Enhanced aniline degradation by Desulfatiglans anilini in a synthetic microbial community with the phototrophic purple sulfur bacterium Thiocapsa roseopersicina.

Desulfatiglans anilini is a sulfate-reducing bacterium (SRB) capable of oxidizing aniline, although growth and aniline turnover rates are slow, making it difficult to analyze the metabolism of the strain. Therefore, this study was designed to investigate the effect of sulfide on growth of D. anilini cultures, in order to improve its growth and aniline turnover rates, and study the biochemical mechanisms of sulfide inhibition. Hydrogen sulfide was found to inhibit growth of D. anilini, regardless of whether the strain was grown with aniline or phenol, and complete inhibition was observed at 20mM hydrogen sulfide. For improving the growth of D. anilini with aniline, the sulfide-consuming phototrophic bacterium Thiocapsa roseopersicina was co-cultured in a synthetic microbial community with D. anilini using a co-cultivation device that continuously removed hydrogen sulfide from the culture. The doubling time of D. anilini with aniline was 15 days in the co-cultivation device, compared to 26 days in the absence of a sulfide-oxidizing partner. Moreover, the aniline degradation rate was significantly increased by a factor of 2.66 during co-cultivation of D. anilini with T. roseopersicina. The initial carboxylation reaction during aniline degradation was measured in cell-free extracts of D. anilini with carbon dioxide (CO2) as a co-substrate in the presence of aniline and ATP. The effects of hydrogen sulfide on this aniline carboxylating system and on phenylphosphate synthase activity for phenol activation were studied, and it was concluded that hydrogen sulfide severely inhibited these enzyme activities.

HupO, a Novel Regulator Involved in Thiosulfate-Responsive Control of HupSL [NiFe]-Hydrogenase Synthesis in Thiocapsa roseopersicina.

[NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacterium Thiocapsa roseopersicina harbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2 in Rhodobacter capsulatus and Cupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional in T. roseopersicina and that thiosulfate is a regulating factor of hup expression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase in T. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in that hup expression was decreased significantly in the hox1 mutant. This reduction in Hup synthesis could be reversed by mutation of hupO, which resulted in strongly elevated hup expression, as well as Hup protein levels, and concomitant in vivo hydrogen uptake activity in the hox1 mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependent hup expression was shown in the hupO mutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network in T. roseopersicina.

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS.

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.

The autocatalytic step is an integral part of the hydrogenase cycle.

We earlier proved the involvement of an autocatalytic step in the oxidation of H(2) by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H(2), we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme-substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.

Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer.

The potential of hydrogen as a clean renewable fuel source and the finite reserves of platinum metal to be utilized in hydrogen production catalysts have provided the motivation for the development of non-noble metal-based solutions for catalytic hydrogen production. There are a number of microorganisms that possess highly efficient hydrogen production catalysts termed hydrogenases that generate hydrogen under certain metabolic conditions. Although hydrogenases occur in photosynthetic microorganisms, the oxygen sensitivity of these enzymes represents a significant barrier in directly coupling hydrogen production to oxygenic photosynthesis. To overcome this barrier, there has been considerable interest in identifying or engineering oxygen tolerant hydrogenases or generating mimetic systems that do not rely on oxygen producing photocatalysts. In this work, we demonstrate photo-induced hydrogen production from a stable [NiFe]-hydrogenase coupled to a [Ru(2,2'-bipyridine)(2)(5-amino-1,10-phenanthroline)](2+) photocatalyst. When the Ru(II) complex is covalently attached to the hydrogenase, photocatalytic hydrogen production occurs more efficiently in the presence of a redox mediator than if the Ru(II) complex is simply present in solution. Furthermore, sustained hydrogen production occurs even in the presence of oxygen by presumably creating a local anoxic environment through the reduction of oxygen similar to what is proposed for oxygen tolerant hydrogenases. These results provide a strong proof of concept for engineering photocatalytic hydrogen production in the presence of oxygen using biohybrid mimetic systems.

The effect of sulfur compounds on H2 evolution/consumption reactions, mediated by various hydrogenases, in the purple sulfur bacterium, Thiocapsa roseopersicina.

The influence of reduced sulfur compounds (including stored S(0)) on H(2) evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S(2)O (3) (2-) , SO (3) (2-) , S(2-) and S(0) as electron donors for light-dependent H(2) production. Dark H(2) evolution from organic substrates via Hox hydrogenase was inhibited by S(0). Under light conditions, endogenous H(2) uptake by Hox or Hup hydrogenases was suppressed by S compounds. CO(2)-dependent H(2) uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H(2) consumption via Hyn hydrogenase was connected to utilization of S(0) as an electron acceptor and resulted in the accumulation of H(2)S. In wild type BBS, with high levels of stored S(0), dark H(2) production from organic substrates was significantly lower, but H(2)S accumulation significantly higher, than in the mutant GB1121(Hox(+)). There is a possibility that H(2) produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S(0).

Modular broad-host-range expression vectors for single-protein and protein complex purification.

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

Genes involved in the biosynthesis of photosynthetic pigments in the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina.

A pigment mutant strain of the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina BBS was isolated by plasposon mutagenesis. Nineteen open reading frame, most of which are thought to be genes involved in the biosynthesis of carotenoids, bacteriochlorophyll, and the photosynthetic reaction center, were identified surrounding the plasposon in a 22-kb-long chromosomal locus. The general arrangement of the photosynthetic genes was similar to that in other purple photosynthetic bacteria; however, the locations of a few genes occurring in this region were unusual. Most of the gene products showed the highest similarity to the corresponding proteins in Rubrivivax gelatinosus. The plasposon was inserted into the crtD gene, likely inactivating crtC as well, and the carotenoid composition of the mutant strain corresponded to the aborted spirilloxanthin pathway. Homologous and heterologous complementation experiments indicated a conserved function of CrtC and CrtD in the purple photosynthetic bacteria. The crtDC and crtE genes were shown to be regulated by oxygen, and a role of CrtJ in aerobic repression was suggested.