Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is "silenced," emanating from the cbb(3) oxidase encoded by the ccoNOQP operon under aerobic conditions. The dominant effect of the ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the cbb(3) oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in conjunction with hemN and hemZ. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.     

From redox flow to gene regulation: role of the PrrC protein of Rhodobacter sphaeroides 2.4.1

Activation of photosynthesis (PS) gene expression by the PrrBA two-component activation system in Rhodobacter sphaeroides 2.4.1 results from the interruption of an inhibitory signal originating from the cbb(3) cytochrome c oxidase via its interaction with oxygen, in conjunction with the Rdx redox proteins. The CcoQ protein, encoded by the ccoNOQP operon, which encodes the cbb(3) cytochrome c oxidase, was shown to act as a "transponder" that conveys the signal derived from reductant flow through cbb(3) to oxygen, to the Prr system. To further define the elements comprising this signal transduction pathway we considered the prrC gene product, which to date possessed no definable role in this signal transduction pathway despite its being part of the prrBCA gene cluster. Similar to mutations in cbb(3) and rdx, suitably constructed prrC deletion mutations lead to PS gene expression in the presence of high oxygen. Unlike mutations that remove cbb(3) terminal oxidase activity or Rdx function, the PrrC deletion mutant shows no effect upon cbb(3) activity, nor does it affect the ratio of the carotenoid (Crt) spheroidene (SE) to spheroidenone (SO). Thus, the PrrC deletion mutant behaves identically to the CcoQ deletion mutant. Taking these and previous results together, we suggest that PrrC is located upstream of the two-component PrrBA activation system in the signal transduction pathway but downstream of the cbb(3) cytochrome c oxidase and its "transponder" CcoQ. The PrrC deletion mutant was also shown to lead to an increase in the DorA protein under aerobic conditions as was shown earlier for the cbb(3) mutant. Finally, PrrC is a member of a highly conserved family of proteins found in both prokaryotes and eukaryotes, and this appears to be the first instance in which a direct regulatory role has been ascribed to a member of this protein family.     

The cbb3 terminal oxidase of Rhodobacter sphaeroides 2.4.1: structural and functional implications for the regulation of spectral complex formation

We have previously shown that the flow of reductant through the cbb3 terminal cytochrome c oxidase of Rhodobacter sphaeroides is essential to the repression of photosynthesis (PS) gene expression in the presence of oxygen by inhibiting the functional activity of the Prr two-component activation system. To gain further insight into the role of the cbb3 oxidase and the cognate ccoNOQP operon in the oxygen regulation of PS gene expression, we constructed nonpolar, in-frame deletions within the ccoN and ccoQ genes. Whereas mutations in ccoN, ccoQ, and ccoP resulted in PS gene expression in the presence of oxygen, only the ccoQ mutation showed both the normal flow of reductant through the cbb3 oxidase and the absence of any alteration in the relative levels of spheroidene and spheroidenone, as is observed for those mutations in the cco operon that result in the loss of terminal oxidase activity. Consistent with these findings is the observation that extra copies of the ccoNOQP operon in trans resulted in the decreased formation of both the B800-850 and B875 spectral complexes under anaerobic growth conditions. These results in conjunction with our earlier findings indicate that (1) the flow of reductant through the cbb3 terminal oxidase is a prerequisite to the regulation of PS gene expression by the Prr two-component regulatory system, (2) the CcoQ protein is involved in conveying the signal derived from reductant flow through the cbb3 terminal oxidase to the Prr regulatory pathway, (3) there is reductant flow through this terminal oxidase under anaerobic conditions, and as a result, the activity of the Prr system is still subject to cbb3 regulation, and (4) the acceptor for reductant flow through cbb3 under anaerobic conditions is in whole or in part involved in the conversion of spheroidene to spheroidenone.     

Effect of mutations of five conserved histidine residues in the catalytic subunit of the cbb3 cytochrome c oxidase on its function

The cbb3 cytochrome c oxidase has the dual function as a terminal oxidase and oxygen sensor in the photosynthetic bacterium, Rhodobacter sphaeroides. The cbb3 oxidase forms a signal transduction pathway together with the PrrBA two-component system that controls photosynthesis gene expression in response to changes in oxygen tension in the environment. Under aerobic conditions the cbb3 oxidase generates an inhibitory signal, which shifts the equilibrium of PrrB kinase/phosphatase activities towards the phosphatase mode. Photosynthesis genes are thereby turned off under aerobic conditions. The catalytic subunit (CcoN) of the R. sphaeroides cbb3 oxidase contains five histidine residues (H214, H233, H303, H320, and H444) that are conserved in all CcoN subunits of the cbb3 oxidase, but not in the catalytic subunits of other members of copper-heme superfamily oxidases. H214A mutation of CcoN affected neither catalytic activity nor sensory (signaling) function of the cbb3 oxidase, whereas H320A mutation led to almost complete loss of both catalytic activity and sensory function of the cbb3 oxidase. H233V and H444A mutations brought about the partial loss of catalytic activity and sensory function of the cbb3 oxidase. Interestingly, the H303A mutant form of the cbb3 oxidase retains the catalytic function as a cytochrome c oxidase as compared to the wild-type oxidase, while it is defective in signaling function as an oxygen sensor. H303 appears to be implicated in either signal sensing or generation of the inhibitory signal to the PrrBA two-component system.     

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.     

Involvement of SenC in assembly of cytochrome c oxidase in Rhodobacter capsulatus

SenC, a Sco1 homolog found in the purple photosynthetic bacteria, has been implicated in affecting photosynthesis and respiratory gene expression, as well as assembly of cytochrome c oxidase. In this study, we show that SenC from Rhodobacter capsulatus is involved in the assembly of a fully functional cbb(3)-type cytochrome c oxidase, as revealed by decreased cytochrome c oxidase activity in a senC mutant. We also show that a putative copper-binding site in SenC is required for activity and that a SenC deletion phenotype can be rescued by the addition of exogenous copper to the growth medium. In addition, we demonstrate that a SenC mutation has an indirect effect on gene expression caused by a reduction in cytochrome c oxidase activity. A model is proposed whereby a reduction in cytochrome c oxidase activity impedes the flow of electrons through the respiratory pathway, thereby affecting the oxidation/reduction state of the ubiquinone pool, leading to alterations of photosystem and respiratory gene expression.     

Differential expression of the CO2 fixation operons of Rhodobacter sphaeroides by the Prr/Reg two-component system during chemoautotrophic growth

In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb(I) and cbb(II) promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbb(I) and cbb(II) promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbb(II) promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbb(I) expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbb(I) promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.     

Bacillus subtilis YdiH is a direct negative regulator of the cydABCD operon

During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa3, caa3, and bd. Cytochrome bd is encoded by the cydABCD operon. We report here the first identification of a regulator for the cydABCD operon, YdiH. While working with DeltaresDE mutant strains, we identified colonies which contained suppressor mutations (cmp) which bypassed the requirement for ResD for all phenotypes not associated with cytochrome aa3 or caa3. Mapping identified a class of Tn10 insertions which were close to the cmp locus (Tn10-2) and a second class (Tn10-1) which was inserted in cydD, a gene which appears to be essential to the cmp phenotype. Sequencing of the cmp loci from four independent DeltaresDE cmp isolates yielded four loss-of-function alleles of ydiH, a gene encoding a protein with homology to AT-rich DNA-binding proteins. Additionally, we determined that cytochrome bd was aberrantly expressed in the DeltaresDE cmp background. Together these data led to the hypothesis that YdiH serves as a negative regulator of cydABCD expression, a hypothesis supported by both gel-shift and DNase I footprinting analyses. YdiH protected the cydA promoter region at three 22-bp repeats located in the long 5' untranslated region (193 bp). Induction of the cydABCD operon in a DeltaresDE background showed that expression of the terminal oxidase bd was responsible for the bypass phenotype observed in a DeltaresDE cmp strain, indicating that cytochrome bd expression complemented the loss of cytochromes aa3 and caa3 in the DeltaresDE strain.     

Expression of Bradyrhizobium japonicum cbb3 terminal oxidase under denitrifying conditions is subjected to redox control

Bradyrhizobium japonicum utilizes cytochrome cbb ₃ oxidase encoded by the fixNOQP operon to support microaerobic respiration under free-living and symbiotic conditions. It has been previously shown that, under denitrifying conditions, inactivation of the cycA gene encoding cytochrome c ₅₅₀, the electron donor to the Cu-containing nitrite reductase, reduces cbb ₃ expression. In order to establish the role of c ₅₅₀ in electron transport to the cbb ₃ oxidase, in this work, we have analyzed cbb ₃ expression and activity in the cycA mutant grown under microaerobic or denitrifying conditions. Under denitrifying conditions, mutation of cycA had a negative effect on cytochrome c oxidase activity, heme c (FixP and FixO) and heme b cytochromes as well as expression of a fixP '–' lacZ fusion. Similarly, cbb ₃ oxidase was expressed very weakly in a napC mutant lacking the c -type cytochrome, which transfers electrons to the NapAB structural subunit of the periplasmic nitrate reductase. These results suggest that a change in the electron flow through the denitrification pathway may affect the cellular redox state, leading to alterations in cbb ₃ expression. In fact, levels of fixP '–' lacZ expression were largely dependent on the oxidized or reduced nature of the carbon source in the medium. Maximal expression observed in cells grown under denitrifying conditions with an oxidized carbon source required the regulatory protein RegR.     

Dominant role of the cbb3 oxidase in regulation of photosynthesis gene expression through the PrrBA system in Rhodobacter sphaeroides 2.4.1

In this study, the H303A mutant form of the cbb(3) oxidase (H303A oxidase), which has the H303A mutation in its catalytic subunit (CcoN), was purified from Rhodobacter sphaeroides. The H303A oxidase showed the same catalytic activity as did the wild-type form of the oxidase (WT oxidase). The heme contents of the mutant and WT forms of the cbb(3) oxidase were also comparable. However, the puf and puc operons, which are under the control of the PrrBA two-component system, were shown to be derepressed aerobically in the R. sphaeroides strain expressing the H303A oxidase. Since the strain harboring the H303A oxidase exhibited the same cytochrome c oxidase activity as the stain harboring the WT oxidase did, the aerobic derepression of photosynthesis gene expression observed in the H303A mutant appears to be the result of a defective signaling function of the H303A oxidase rather than reflecting any redox changes in the ubiquinone/ubiquinol pool. It was also demonstrated that ubiquinone inhibits not only the autokinase activity of full-length PrrB but also that of the truncated form of PrrB lacking its transmembrane domain, including the proposed quinone binding sequence. These results imply that the suggested ubiquinone binding site within the PrrB transmembrane domain is not necessary for the inhibition of PrrB kinase activity by ubiquinone. Instead, it is probable that signaling through H303 of the CcoN subunit of the cbb(3) oxidase is part of the pathway through which the cbb(3) oxidase affects the relative kinase/phosphatase activity of the membrane-bound PrrB.     

Transcriptome of a Nitrosomonas europaea mutant with a disrupted nitrite reductase gene (nirK)

Global gene expression was compared between the Nitrosomonas europaea wild type and a nitrite reductase-deficient mutant using a genomic microarray. Forty-one genes were differentially regulated between the wild type and the nirK mutant, including the nirK operon, genes for cytochrome c oxidase, and seven iron uptake genes. Relationships of differentially regulated genes to the nirK mutant phenotype are discussed.     

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.     

Control of catechol meta-cleavage pathway in Alcaligenes eutrophus

Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway. Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate. Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon. From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon). Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.     

Cloning of the psbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K

We cloned and sequenced the psbK gene, coding for a small photosystem II component (PSII-K), from the transformable cyanobacterium, Synechocystis sp. PCC 6803, and determined the N-terminal sequence of mature PSII-K. The psbK gene product is processed by cleaving off eight amino acid residues from the N terminus. A mutant lacking psbK was constructed; this mutant grew photoautotrophically, but its growth rate was reduced. The number of photosystem II reaction centers on a chlorophyll basis was decreased by less than a factor of 2 in the psbK-deletion mutant. In Synechocystis sp. PCC 6803, the psbK gene is transcribed as a single gene and is not part of an operon. Single-site mutations were introduced into psbK leading to early termination or deletion of the presequence. The phenotype of these mutants strongly resembles that of the psbK deletion mutant, indicating that indeed the change in phenotype in the deletion mutant is directly correlated with PSII-K. PSII-K is not essential for photosystem II assembly or activity but is needed for optimal photosystem II function.