Phototrophic Nonsulfur Bacterium Research Articles

Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(β-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly(β-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H2 production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Development of a low-cost culture medium for the rapid production of plant growth-promoting Rhodopseudomonas palustris strain PS3.

Rhodopseudomonas palustris PS3 is one of the purple phototrophic non-sulfur bacteria (PNSB), which have plant growth-promoting effects on various plants. To expand the scale of PS3 fermentation in a time- and cost-effective fashion, the purpose of this work was to evaluate the use of low-cost materials as culture media and to optimize the culture conditions via response surface methodology. Corn steep liquor (CSL) and molasses were identified as potential materials to replace the nitrogen and carbon sources, respectively, in the conventional growth medium. The optimum culture conditions identified through central composite design were CSL, 39.41 mL/L; molasses, 32.35 g/L; temperature, 37.9°C; pH, 7.0; and DO 30%. Under the optimized conditions, the biomass yield reached 2.18 ± 0.01 g/L at 24 hours, which was 7.8-fold higher than that under the original medium (0.28 ± 0.01 g/L). The correlation between the predicted and experimental values of the model was over 98%, which verified the validity of the response models. Furthermore, we verified the effectiveness of the R. palustris PS3 inoculant grown under the newly developed culture conditions for plant growth promotion. This study provides a potential strategy for improving the fermentation of R. palustris PS3 in low-cost media for large-scale industrial production.

Whole-genome sequencing and comparative analysis of two plant-associated strains of Rhodopseudomonas palustris (PS3 and YSC3)

Rhodopseudomonas palustris strains PS3 and YSC3 are purple non-sulfur phototrophic bacteria isolated from Taiwanese paddy soils. PS3 has beneficial effects on plant growth and enhances the uptake efficiency of applied fertilizer nutrients. In contrast, YSC3 has no significant effect on plant growth. The genomic structures of PS3 and YSC3 are similar; each contains one circular chromosome that is 5,269,926 or 5,371,816 bp in size, with 4,799 or 4,907 protein-coding genes, respectively. In this study, a large class of genes involved in chemotaxis and motility was identified in both strains, and genes associated with plant growth promotion, such as nitrogen fixation-, IAA synthesis- and ACC deamination-associated genes, were also identified. We noticed that the growth rate, the amount of biofilm formation, and the relative expression levels of several chemotaxis-associated genes were significantly higher for PS3 than for YSC3 upon treatment with root exudates. These results indicate that PS3 responds better to the presence of plant hosts, which may contribute to the successful interactions of PS3 with plant hosts. Moreover, these findings indicate that the existence of gene clusters associated with plant growth promotion is required but not sufficient for a bacterium to exhibit phenotypes associated with plant growth promotion.

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD+ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.

Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium Rhodovulum sulfidophilum.

The marine bacterium Rhodovulum sulfidophilum is a nonsulfur phototrophic bacterium, which is known to produce extracellular nucleic acids in soluble form in culture medium. In the present paper, constructing the response regulator ctrA-deficient mutant of R. sulfidophilum, we found that this mutation causes a significant decrease in the extracellular DNA production. However, by the introduction of a plasmid containing the wild type ctrA gene into the mutant, the amount of extracellular DNA produced was recovered. This is the first and clear evidence that the extracellular DNA production is actively controlled by the CtrA in R. sulfidophilum.

Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described.

ЕКОЛОГО-ТРОФІЧНІ ГРУПИ МІКРООРГАНІЗМІВ ВОДИ ОЗЕРА ЯВОРІВСЬКЕ

The chemical composition of Yavorivskе lake water in the autumn of 2016 at different depths (0−70 m) was investigated and the main ecological trophic groups of microorganisms, involved in transformation of carbon, nitrogen and sulfur compounds, were studied. Acidity, mineralization and Na + , Ca 2+ , Mg 2+ , HCO 3 - , Cl - and NO 3 - content at all depths, K + , P 2 0 5 and NH 4 + at depths up to 20–50 m were insignificant and did not exceed the permissible norms. At all depths the content of SO 4 2- (913–1530 mg/l) and H 2 S (30–34 mg/l) at depths over 30 m significantly exceeded the norms. It was established that the change in the quantity of different ecological trophic groups of microorganisms and their ratio in the lake water, compared with the source type reservoir of Roztochya reserve, is an adaptive response of the microbial community to stress environmental factors. The total quantity of all ecological trophic groups of microorganisms in water of Yavorivske lake with depth decreases from 5.24×10 6 to 2.45×10 6 CFU/ml of water and is 2–5 times lower than in the control: 1.28×10 7 CFU/ml of water. In Yavorivske lake is formed new microbocenosis, in which on the surface and at depth of 30 m the most numerous ecological trophic groups are neutrophilic colorless sulfur oxidizing bacteria (39.3 and 13.3 %), microorganisms that use organic forms of nitrogen (21.6 and 42.9 %), nitrifying (7.3 and 9.4 % of phase I, 13.5 and 4.9 % of phase II of nitrification), denitrifying (7.1 and 11.3 %), oligonitrophilic bacteria (4.4 and 5.1 %), microorganisms that use mineral forms of nitrogen (5.8 and 1.4 % respectively) and phototrophic nonsulfur bacteria (4.9 % at depth of 30 m). Sulfate and sulfur reducing (42.9 and 44.9 %), phototropic sulfur (2.7 and 4.5 %) and nonsulfur bacteria (4.4 and 3.7 % respectively) are the most numerous ecological trophic groups of microorganisms at depths of 50 and 70 m of the lake. The quantity of acidophilic colorless sulfur oxidizing bacteria, cyanobacteria, cellulose degrading microorganisms and microscopic fungi at all depths was insignificant. In control the most numerous ecological trophic groups are microorganisms that use organic forms of nitrogen (28.3 %), oligonitrophilic (25.6 %), nitrifying (23.5 % of phase II and 15.7 % of phase I of nitrification), neutrophilic colorless sulfur oxidizing bacteria (3.0 %), microscopic fungi (1.9 %). Their number, besides neutrophilic colorless sulfur oxidizing bacteria, is much higher than in Yavorivske lake water.

Agrobacterial ipt gene for cytokinin biosynthesis is found in phototrophic non-sulfur purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris

Phototrophic non-sulfur bacteria (PNSB) are close relatives of cytokinin producing agrobacteria. However, the sequenced PNSB genomes do not have the ipt gene sequences involved in the cytokinin biosynthesis. In Rhodobacter sphaeroides and Rhodopseudomonas palustris transformed with ipt, we previously observed the substantial morphological and physiological changes as compared with the original strains. To find associations of these changes with the ipt function, its expression levels were studied in the transformants and the parent strains. Unexpectedly, the ipt transcripts were found in both the transformants and the original untransformed strains, although the ipt expression level was several orders of magnitude lower in the latter compared to the transformants. The ipt sequences amplified from the plasmid DNA templates of the parent PNSB strains showed that they were identical to each other but differed in four nucleotides compared to the ipt gene from the Agrobacterium tumefaciens pTiBo 542 plasmid. The in silico comparison amongst the ipt sequences from the other agrobacterial plasmids, as well as from the other PNSB, demonstrated their low identity. This is the first report on the active gene of the cytokinin biosynthesis found in Rba. sphaeroides and Rps. palustris which is homologous to the agrobacterial ipt gene.

Posttranslational modification of a vanadium nitrogenase.

In microbes that fix nitrogen, nitrogenase catalyzes the conversion of N2 to ammonia in an ATP-demanding reaction. To help conserve energy some bacteria inhibit nitrogenase activity upon exposure to ammonium. The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 can synthesize three functional nitrogenase isoenzymes: a molybdenum nitrogenase, a vanadium nitrogenase, and an iron nitrogenase. Previous studies showed that in some alphaproteobacteria, including R. palustris, molybdenum nitrogenase activity is inhibited by ADP-ribosylation when cells are exposed to ammonium. Some iron nitrogenases are also posttranslationally modified. However, the posttranslational modification of vanadium nitrogenase has not been reported. Here, we investigated the regulation of the alternative nitrogenases of R. palustris and determined that both its vanadium nitrogenase and its iron nitrogenase activities were inhibited and posttranslationally modified when cells are exposed to ammonium. Vanadium nitrogenase is not found in all strains of R. palustris, suggesting that it may have been acquired by horizontal gene transfer. Also, phylogenetic analyses of the three nitrogenases suggest that VnfH, the target of ADP-ribosylation, may be the product of a gene duplication of nifH, the molybdenum nitrogenase homolog.

Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent

Hydrogen production (HP) from dark fermentation effluent of starch wastewater via vertical tubular photo-bioreactor was investigated. The reactor was inoculated with mixed culture of bacteria and operated at light intensity of 190W/m2. Hydraulic retention time (HRT) and organic loading rate (OLR) was varied between 0.9 to 4.0h and 3.2 to 16gCOD/l.d., respectively. Increasing the HRT from 0.9 to 2.5h, significantly (P<0.05) increased HP from 1±0.04 to 3.05±0.19l/d, respectively. However, minimal increase in HP occurred when increasing the HRT up to 4.0h. The HP remained unaffected when increasing the OLR from 3.2 to 6.4gCOD/l.d. Further increase in the OLR up to 8.2 and 16gCOD/l.d., resulted in a drop in HP i.e. 0.96 and 0.19l/d, respectively. Microbial community analysis of the reactor samples showed the presence and dominance of hydrogen producing purple non-sulfur phototrophic (PNS) bacterium, Rhodopseudomonas palustris in the reactor.

Distribution and Origin of Oxygen-Dependent and Oxygen-Independent Forms of Mg-Protoporphyrin Monomethylester Cyclase among Phototrophic Proteobacteria

Magnesium-protoporphyrin IX monomethylester cyclase is one of the key enzymes of the bacteriochlorophyll biosynthesis pathway. There exist two fundamentally different forms of this enzyme. The oxygen-dependent form, encoded by the gene acsF, catalyzes the formation of the bacteriochlorophyll fifth ring using oxygen, whereas the oxygen-independent form encoded by the gene bchE utilizes an oxygen atom extracted from water. The presence of acsF and bchE genes was surveyed in various phototrophic Proteobacteria using the available genomic data and newly designed degenerated primers. It was found that while the majority of purple nonsulfur bacteria contained both forms of the cyclase, the purple sulfur bacteria contained only the oxygen-independent form. All tested species of aerobic anoxygenic phototrophs contained acsF genes, but some of them also retained the bchE gene. In contrast to bchE phylogeny, the acsF phylogeny was in good agreement with 16S inferred phylogeny. Moreover, the survey of the genome data documented that the acsF gene occupies a conserved position inside the photosynthesis gene cluster, whereas the bchE location in the genome varied largely between the species. This suggests that the oxygen-dependent cyclase was recruited by purple phototrophic bacteria very early during their evolution. The primary sequence and immunochemical similarity with its cyanobacterial counterparts suggests that acsF may have been acquired by Proteobacteria via horizontal gene transfer from cyanobacteria. The acquisition of the gene allowed purple nonsulfur phototrophic bacteria to proliferate in the mildly oxygenated conditions of the Proterozoic era.

Improvement of hydrogen production with Rhodopseudomonas palustris CQK-01 by Ar gas sparging

For improving photo-biohydrogen production, a novel gas bubble column photobioreactor with Ar gas sparging was developed for biohydrogen production by purple non-sulfur phototrophic bacteria, Rhodoseudomonas palustris CQK-01. The dissolved hydrogen concentration was in-situ measured by a hydrogen microsensor. Experimental results demonstrated that Ar gas sparging dramatically decreased the dissolved hydrogen concentration, resulting in an improvement in the photo-biohydrogen production performance. Furthermore, effects of the gas flow rate and the time interval of gas sparging were investigated. The results showed that with an increase in the gas flow rate, the hydrogen production performance increased initially due to the reduced dissolved hydrogen concentration and enhanced mass transport, and then it decreased as a result of an increased shear stress. Meanwhile, the short sparging time interval resulted in a low accumulation of dissolved hydrogen in the bioreactor, hence high hydrogen production performance. The optimal hydrogen production rate (5.86 mmol/l/h) and hydrogen yield (3.38 mol H2/mol glucose) were obtained at the gas flow rate of 10 ml/min, respectively.

Effect of Substrate pH Value on Hydrogen Production by Photosynthetic Bacteria*

For exploring the hydrogen production mechanism of photosynthetic bacteria,hydrogen production by the purple non-sulfur phototrophic bacterium Rhodoseudomonas palustris CQK-01 illuminated by light-emitting diodes with light wavelength of 470 nm was experimentally investigated in batch culture mode under anaerobic conditions,and the effect of initial substrate pH value on hydrogen production performance and energy conversion efficiencies were discussed.Glucose with concentration of 75 mmol/L was used as carbon source in medium,and the initial substrate pH values were set at 5.5,6.0,7.0,8.0 and 9.0.The light intensity was set at 1 000 lx and operation temperature was 30 ℃.The experimental results showed that the weak alkaline condition was favorable for the growth of photosynthetic bacteria,and after 96 hours' operation for hydrogen production,the total hydrogen production and the maximum hydrogen production rate was 8.0 mmol and 3.39 mmol g-1(cell dry weight) h-1 at the optimal pH 7.0,respectively.The produced hydrogen purity was 70%,and the substrate energy conversion efficiency and light energy conversion efficiency were 1.98% and 7.7%,respectively.It was also found that the substrate energy conversion efficiency,light energy conversion efficiency,hydrogen production and hydrogen production rate were higher in acidic substrate than in alkaline substrate,while the hydrogen purity increased with the increase of initial substrate pH value

Growth and hydrogen production of &amp;lt;italic&amp;gt;Rhodoseudomonas palustris&amp;lt;/italic&amp;gt; CQK-01 in a gas sparging photobioreactor

Batch culture hydrogen production by the purple non-sulfur phototrophic bacteria Rhodoseudomonas palustris CQK-01 was experimentally investigated in a novel gas sparging photobioreactor. The effects of sparging frequency on the growth and hydrogen production performance of Rhodoseudomonas palustris CQK-01 are discussed. The experimental results showed that sparging Ar gas into the reactor effectively reduced hydrogen concentration in the culture and hence weakened the product feedback inhibition, resulting in enhancement of hydrogen production performance. The gas sparging frequency was found to be an important factor for bacterial growth and hydrogen production by photosynthetic bacteria(PSB), and the optimum hydrogen production performance of the bioreactor was achieved under gas sparging with an interval of 3 h. The total hydrogen production, light conversion efficiency, hydrogen yield, average substrate consumption rate and average hydrogen production rate were 33.25 mmol, 18.46%, 1.79 mol (H2)/mol (glucose), 0.28 mmol L-1 h-1 and 0.50 mmol L-1 h-1, respectively.

Production of Hydrogen Gas from Light and the Inorganic Electron Donor Thiosulfate by Rhodopseudomonas palustris

A challenge for photobiological production of hydrogen gas (H(2)) as a potential biofuel is to find suitable electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron donor for nitrogenase-catalyzed H(2) production by the purple nonsulfur phototrophic bacterium (PNSB) Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate and bicarbonate and produced H(2) when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions). In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate to H(2). H(2) production with acetate and succinate as electron donors was less efficient (40 to 60%), partly because nongrowing cells excreted the intermediary metabolite α-ketoglutarate into the culture medium. The fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of H(2) production. The possibility to use thiosulfate expands the range of electron-donating compounds for H(2) production by PNSBs beyond biomass-based electron donors.

Regulation of carbon flows in the tricarboxylic acid cycle-glyoxylate bypass system in Rhodopseudomonas palustris under different growth conditions

The functional roles of the malate dehydrogenase (MDH) tetrameric and dimeric isoforms in the metabolism of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris, strain f-8pt, was studied with the use of specific inhibitors. It was shown that the enzyme tetrameric form allows the functioning of the glyoxylate cycle and the dimeric form provides for the operation of the tricarboxylic acid cycle.

High resolution crystal structure of Rubrivivax gelatinosus cytochrome c′

The structure of the cytochrome c' from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus was determined using two crystals grown independently at pH 6.3 and pH 8. The resolution attained for the two structures (1.29 A and 1.50 A for the crystals at high and low pH, respectively) is the highest to date for this class of proteins. The two structures were compared in detail in an attempt to investigate the influence of pH on the geometry of the haem and of the coordination environment of the Fe(III) ion. However, while the results suggest some small propensity for the movement of the metal atom out of the plane of the haem ring upon pH increase, the accuracy of the measurements at these two pH below the pK of the axial histidine is not sufficient to provide hard evidence of a shift in the iron position and associated changes.

EFFECTS OF THE HERBICIDE, BUTACHLOR, ON NITROGEN FIXATION IN PHOTOTROPHIC NONSULFUR BACTERIA

In an effort to identify possible microbes for seeking bioagents for remediation of herbicide-contaminated soils, seven species of phototrophic nonsulfur bacteria (Rhodobacter capsulatus and sphaeroides, Rhodospirillum rubrum, Rhodopseudomonas acidophila, blastica and viridis, Rhodomicrobium vannielii) were grown in the presence of the herbicide, butachlor, and bacterial growth rates and nitrogen fixation were measured with different carbon sources. Under general conditions, all species showed 17-53% reductions in growth rate following butachlor treatment. Under nitrogen-fixing conditions, Rb. capsulatus and Rs. rubrum showed 1-4% increases in the growth rates and 2-10% increases in nitrogen-fixing abilities, while the other 5 species showed decreases of 17-47% and 17-85%, respectively. The finding that Rp. acidophila, Rp. blastica, Rp. viridis and Rm. vannielii showed stronger inhibitions of nitrogenase activity seems to indicate that species in genera Rhodobacter and Rhodospirillum are less influenced by butachlor than those in Rhodopseudomonas and Rhodomicrobium in terms of nitrogen-fixing ability. Overall, nitrogenase activity was closely correlated with both growth rate and glutamine synthetase activity (representing nitrogen metabolism). When the carbon sources were compared, pyruvate (three carbons) was best for all species in terms of growth rate and nitrogen fixation, with malate (four carbons) showing intermediate values and ribose (five carbons) showing the lowest; these trends did not change in response to butachlor treatment. We verified that each of the 7 species had a plasmid (12.2~23.5 Kb). We found that all 7 species could use butachlor as a sole carbon source and 3 species were controlled by plasmid-born genes, but it is doubtful whether plasmid-born genes were responsible to nitrogen fixation.

Involvement of the C-terminal extension of the alpha polypeptide and of the PucC protein in LH2 complex biosynthesis in Rubrivivax gelatinosus.

The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes. In particular, the puc operon contains only the pucB and pucA genes encoding the beta and alpha polypeptides of the light-harvesting 2 (LH2) complex. Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation. The transcription of pucBA and pucC has been studied. No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis. The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb. The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable. It was reported that the alpha polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro. The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated. Two mutants with C-terminal deletions of 13 and 18 residues have been constructed. Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2-, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis. Another important factor involved in the LH2 biogenesis is the PucC protein. To gain insight into the function of this protein in R. gelatinosus, we constructed and characterized a PucC mutant. The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions. This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus,

In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (1 microM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (1 mM), was capable of partially (approximately 25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of 1 W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N(2) and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H(2) from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E(m) < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E(m) approximately -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20% decrease in the intensity of the FeWco signal, indicating that the cofactor can be enzymatically reduced only to a small extent. The data presented in the current study demonstrate the pivotal role of molybdenum in optimal N(2) fixation and provides direct evidence that the inability of a tungsten-substituted nitrogenase to reduce N(2) is due to the difficulty to effectively reduce the FeW cofactor beyond its semi-reduced state.