediate of the biochemical pathway they regulate
[133]. Accumulating physiological and in vitro biochemical evidence indicates that this is true for CbbRmediated regulation of cbb gene expression in both
R. sphaeroides and R. capsulatus. Clear indications that
a CBB cycle intermediate(s) acts as an inducer of cbb
gene expression first came from observations in R.
capsulatus. In this organism form II RubisCO, encoded
by cbbM (Fig. 1), is expressed during photoautotrophic
and photoheterotrophic growth in the presence of malate while form I RubisCO, encoded by cbbLS, is only
expressed under photoautotrophic conditions and not
during photoheterotrophic growth on malate [72,134].
Inactivation of cbbM in R. capsulatus results in the expression of form I RubisCO and an increase in both cbbI
and cbbII promoter activity during photoheterotrophic
growth on malate [74,135]. This compensatory expression of form I RubisCO was shown to be dependent on
the presence of functional phosphoribulokinase, encoded by cbbP, suggesting that a CBB cycle intermediate
may be involved in the photoheterotrophic upregulation
of form I RubisCO synthesis in this strain [73]. These
findings were extended using a R. capsulatus cbbL
cbbM (SBI/II) double RubisCO mutant. Due to the
absence of a functional CBB cycle, this strain can only
grow photoheterotrophically when supplied with an alternative electron acceptor such as DMSO [73]. Photoheterotrophic growth under these conditions results in
dramatically increased levels of cbbI and cbbII promoter
activity, as well as increased activity levels of the CBB
cycle enzymes phosphoribulokinase (PRK) and fructose
1,6/sedoheptulose 1,7-bisphosphatase (FBPase) [135].
As with earlier studies, the increased cbbI and cbbII expression in SBI/II was dependent, on a functional cbbP
while inactivation of cbbT, immediately downstream of
cbbP, had no effect [135]. Inactivation of cbbRII in the
SBI/II background (SBRI/IIRII) resulted in almost no
expression of the cbbII operon while cbbI expression,
albeit lower than strain SBI/II, was still elevated presumably through the action of CbbRI [135], suggesting
that the signal responsible for the increased cbb expression in SBI/II is sensed by both CbbRI and CbbRII.
All of this evidence indicates that in R. capsulatus cbb
gene expression is activated in response to the build up
of a CBB cycle intermediate or byproduct, possibly
RuBP, ADP or a derivative there of, that is generated as
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a result of PRK function. Some of the physiological
evidence also indicated that there is a separate metabolic
signal, unrelated to PRK function, controlling photoheterotrophic cbbII expression since inactivation of cbbP
in both wild-type and SBI/II strains resulted in regulated
expression of the cbbII promoter at wild-type levels
[135,136]. A number of lines of evidence indicate that
CbbR-mediated control of cbb expression in R. sphaeroides responds to the same PRK-derived signal as
that in R. capsulatus. Like R. capsulatus, inactivation of
one of the two RubisCO genes in R. sphaeroides results
in the compensatory CbbR-dependent increase in expression of the remaining RubisCO [58,62,64,65,67]. In
addition, an R. sphaeroides double RubisCO mutant
also shows dramatically increased levels of both cbbI
and cbbII promoter activity when grown photoheterotrophically in the presence of DMSO [137]. Finally,
expression of a R. sphaeroides cbbI promoter lacZ fusion
was also upregulated in a R. capsulatus SBI/II background that was dependent on both the presence of
R. sphaeroides CbbR and functional cbbPII [135].
In vitro studies testing the effect of metabolites on the
DNA-binding properties of purified R. capsulatus
CbbRI and CbbRII , also point to RuBP as the inducer
sensed by both CbbRI and CbbRII , thus reinforcing the
in vivo studies. The presence of RuBP was found to
increase the binding affinity of both CbbRI and CbbRII
to their DNA target sites in gel mobility shift assays
[P. Vichivanives,. J.M. Dubbs and F.R. Tabita, unpublished results] and a similar RuBP effect was also
observed with R. sphaeroides CbbR (D. Dangel, J.M.
Dubbs and F.R. Tabita, unpublished results). As predicted by the in vivo studies, additional metabolites were
found to affect CbbR binding to DNA. The CBB cycle
derived metabolites phosphenolpyruvate, 3-phosphoglycerate, 2-phosphoglycerate and 2-phosphoglycolate
have been shown to increase CbbRI and CbbRII
DNA-binding affinity in gel mobility shift assays
(P. Vichivanives, J.M. Dubbs and F.R. Tabita, unpublished results). Moreover, a number of metabolites have
been found to specifically affect the DNA binding of
only one of the two CbbRs in R. capsulatus. K2 HPO4
and to a lesser degree ATP enhanced the binding of
CbbRI to its DNA target site but had no affect on
CbbRII , while fructose 1,6-bisphosphate increased the
DNA-binding affinity of CbbRII only [123]. Different
effector molecules have been identified for the CbbRs
from Xanthobacter flavis (NADPH) and Ralstonia eutropha (phosphenolpyruvate) [138,139] leading to the
suggestion that the effector molecule(s) that are sensed
by CbbR may be organism-specific [2]. Some of the
metabolites that affected the DNA-binding characteristics of R. capsulatus CbbRI and CbbRII in gel moility
shifts were also shown to cause alterations in the
DNaseI footprint of CbbR. The presence of RuBP
caused a reduction in the extent of DNasel protection by
CbbRI on the 30 side along with a disappearance of the
central DNasel hypersensitive sites. Similar reductions
in the CbbRII DNaseI footprint were induced by the
presence of both fructose 1,6-bisphosphate and 3phosphoglycerate. Changes in DNaseI footprints in response to the presence of inducer molecules have been
observed for other LTTR regulators [133]. The accumulated data to this point indicate that in R. capsulatus,
both CbbRI and CbbRII are likely to respond to more
than one effector molecule, some of which are specific
for a given CbbR, resulting in multiple regulatory inputs
governing the control of cbb expression even at this level. While evidence implicating RuBP as an inducer of
CbbR-mediated activation of cbb expression is strong,
the function of the other potential effector molecules is
unclear. In R. eutropha (Alcaligenes eutrophus), phosphenolpyruvate has been shown to increase the affinity
of CbbR for its DNA-binding site as well as negatively
affect CbbR-mediated activation of cbb expression [139].
This raises the possibility that one or more of the additional molecules that affect R. capsulatus CbbR binding may be negative effectors. It could be envisioned that
CbbR-mediated cbb expression in R. capsulatus is under
the positive control of the CBB cycle-specific intermediate RuBP, and possibly fructose 1,6-bisphosphate, and
negatively affected by one or more two- or three-carbon
phosphorylated compounds that are either CBB cycle
end products or derived from them. Thus, CbbR could
be sensing both the efficiency of CBB cycle function as
well as the overall fixed carbon status of the cell.
Finally, in recent studies, the bioselection of randomly mutagenized cbbR genes that both negatively and
positively affect CbbR-mediated transcription has lead
to a wealth of information on the structural basis for
effector-mediated CbbR function (D. Dangel, J.L.
Gibson, A.P. Jannsen and F.R. Tabita, manuscript in
preparation). In this study, a cbbI promoter-lacZ fusion
was inserted into the chromosome of a host strain of
R. sphaeroides in which the endogenous cbbR gene was
inactivated. This strain (strain 87) was subsequently
used to detect mutations in CbbR that affect its function, assayed after separately mutagenizing cbbR sequences, followed by noting the ability of plasmid borne
copies of these sequences to complement strain 87 and
yield a color change on X-gal plates. This protocol resulted in single residue mutant forms of CbbR that were
more highly active in transcription than the wild-type
protein under all growth conditions and also activated
transcription under normally repressive growth conditions. In addition, some mutant CbbR proteins either
responded or did not respond to effectors (RuBP) in
vivo and in vitro, and some mutant proteins were poorly
able to activate transcription. Thus, specific single residues on the protein that are important for cbbR specificity, activity and interaction with effectors were
mapped for the first time and residues that appeared to
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
act synergistically were also noted. Clearly, as we learn
more about the structure and function of CbbR, a
deeper understanding of the regulatory mechanism will
result.
5. Other potential regulators of cbb gene expression
There is compelling evidence to indicate that other
regulators are important for the control of either cbbI
[43] or cbbII [48] gene expression in R. sphaeroides
(Fig. 4). R. palustris represents an interesting situation,
in which the involvement of the Pas gene, and the potential role of the RR1 and RR2 genes, was discussed
earlier (Fig. 1). A consortium of investigators, under the
auspices of the Genomes to Life Program of the US
Department of Energy, has been examining aspects of
the integrative control of gene expression in this organism, which clearly has more diverse metabolic
capabilities than any other organism, including R. sphaeroides and R. capsulatus. Included are studies of CO2 ,
N2 , H2 , sulfur, aromatic hydrocarbon and energy metabolism. This is the first NSP organism for which
the entire genomic sequence was determined (http://
genome.jgi-psf.org/finished_microbes/rhopa/rhopa.home.
html). Interestingly, this organism most closely resembles B. japonicum, while showing interesting differences
from, and some similarities to, the typical Rhodobacter
paradigm. A mini-Tn5-lacZ library, prepared by Professor Carrie Harwood’s laboratory, in which nearly
every ORF was disrupted, allowed the Harwood and
Tabita laboratories to isolate and identify several unique
ORFs involved in the metabolic control of the key
processes (mentioned above) catalyzed by this organism.
The site of the Tn5 disruption was identified via arbitrary PCR methods. As far as CO2 fixation and autotrophy, several potential regulator molecules, as well as
other loci, were shown to be specifically involved with
CO2 -dependent growth. This approach, combined with
microarrays available with this organism, and ongoing
proteomics studies by members of the consortium,
should point the way to a greater understanding of
regulators that both globally and specifically influence
these-key processes in R. palustris; in many instances
these findings might then be applied to Rhodobacter and
other NSP bacteria as well.
6. A final word on integrative control of gene expression
It is apparent that global regulators such as the Reg/
Prr system have the ability to control many of the interesting modes of metabolism that these organisms
undergo. However, it will be interesting to determine
whether additional processes such as aromatic hydrocarbon degradation and sulfur oxidation, which are
catalyzed by the most versatile of these organisms,
371
R. palustris, are also regulated by the Reg/Prr system.
Such studies are currently underway by members of the
R. palustris research consortium. In addition, microarray and proteomic studies in all NSP organisms for
which a complete genome is available will likely provide
revealing insights as to the importance of additional
regulators that contribute to the control of key metabolic processes. However, it might be useful to stress
that the crux of all control relates to the means by which
these organisms respond to and handle exposure to the
relative oxidation–reduction or redox potential of the
surrounding environment. All processes, from basic
carbon and nitrogen metabolism to energetics and synthesis of components of the photosystem, are exquisitely
controlled by the relative success by which the organisms either remove or sequester reducing equivalents.
Under phototrophic growth conditions, the preferential
means to remove, dissipate or sequester excess reducing
equivalents is through the use of CO2 as the electron
sink. Thus, when NSP bacteria grow in their preferred
mode, using organic carbon as the electron donor in the
light, excess reducing equivalents must be removed or
phototrophic growth is impossible. A product of organic
carbon photometabolism is CO2 and this ‘‘metabolic’’
CO2 serves as the preferred electron acceptor for the
excess reducing equivalents. Experimentally, one can
show the importance of CO2 as a required electron sink
by interfering with the CO2 reduction process, i.e., by
knocking out the CBB pathway, which is the means by
which NSP bacteria reduce CO2 and thus balance their
redox potential [3]. Clearly then, metabolic success as a
phototroph directly depends on the organism’s ability to
reduce CO2 . Only if an external electron acceptor such
as DMSO is added will such CBB-deficient strains grow
under phototrophic conditions [140]. However, the great
adaptability of these organisms has led to important
findings relative to how other important processes are
influenced by the CBB system. Thus, it was shown
that CBB-deficient strains of R. sphaeroides [140] and
R. capsulatus [61,136], and now R. palustris (T.E. Hanson, J.L. Gibson and F.R. Tabita, unpublished results)
could each be used to select for strains that would
eventually grow photoheterotrophically in the absence
of added electron acceptors. In each instance, it was
shown that these adaptive mutants, resulting in what we
have termed the photoheterotrophic competent or PHC
phenotype [140], had turned on alternative systems to
remove the reducing equivalents generated from the
oxidation of the organic carbon. The PHC strains basically ‘‘learned’’ to cope with the absence of the CBB
system. One very important adaptation was the derepression of the nitrogenase system [103,136] that allows
the organism to use the nitrogenase complex as a hydrogenase such that protons are reduced to molecular
hydrogen, which is then dissipated to balance the redox
potential of the cell. This is a very powerful and
372
J.M. Dubbs, F. Robert Tabita / FEMS Mircobiology Reviews 28 (2004) 353–376
important adaptation as somehow the normal control
by ammonia is abrogated and the cells produce H2 gas
in an unfettered fashion. This has also been shown with
R. rubrum as well [103] and it is clear that a very fundamental change in the overall intracellular regulatory
network has occurred in all four organisms. To show
that the CBB system is the preferred redox balancing
mechanism, complementation of these PHC mutants
with wild-type CBB genes results in the organism returning to its preferred life style, that is the use of CO2
as electron acceptor along with normal ammonia control over nitrogenase synthesis [103,136]. What is interesting is that all four organisms behave the same way, as
if there is some fundamental change that must occur to
allow NSP bacteria to accrue the PHC phenotype and
derepress nitrogenase synthesis. As we home in on this
fundamental locus of control that allows this adaptation
it is apparent that we will learn much about the molecular events that occur to integrate the control of
carbon and nitrogen metabolism. Finally, it should be
stressed that the PHC nif derepressing strains are not the
only genetic adaptations that may be selected after interfering with the CBB system. CBB-deficient strains
that do not derepress nitrogenase have been isolated
that are capable of growth under photoheterotrophic
conditions [137,140]. However, the basis for the ability
of these strains to balance their redox potential in the
absence of either the CBB or the nitrogenase system is
unknown at this time. Yet the same over-arching influence of the CBB pathway on the expression of these
alternative redox balancing mechanisms is apparent.
The next few years should result in a deeper understanding of how control of all redox balancing systems is
integrated in the NSP bacteria.
Acknowledgements
Research in the FRT laboratory was supported by
grants from the National Institutes of General Medical
Sciences of the US National Institutes of Health/Public
Health Service and from the Genomes to Life Program
of the US Department of Energy.
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