Induction of carbon monoxide dehydrogenase to facilitate redox balancing in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant strain of Rhodospirillum rubrum

A ribulose-1,5-bisphosphate carboxylase/oxygenase-deficient mutant strain (strain I-19) of Rhodospirillum rubrum was capable of growth under photoheterotrophic conditions in the absence of exogenous electron acceptors. These results suggested that alternative means of removing reducing equivalents have been acquired that allow this strain to remove reducing equivalents in the absence of a functional Calvin-Benson-Bassham reductive pentose phosphate pathway. Previously, the proton-reducing activity of the dinitrogenase complex was implicated in helping to maintain redox balance. However, since considerable amounts of CO2 were still fixed in this strain, the complete profile of enzymes involved in alternative CO2 fixation schemes was assessed. A specific and substantial induction of carbon monoxide dehydrogenase (CO dehydrogenase) synthesis was found in the mutant strain; although none of the other CO2 fixation pathways or enzyme activities were altered. These results suggested that CO dehydrogenase contributes to the photoheterotrophic success of strain I-19. Furthermore, the data implicate interacting and complex regulatory processes required to maintain the proper redox balance of this organism and other nonsulfur purple bacteria.     

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.     

Interactive control of Rhodobacter capsulatus redox-balancing systems during phototrophic metabolism

In nonsulfur purple bacteria, redox homeostasis is achieved by the coordinate control of various oxidation-reduction balancing mechanisms during phototrophic anaerobic respiration. In this study, the ability of Rhodobacter capsulatus to maintain a balanced intracellular oxidation-reduction potential was considered; in addition, interrelationships between the control of known redox-balancing systems, the Calvin-Benson-Bassham, dinitrogenase and dimethyl sulfoxide reductase systems, were probed in strains grown under both photoheterotrophic and photoautotrophic growth conditions. By using cbb(I) (cbb form I operon)-, cbb(II)-, nifH-, and dorC-reporter gene fusions, it was demonstrated that each redox-balancing system responds to specific metabolic circumstances under phototrophic growth conditions. In specific mutant strains of R. capsulatus, expression of both the Calvin-Benson-Bassham and dinitrogenase systems was influenced by dimethyl sulfoxide respiration. Under photoheterotrophic growth conditions, coordinate control of redox-balancing systems was further manifested in ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase deletion strains. These findings demonstrated the existence of interactive control mechanisms that govern the diverse means by which R. capsulatus maintains redox poise during photoheterotrophic and photoautotrophic growth.     

Calvin Cycle Mutants of Photoheterotrophic Purple Nonsulfur Bacteria Fail To Grow Due to an Electron Imbalance Rather than Toxic Metabolite Accumulation

Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO₂-fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO), prevents photoheterotrophic growth unless an electron acceptor is provided or if cells can dispose of electrons as H₂. Such observations led to the long-standing model wherein the Calvin cycle is necessary during photoheterotrophic growth to maintain a pool of oxidized electron carriers. This model was recently challenged with an alternative model wherein disrupting RubisCO activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate (RuBP) (D. Wang, Y. Zhang, E. L. Pohlmann, J. Li, and G. P. Roberts, J. Bacteriol. 193:3293-3303, 2011, http://dx.doi.org/10.1128/JB.00265-11). Here, we confirm that RuBP accumulation can impede the growth of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) RubisCO-deficient (ΔRubisCO) mutants under conditions where electron carrier oxidation is coupled to H₂ production. However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H₂ production is permitted. Thus, the Calvin cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H₂ production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated.     

Rapid heating of intact leaves reveals initial effects of stromal oxidation on photosynthesis

Heat stress in leaves under natural conditions is characterized by rapid fluctuations in temperature. These fluctuations can be on the order of 10 degrees C in 7 s. By using a specially modified gas-exchange chamber, these conditions were mimicked in the laboratory to analyse the biochemical response to heat spikes. The decline in ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activity during prolonged heat stress is generally associated with an increase in ribulose 1,5-bisphosphate (RuBP) levels. However, rapid heating caused an initial decline in RuBP which was subsequently followed by a small decline in Rubisco carbamylation. The ratio of RuBP to Rubisco sites declined from a saturating concentration to a sub-saturating concentration, providing a possible mechanism for the decarbamylation of Rubisco. If RuBP is saturating (>1.8 RuBP Rubisco site(-1)), it acts as a cap on the catalytic site and keeps Rubisco activated. Measurements of triose-phosphate levels and NADP-malate dehydrogenase activation (a stromal redox proxy) indicated that the regeneration of RuBP by the Calvin cycle was limited by the availability of redox power.     

Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.     

Roles of CfxA, CfxB, and external electron acceptors in regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase expression in Rhodobacter sphaeroides.

The Rhodobacter sphaeroides genome contains two unlinked genetic regions each encoding a series of proteins involved in CO2 fixation which include phosphoribulokinase (prkA and prkB) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcLS and rbcR) (P. L. Hallenbeck and S. Kaplan, Photosynth. Res. 19:63-71, 1988; F. R. Tabita, Microbiol. Rev. 52:155-189, 1988). We examined the effect of CO2 in the presence and absence of an alternate electron acceptor, dimethyl sulfoxide, on the expression of rbcR and rbcLS in photoheterotrophically grown R. sphaeroides. The expression of both rbcR and rbcLS was shown to depend on the CO2 concentration when succinate was used as the carbon source. It was also demonstrated that CO2 fixation is critical for photoheterotrophic growth but could be replaced by the alternative reduction of dimethyl sulfoxide to dimethyl sulfide. Dimethyl sulfoxide severely depressed both rbcR and rbcLS expression in cells grown photoheterotrophically at CO2 concentrations of 0.05% or greater. However, cells grown photoheterotrophically in the absence of exogenous CO2 but in the presence of dimethyl sulfoxide had intermediate levels of expression of rbcL and rbcR, suggesting partially independent control by limiting CO2 tension. We also present evidence for the existence of two gene products, namely, CfxA and CfxB, which are encoded by genes immediately upstream of rbcLS and rbcR, respectively. Strains were constructed which contained null mutations in cfxA and/or cfxB. Each mutation eliminated expression of the linked downstream rbc operon. Further, studies utilizing these strains demonstrated that each form of ribulose 1,5-bisphosphate carboxylase/oxygenase plays an essential role in maintaining the cellular redox balance during photoheterotrophic growth at differing CO2 concentrations.     

Modified pathway to synthesize ribulose 1,5-bisphosphate in methanogenic archaea

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with M. jannaschii, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of M. jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M. jannaschii and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M. jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli. Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.     

Effect of growth condition on enzymes of the citric acid cycle and the glyoxylate cycle in the photosynthetic bacterium Rhodopseudomonas palustris

The enzymes of the citric acid and glyoxylate cycles as well as RuBP carboxylase were measured in cell-free extracts from Rhodopseudomonas palustris after growth under chemoheterotrophic, photoheterotrophic and photolithotrophic conditions. Although the citric acid cycle was found to be complete under all growth conditions, significant differences in certain enzyme activities occurred as a function of the different energy sources applied. The glyoxylate cycle also was complete under all growth conditions with highest isocitrate lyase activity seen after photoheterotrophic growth on acetate. Photo- and chemoheterotrophic growth on malate reduced the isocitrate lyase. The activity was not repressed further by photolithotrophic growth on thiosulfate. RuBP carboxylase activity, present under photolithotrophic conditions, was repressed by chemoheterotrophic growth but was not decreased by the presence of organic substrates during photoheterotrophic growth.

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Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Effect of growth conditions on carboxylating enzymes of Zea mays plants

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and ribulose-1,5-bisphospate (RuBP) carboxylase (EC 4.1.1.39) activities in leaves of different maize hybrids grown under field conditions (high light intensity) and in a growth chamber (low light intensity) were determined. Light intensity and leaf age affected PEP carboxylase activity, whereas RuBP carboxylase was affected by leaf age only at low light intensity. PEP carboxylase/RuBP carboxylase activity ratio decreased according to light intensity and leaf age. Results demonstrate that Zea mays grown under field conditions is a typical C₄ species in all leaves independently from their position on the stem, whereas it may be a C₃ plant when it is grown in a growth chamber at low light intensityAbbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-Benson-Bassham pathway

Carbon dioxide serves as the preferred electron acceptor during photoheterotrophic growth of nonsulfur purple photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. This CO2, produced as a result of the oxidation of preferred organic carbon sources, is reduced through reactions of the Calvin-Benson-Bassham reductive pentose phosphate pathway. This pathway is thus crucial to maintain a balanced intracellular oxidation-reduction potential (or redox poise) under photoheterotrophic growth conditions. In the absence of a functional Calvin-Benson-Bassham pathway, either an exogenous electron acceptor, such as dimethylsulfoxide, must be supplied or the organism must somehow develop alternative electron acceptor pathways to preserve the intracellular redox state of the cell. Spontaneous variants of Rba. capsulatus strains deficient in the Calvin-Benson-Bassham pathway that have become photoheterotrophically competent (in the absence of an exogenous electron acceptor) were isolated. These strains (SBP-PHC and RCNd1, RCNd3, and RCNd4) were shown to obviate normal ammonia control and derepress synthesis of the dinitrogenase enzyme complex for the dissipation of excess reducing equivalents and generation of H2 gas via proton reduction. In contrast to previous studies with other organisms, the dinitrogenase reductase polypeptides were maintained in an active and unmodified form in strain SBP-PHC and the respective RCNd strains. Unlike the situation in Rba. sphaeroides, the Rba. capsulatus strains did not regain full ammonia control when complemented with plasmids that reconstituted a functional Calvin-Benson-Bassham pathway. Moreover, dinitrogenase derepression in Rba. capsulatas was responsive to the addition of the auxiliary electron acceptor dimethylsulfoxide. These results indicated a hierarchical control over the removal of reducing equivalents during photoheterotrophic growth that differs from strains of Rba. sphaeroides and Rhodospirillum rubrum deficient in the Calvin-Benson-Bassham pathway.     

Redox homeostasis phenotypes in RubisCO-deficient Rhodobacter sphaeroides via ensemble modeling

Photosynthetic bacteria are capable of carrying out the fundamental biological processes of carbon dioxide assimilation and photosynthesis. In this work, ensemble modeling (EM) was used to examine the behavior of mutant strains of the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides containing a blockage in the primary CO(2) assimilatory pathway, which is responsible for cellular redox balance. When the Calvin-Benson-Bassham (CBB) pathway is nonfunctional, spontaneous adaptive mutations have evolved allowing for the use of at least two separate alternative redox balancing routes enabling photoheterotrophic growth to occur. The first of these routes expresses the nitrogenase complex, even in the presence of normal repressing ammonia levels, dissipating excess reducing power via its inherent hydrogenase activity to produce large quantities of hydrogen gas. The second of these routes may dissipate excess reducing power through reduction of sulfate by the formation of hydrogen sulfide. EM was used here to investigate metabolism of R. sphaeroides and clearly shows that inactivation of the CBB pathway affects the organism's ability to achieve redox balance, which can be restored via the above-mentioned alternative redox routes. This work demonstrates that R. sphaeroides is capable of adapting alternative ways via mutation to dissipate excess reducing power when the CBB pathway is inactive, and that EM is successful in describing this behavior.     

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase)

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.