New perspectives on butyrate assimilation in Rhodospirillum rubrum S1H under photoheterotrophic conditions.

BACKGROUND: The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. RESULTS: In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. CONCLUSION: Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.

Reductive tricarboxylic acid cycle enzymes and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin-cycle mutant.

Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin-cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.

Inhibition of bacteriochlorophyll biosynthesis in the purple phototrophic bacteria Rhodospirillumrubrum and Rhodobacter capsulatus grown in the presence of a toxic concentration of selenite.

Background In many works, the chemical composition of bacterially-produced elemental selenium nanoparticles (Se0-nanoparticles) was investigated using electron dispersive X-ray analysis. The results suggest that these particles should be associated with organic compounds. However, a complete analysis of their chemical composition is still missing. Aiming at identifying organic compounds associated with the Se0-nanoparticles produced by the purple phototrophic bacteria Rhodospirillum rubrum and Rhodobacter capsulatus (α group of the proteobacteria), we used MALDI-TOF spectrometry.Results This technic revealed that numerous signals obtained from particles produced by both species of bacteria were from metabolites of the photosynthetic system. Furthermore, not only bacteriochlorophyll a, bacteriopheophytin a, and bacteriopheophorbide a, which are known to accumulate in stationary phase cultures of these bacteria grown phototrophically in the absence of selenite, were identified. The particles were also associated with intermediary metabolites of the bacteriochlorophyll a biosynthesis pathway such as protoporphyrin IX, protoporphyrin IX monomethyl ester, bacteriochlorophyllide a and, most likely, Mg-protoporphyrin IX-monomethyl ester, as well as with oxidation products of the substrates of protochlorophyllide reductase and chlorin reductase.Conclusion Accumulation of intermediary metabolites of the bacteriochlorophyll biosynthesis pathway in these purple phototrophic bacteria was attributed to inhibition of oxygen-sensitive enzymes involved in this pathway. Consistent with this interpretation it has been reported that these bacteria reduce selenite intracellularly, that they contain high levels of glutathione and that the reduction of selenite with glutathione is a very fast reaction accompanied by the production of reactive oxygen species. As many enzymes involved in the biosynthesis of bacteriochlorophyll contain [Fe-S] clusters in their active site, which are known to be degraded in the presence of reactive oxygen species as well as in the presence of molecular oxygen, we concluded that the substrates of these enzymes accumulate in cells during selenite reduction.Association of metabolites of bacteriochlorophyll biosynthesis and degradation with the Se0-nanoparticles produced by Rhodospirillum rubrum and Rhodobacter capsulatus is proposed to result from coating of the nanoparticles with the intracytoplasmic membrane of these bacteria, where the photochemical apparatus is concentrated.

Genetic Plasticity and Ethylmalonyl Coenzyme A Pathway during Acetate Assimilation in Rhodospirillum rubrum S1H under Photoheterotrophic Conditions.

Purple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility. Rhodospirillum rubrum has been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable in Rs. rubrum, the citramalate cycle pathway and the ethylmalonyl-coenzyme A (CoA) pathway are proposed as alternative anaplerotic pathways for acetate assimilation. However, despite years of debate, neither has been confirmed to be essential. Here, using functional genomics, we demonstrate that the ethylmalonyl-CoA pathway is required for acetate photoassimilation. Moreover, an unexpected reversible long-term adaptation is observed, leading to a drastic decrease in the lag phase characterizing the growth of Rs. rubrum in the presence of acetate. Using proteomic and genomic analyses, we present evidence that the adaptation phenomenon is associated with reversible amplification and overexpression of a 60-kb genome fragment containing key enzymes of the ethylmalonyl-CoA pathway. Our observations suggest that a genome duplication and amplification phenomenon is not only involved in adaptation to acute stress but can also be important for basic carbon metabolism and the redox balance.IMPORTANCE Purple nonsulfur bacteria represent a major group of anoxygenic photosynthetic bacteria that emerged as a promising resource for biotechnology because of their great metabolic versatility and ability to grow under various conditions. Rhodospirillum rubrum S1H has notably been selected by the European Space Agency to colonize its life support system, called MELiSSA, due to its capacity to perform photoheterotrophic assimilation of volatile fatty acids (VFAs), mainly acetate. VFAs are valuable carbon sources for many applications, combining bioremediation of contaminated environments with the generation of added-value products. Acetate is one of the major volatile fatty acids generated as a by-product of fermentation processes. In Rs. rubrum, purple nonsulfur bacteria, the assimilation of acetate is still under debate since two different pathways have been proposed. Here, we clearly demonstrate that the ethylmalonyl-CoA pathway is the major anaplerotic pathway for acetate assimilation in this strain. Interestingly, we further observed that gene duplication and amplification, which represent a well-known phenomenon in antibiotic resistance, also play a regulatory function in carbon metabolism and redox homeostasis.

An aerobic detoxification photofermentation by Rhodospirillum rubrum for converting soy sauce residue into feed with moderate pretreatment.

This paper reports an effective process for converting soy sauce residue into feeds by combining moderate acid hydrolysis and ammonization with Rhodospirillum rubrum fermentation. After pretreatment with dilute sulfuric or phosphoric acid (1%, w/w) at 100 °C, materials were subjected to fermentation under several gases (N2, CO2, and air) and different light intensities in a 2-L fermentor. Following sulfuric acid treatment, the true protein increased from 188 to 362 g kg-1 and the crude fiber decreased from 226 to 66 g kg-1 after fermentation at 0.5 L min-1 L-1 of air flow and a light intensity of 750 lx and following phosphoric acid treatment, the true protein increased by 90% and the crude fiber decreased by 67% after fermentation at 0.6 L min-1 L-1 of air flow and a light intensity of 600 lx Other contents, including crude fat, crude ash, phosphorus, sulfur, sulfur-containing amino acids, sodium chloride, and calcium, were also improved for use as feed. Meantime, some toxic substances, including furfural, hydroxymethylfurfural (5-HMF), acetic acid, phenol, and cresol, which were produced by the pretreatments, could be removed by 12-32, 5-8, 49-53, 7-8, and 7-8%, respectively; and total sugars, glucose, and xylose could be utilized by 68-69, 71-72, and 63-67% respectively. The quality of soy sauce residue is improved for use as feed and some toxic substances can be decreased via the R. rubrum fermentation.

Design of a tailor-made platform for syngas bioconversion into polyhydroxybutyrate.

Biodegradable polymers such as polyhydroxybutyrate (PHB) are part of the emerging portfolio of renewable materials, which are addressing the issue of plastic waste. Syngas, as a cheap, renewable and sustainable resource that can be obtained from biomass or waste, is viewed as an excellent feedstock for different bioprocesses, including syngas to PHB bioconversion. However, due to the hazardous nature of syngas, it is of utmost importance to consider safety aspects of the process. This recently developed tailor-made platform for safe syngas fermentation and PHB production addresses safety aspects and demonstrates the importance of robust online and in-line analytical tools allowing for monitoring and controlling of this bioprocess.

Syngas obtained by microwave pyrolysis of household wastes as feedstock for polyhydroxyalkanoate production in Rhodospirillum rubrum.

The massive production of urban and agricultural wastes has promoted a clear need for alternative processes of disposal and waste management. The potential use of municipal solid wastes (MSW) as feedstock for the production of polyhydroxyalkanoates (PHA) by a process known as syngas fermentation is considered herein as an attractive bio-economic strategy to reduce these wastes. In this work, we have evaluated the potential of Rhodospirillum rubrum as microbial cell factory for the synthesis of PHA from syngas produced by microwave pyrolysis of the MSW organic fraction from a European city (Seville). Growth rate, uptake rate, biomass yield and PHA production from syngas in R. rubrum have been analysed. The results revealed the strong robustness of this syngas fermentation where the purity of the syngas is not a critical constraint for PHA production. Microwave-induced pyrolysis is a tangible alternative to standard pyrolysis, because it can reduce cost in terms of energy and time as well as increase syngas production, providing a satisfactory PHA yield.

New waves underneath the purple strain.

Successful merging of chemical and biotechnological operations is essential to achieve cost-efficient industrialization of bio-based processes. The demonstration of the use of syngas, derived from microwave assisted pyrolysis of municipal solid waste, for the improved growth and poly-3-hydroxybutyrate production in Rhodospirillium rubrum, stands out as an example of the synergistic contribution of chemical engineering and applied microbiology to sustainable biomaterial manufacturing, paving the way to similar applications for other syngas derived bioproducts.

Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining.

Poly(3-hydroxyalkanoates) (PHAs) are bio-based and biodegradable polyesters which have been considered as a promising alternative to petrol-based plastics. Their bacterial production is a dynamic process in which intracellular polymerization and depolymerization are closely linked and depend on the availability of carbon substrates and other nutrients. These dynamics require a fast and quantitative method to determine the optimal harvest-time of PHA containing cells or to adjust carbon supply. In principle, flow cytometry (FCM) is an ideal tool that suits these requirements and, in addition, provides data on the PHA content of different cell populations. However, FCM-based PHA quantification methods have often relied on laborious sample preparation including washing steps and long incubation times. Here, we introduce a fast method based on double-staining using BODIPY 493/503 for PHA staining and SYTO 62 for DNA that allows acquiring reliable fluorescence and cell count data in <10min. Finally, fed-batch experiments with Pseudomonas putida KT2440 and Rhodospirillum rubrum S1 revealed that the method was robust and independent of the strain and type of PHA (medium-chain-length [mcl-] and short-chain-length [scl-] PHA, respectively). Interestingly, the specific PHA fluorescence was in case of mcl-PHA larger than for scl-PHA, probably reflecting the different material properties (e.g., specific density, hydrophilicity and crystallinity).

Metabolic Regulation as a Consequence of Anaerobic 5-Methylthioadenosine Recycling in Rhodospirillum rubrum.

UNLABELLED: Rhodospirillum rubrum possesses a novel oxygen-independent, aerobic methionine salvage pathway (MSP) for recycling methionine from 5-methylthioadenosine (MTA), the MTA-isoprenoid shunt. This organism can also metabolize MTA as a sulfur source under anaerobic conditions, suggesting that the MTA-isoprenoid shunt may also function anaerobically as well. In this study, deep proteomics profiling, directed metabolite analysis, and reverse transcriptase quantitative PCR (RT-qPCR) revealed metabolic changes in response to anaerobic growth on MTA versus sulfate as sole sulfur source. The abundance of protein levels associated with methionine transport, cell motility, and chemotaxis increased in the presence of MTA over that in the presence of sulfate. Purine salvage from MTA resulted primarily in hypoxanthine accumulation and a decrease in protein levels involved in GMP-to-AMP conversion to balance purine pools. Acyl coenzyme A (acyl-CoA) metabolic protein levels for lipid metabolism were lower in abundance, whereas poly-ß-hydroxybutyrate synthesis and storage were increased nearly 10-fold. The known R. rubrum aerobic MSP was also shown to be upregulated, to function anaerobically, and to recycle MTA. This suggested that other organisms with gene homologues for the MTA-isoprenoid shunt may also possess a functioning anaerobic MSP. In support of our previous findings that ribulose-1,5-carboxylase/oxygenase (RubisCO) is required for an apparently purely anaerobic MSP, RubisCO transcript and protein levels both increased in abundance by over 10-fold in cells grown anaerobically on MTA over those in cells grown on sulfate, resulting in increased intracellular RubisCO activity. These results reveal for the first time global metabolic responses as a consequence of anaerobic MTA metabolism compared to using sulfate as the sulfur source. IMPORTANCE: In nearly all organisms, sulfur-containing byproducts result from many metabolic reactions. Unless these compounds are further metabolized, valuable organic sulfur is lost and can become limiting. To regenerate the sulfur-containing amino acid methionine, organisms typically employ one of several variations of a "universal" methionine salvage pathway (MSP). A common aspect of the universal MSP is a final oxygenation step. This work establishes that the metabolically versatile bacterium Rhodospirillum rubrum employs a novel MSP that does not require oxygen under either aerobic or anaerobic conditions. There is also a separate, dedicated anaerobic MTA metabolic route in R. rubrum This work reveals global changes in cellular metabolism in response to anaerobic MTA metabolism compared to using sulfate as a sulfur source. We found that cell mobility and transport were enhanced, along with lipid, nucleotide, and carbohydrate metabolism, when cells were grown in the presence of MTA.

Magnesium ions improving the growth and organics reduction of Rhodospirillum rubrum cultivated in sewage through regulating energy metabolism pathways.

Rhodospirillum rubrum has the potential for biomass resource recycling combined with sewage purification. However, low biomass production and yield restricts the potential for sewage purification. This research investigated the improvement of biomass production, yield and organics reduction by Mg²âº in R. rubrum wastewater treatment. Results showed that with optimal dosage (120 mg/L), biomass production reached 4,000 mg/L, which was 1.5 times of that of the control group. Biomass yield was improved by 43.3%. Chemical oxygen demand (COD) removal reached over 90%. Hydraulic retention time was shortened by 25%. Mechanism analysis indicated that Mg²âº enhanced the isocitrate dehydrogenase and Ca²âº/Mg²âº-ATPase activities, bacteriochlorophyll content on respiration and photophosphorylation. These effects then enhanced ATP production, which led to more biomass accumulation and COD removal. With 120 mg/L Mg²âº dosage, the isocitrate dehydrogenase and Ca²âº/Mg²âº-ATPase activities, bacteriochlorophyll content, ATP production were improved, respectively, by 33.3%, 50%, 67%, 41.3% compared to those of the control group.

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from unrelated carbon sources in engineered Rhodospirillum rubrum.

Different genes encoding pyridine nucleotide transhydrogenases (pntAB, udhA) and acetoacetyl-CoA reductases (phaB) were heterologously overexpressed in Rhodospirillum rubrum S1. A recombinant strain, which harbored the gene encoding the membrane-bound transhydrogenase PntAB from Escherichia coli MG1655 and the phaB1 gene coding for an NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha H16, accumulated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [Poly(3HB-co-3HV)] with a 3HV fraction of up to 13 mol% from fructose. This was a 13-fold increase of the 3HV content when compared to the wild-type strain. Higher contents of 3HV are known to reduce the brittleness of this polymer, which is advantageous for most applications. The engineered R. rubrum strain was also able to synthesize this industrially relevant copolymer from CO2 and CO from artificial synthesis gas (syngas) with a 3HV content of 56 mol%. The increased incorporation of 3HV was attributed to an excess of propionyl-CoA, which was generated from threonine and related amino acids to compensate for the intracellular redox imbalance resulting from the transhydrogenase reaction. Thereby, our study presents a novel, molecular approach to alter the composition of bacterial PHAs independently from external precursor supply. Moreover, this study also provides a promising production strain for syngas-derived second-generation biopolymers.

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.

Quorum sensing influences growth and photosynthetic membrane production in high-cell-density cultivations of Rhodospirillum rubrum.

BACKGROUND: The facultative anoxygenic photosynthetic bacterium Rhodospirillum rubrum exhibits versatile metabolic activity allowing the adaptation to rapidly changing growth conditions in its natural habitat, the microaerobic and anoxic zones of stagnant waters. The microaerobic growth mode is of special interest as it allows the high-level expression of photosynthetic membranes when grown on succinate and fructose in the dark, which could significantly simplify the industrial production of compounds associated with PM formation. However, recently we showed that PM synthesis is no longer inducible when R. rubrum cultures are grown to high cell densities under aerobic conditions. In addition a reduction of the growth rate and the continued accumulation of precursor molecules for bacteriochlorophyll synthesis were observed under high cell densities conditions. RESULTS: In the present work, we demonstrate that the cell density-dependent effects are reversible if the culture supernatant is replaced by fresh medium. We identified six N-acylhomoserine lactones and show that four of them are produced in varying amounts according to the growth phase and the applied growth conditions. Further, we demonstrate that N-acylhomoserine lactones and tetrapyrrole compounds released into the growth medium affect the growth rate and PM expression in high cell density cultures. CONCLUSIONS: In summary, we provide evidence that R. rubrum possesses a Lux-type quorum sensing system which influences the biosynthesis of PM and the growth rate and is thus likely to be involved in the phenotypes of high cell density cultures and the rapid adaptation to changing environmental conditions.

Role of genetic redundancy in polyhydroxyalkanoate (PHA) polymerases in PHA biosynthesis in Rhodospirillum rubrum.

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C(5) and C(6) monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.

Fructose metabolism of the purple non-sulfur bacterium Rhodospirillum rubrum: effect of carbon dioxide on growth, and production of bacteriochlorophyll and organic acids.

During fermentative metabolism, carbon dioxide fixation plays a key role in many bacteria regarding growth and production of organic acids. The present contribution, dealing with the facultative photosynthetic bacterium Rhodospirillum rubrum, reveals not only the strong influence of ambient carbon dioxide on the fermentative break-down of fructose but also a high impact on aerobic growth with fructose as sole carbon source. Both growth rates and biomass yield increased with increasing carbon dioxide supply in chemoheterotrophic aerobic cultures. Furthermore, intracellular metabolite concentration measurements showed almost negligible concentrations of the tricarboxylic acid cycle intermediates succinate, fumarate and malate under aerobic growth, in contrast to several metabolites of the glycolysis. In addition, we present a dual phase fed-batch process, where an aerobic growth phase is followed by an anaerobic production phase. The biosynthesis of bacteriochlorophyll and the secretion of organic acids were both affected by the carbon dioxide supply, the pH value and by the cell density at the time of switching from aerobic to anaerobic conditions. The formation of pigmented photosynthetic membranes and the amount of bacteriochlorophyll were inversely correlated to the secretion of succinate. Accounting the high biotechnological potential of R. rubrum, optimization of carbon dioxide supply is important because of the favored application of fructose-containing fermentable feedstock solutions in bio-industrial processes.

Identification of a new gene required for the biosynthesis of rhodoquinone in Rhodospirillum rubrum.

Rhodoquinone (RQ) is a required cofactor for anaerobic respiration in Rhodospirillum rubrum, and it is also found in several helminth parasites that utilize a fumarate reductase pathway. RQ is an aminoquinone that is structurally similar to ubiquinone (Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is not found in humans or other mammals, and therefore, the inhibition of its biosynthesis may provide a novel antiparasitic drug target. To identify a gene specifically required for RQ biosynthesis, we determined the complete genome sequence of a mutant strain of R. rubrum (F11), which cannot grow anaerobically and does not synthesize RQ, and compared it with that of a spontaneous revertant (RF111). RF111 can grow anaerobically and has recovered the ability to synthesize RQ. The two strains differ by a single base pair, which causes a nonsense mutation in the putative methyltransferase gene rquA. To test whether this mutation is important for the F11 phenotype, the wild-type rquA gene was cloned into the pRK404E1 vector and conjugated into F11. Complementation of the anaerobic growth defect in F11 was observed, and liquid chromatography-time of flight mass spectrometry (LC-TOF-MS) analysis of lipid extracts confirmed that plasmid-complemented F11 was able to synthesize RQ. To further validate the requirement of rquA for RQ biosynthesis, we generated a deletion mutant from wild-type R. rubrum by the targeted replacement of rquA with a gentamicin resistance cassette. The ΔrquA mutant exhibited the same phenotype as that of F11. These results are significant because rquA is the first gene to be discovered that is required for RQ biosynthesis.

Novel substrate (algal protein) for cultivation of Rhodospirillum rubrum.

Rhodospirillum rubrum was grown under light anaerobic conditions with phycocyanin (C-pc) extracted from Spirulina platensis as the sole source of carbon and nitrogen. When grown under these conditions cellular components like lipids, carbohydrates, protein, carotenoids, bacteriochlorophyll were similar to the one grown with malic acid and ammonium chloride. Growth of R. rubrum increased with increase in concentration of C-pc (200 to 1000 mg/l). R. rubrum also utilized C-pc under dark anaerobic condition. With both malic acid and C-pc as carbon sources C-pc was consumed only after exhaustion of malic acid under light anaerobic condition. No aberration of cell morphology was seen under scanning electron microscope (SEM). R. rubrum utilized both phycocyanobilin and phycoprotein individually as well as in combination. When grown with 1000 mg/l of phycoprotein 450 mg/l of biomass was obtained, and with combination of phycocyanobilin (75 mg/l) and phycoprotein (925 mg/l) 610 mg/l of biomass was obtained. Phycocyanobilin alone did not inhibit the growth of R. rubrum. Utilization of C-pc with protease like activity was observed in plate assay. Protease like activity was also observed as zones around the colonies in plates containing sterilized casein, gelatin and filter sterilized bovine serum albumin. No amino acids were detected in the supernatant when analyzed with ninhydrin. Extracellular protease like activity was highest when C-pc was used as substrate (2.8 U/ml). Intracellular protease like activity was not detected in cell free extracts.

The poor growth of Rhodospirillum rubrum mutants lacking RubisCO is due to the accumulation of ribulose-1,5-bisphosphate.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO(2) fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO(2) to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of cbbM mutants in Rhodospirillum rubrum that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for R. rubrum to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the cbb operon (cbbEFPT) in R. rubrum, as seen with related organisms, and define the transcriptional organization of the cbb genes.

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum.

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.