Thiocapsa Roseopersicina BBS Research Articles

The Complete Genome of a Novel Typical Species Thiocapsa bogorovii and Analysis of Its Central Metabolic Pathways.

The purple sulfur bacterium Thiocapsa roseopersicina BBS is interesting from both fundamental and practical points of view. It possesses a thermostable HydSL hydrogenase, which is involved in the reaction of reversible hydrogen activation and a unique reaction of sulfur reduction to hydrogen sulfide. It is a very promising enzyme for enzymatic hydrogenase electrodes. There are speculations that HydSL hydrogenase of purple bacteria is closely related to sulfur metabolism, but confirmation is required. For that, the full genome sequence is necessary. Here, we sequenced and assembled the complete genome of this bacterium. The analysis of the obtained whole genome, through an integrative approach that comprised estimating the Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (DDH) parameters, allowed for validation of the systematic position of T. roseopersicina as T. bogorovii BBS. For the first time, we have assembled the whole genome of this typical strain of a new bacterial species and carried out its functional description against another purple sulfur bacterium: Allochromatium vinosum DSM 180T. We refined the automatic annotation of the whole genome of the bacteria T. bogorovii BBS and localized the genomic positions of several studied genes, including those involved in sulfur metabolism and genes encoding the enzymes required for the TCA and glyoxylate cycles and other central metabolic pathways. Eleven additional genes coding proteins involved in pigment biosynthesis was found.

Effect of Hg2+ on HydSL Hydrogenase of the Purple Sulfur Bacteria Thiocapsa roseopersicina BBS

The inhibitory effect of mercury(II) chloride on the activity and structure of the hydrogenase Thiocapsa roseopersicina BBS was studied. The kinetics of hydrogenase inactivation in the presence of different inhibitor concentrations was determined. The irreversible nature of Hg²⁺ action was established, and hydrogenase inhibition constants at different temperatures were determined. The presence of this inhibitor in enzyme solution significantly reduced its stability and caused denaturation at temperatures above 50°C. In the process of enzyme incubation with Hg²⁺, the absorption band fades in the visible region of the spectrum, indicating the destruction of iron–sulfur clusters. Comparative analysis of the infrared Fourier spectra of hydrogenase without the addition and after incubation with inhibitor indicates the destruction of the NiFe active center.

Interaction of HydSL hydrogenase from Thiocapsa roseopersicina with cyanide leads to destruction of iron-sulfur clusters

The effects of cyanide on enzymatic activity and absorption spectra in the visible and mid-IR (2150–1850cm−1) regions were characterized for purified HydSL hydrogenase from the purple sulfur bacterium Thiocapsa (T.) roseopersicina BBS. Prolonged incubation (over hours) of T. roseopersicina hydrogenase with exogenous cyanide was shown to result in an irreversible loss of activity of the enzyme in both the oxidized (as isolated) and H2-reduced states. The frequency position of the active site CO and CN− ligand stretching bands in the Fourier transform infrared (FTIR) spectrum of the oxidized form of hydrogenase was not influenced by cyanide treatment. The 410-nm absorption band characteristic of hydrogenase iron‑sulfur clusters showed a bleaching concomitantly with cyanide inactivation. A new band at 2038cm−1 was present in the FTIR spectrum of the cyanide-inactivated preparation, which band is assignable to ferrocyanide as a possible product of a destructive interaction of hydrogenase with cyanide. The results are interpreted in terms of a slow destruction of iron‑sulfur clusters of hydrogenase in the presence of cyanide accompanied by a release of iron ions in the form of ferrocyanide into the surrounding solution. Such a slow and irreversible cyanide-dependent inactivation seems to be complementary to a recently described rapid, reversible inhibitory reaction of cyanide with the active site of hydrogenases [S.V. Hexter, M.-W. Chung, K.A. Vincent, F.A. Armstrong, J. Am. Chem. Soc. 136 (2014) 10470–10477].

Modeling three-dimensional structure of two closely related Ni-Fe hydrogenases.

The results of homology modeling of HydSL, a NiFe-hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina BBS, and deep-water bacterium Alteromonas macleodii deep ecotype are presented in this work. It is shown that the models have larger confidence level than earlier published ones; full-size models of these enzymes are presented for the first time. The C-end fragment of small subunit of T. roseopersicina hydrogenase is shown to have random orientation in relation to the main protein globule. The obtained models of this enzyme have a large number of ion pairs, as well as thermostable HydSL hydrogenase from Allochromatium vinosum, in contrast to thermostable HydSL hydrogenase from Alt. macleodii and thermolabile HydAB hydrogenase from Desulfovibrio vulgaris. The possible determinant of oxygen stability of studied hydrogenases could be the lack of several intramolecular tunnels. Hydrophobic and electrostatic surfaces were mapped in order to find out possible pathways of coupling hydrogenase to electron-transferring chains, as well as methods for construction of artificial photobiohydrogen-producing systems.

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.

Моделирование пространственной структуры гидрогеназы HydSL пурпурной серной бактерии Thiocapsa roseopersicina BBS

Моделирование пространственной структуры гидрогеназы HydSL пурпурной серной бактерии Thiocapsa roseopersicina BBS

Relationship between PHA and hydrogen metabolism in the purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS

Purple sulfur phototrophic bacteria accumulate various storage materials, such as sulfur globules, glycogen or polyhydroxy alkanoates (PHAs) under appropriate conditions. The formation of these materials requires reducing power which might be recovered upon their breakdown. This work aims at the understanding of the metabolism of PHA and its link to the nitrogenase mediated in vivo H2 evolution in Thiocapsa roseopersicina BBS. The strain could accumulate 30% of the dry cell weight in the form of PHAs. Analysis of the genome sequence revealed the loci involved in PHAs synthesis and degradation. Phylogenetic analysis indicated independent evolution of the anabolic and catabolic proteins. A mutant carrying deleted PHA biosynthesis genes has been created in a host containing nitrogenase but none of the hydrogenases. Determination of the H2 evolving capacity of the mutant revealed significantly reduced H2 production in PHA deficient cells. Addition of excess electron sources such as thiosulfate stimulated the H2 production via multiple effects.

Electron‐transfer subunits of the NiFe hydrogenases in Thiocapsa roseopersicina BBS

Thiocapsa roseopersicina BBS contains at least three different active NiFe hydrogenases: two membrane-bound enzymes and one apparently localized in the cytoplasm. In addition to the small and large structural subunits, additional proteins are usually associated with the NiFe hydrogenases, connecting their activity to other redox processes in the cells. The operon of the membrane-associated hydrogenase, HynSL, has an unusual gene arrangement: between the genes coding for the large and small subunits, there are two open reading frames, namely isp1 and isp2. Isp1 is a b-type haem-containing transmembrane protein, whereas Isp2 displays marked sequence similarity to the heterodisulfide reductases. The other membrane-bound (Hup) NiFe hydrogenase contains the hupC gene, which codes for a cytochrome b-type protein that probably plays a role in electron transport. The operon of the NAD(+)-reducing Hox hydrogenase contains a hoxE gene. In addition to the hydrogenase and diaphorase parts of the complex, the fifth HoxE subunit may serve as a third redox gate of this enzyme. The physiological functions of these putative electron-mediating subunits were studied by disruption of their genes. The deletion of some accessory proteins dramatically reduced the in vivo activities of the hydrogenases, although they were fully active in vitro. The absence of HupC resulted in a decrease in HupSL activity in the membrane, but removal of the Isp1 and Isp2 proteins did not have any significant effect on the location of HynSL activity. Through the use of a tagged HoxE protein, the whole Hox hydrogenase pentamer could be purified as an intact complex.

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).

A novel approach for biohydrogen production

H 2 is considered as the ultimate cleanest energy carrier to be generated from renewable sources. Biohydrogen production is carried out using the enzymes nitrogenase or hydrogenase. The purple sulphur phototrophic bacterium, Thiocapsa roseopersicina BBS contains a nitrogenase and several NiFe hydrogenases. Hydrogenases can be used as fuel cell H 2 splitting catalyst and nitrogenase(s) and hydrogenases in biological H 2 production. The membrane-associated Hyn and Hup hydrogenases preferentially work in the H 2 uptake direction in vivo, the cytoplasmic Hox enzyme is a bidirectional enzyme. Non-nitrogen-fixing conditions, which render Hup and Hyn non-functioning, lead to sustained net hydrogen production via the Hox enzyme. The cumulative H 2 production by the apparently constitutive Hox and the inducible nitrogenase may become comparable under appropriate conditions.

Hydrogen independent expression of hupSL genes in Thiocapsa roseopersicina BBS

The expression of many membrane bound [NiFe] hydrogenases is regulated by their substrate molecule, hydrogen. The HupSL hydrogenase, encoded in the hupSLCDHIR operon, probably plays a role in hydrogen recycling in the phototrophic purple bacterium, Thiocapsa roseopersicina BBS. RpoN, coding for sigma factor 54, was shown to be important for expression, suggesting a regulated biosynthsis from the hup gene cluster. The response regulator gene, hupR, has been identified in the hup operon and expression of hupSL was reduced in a chromosomal hupR mutant, which indicated that HupR was implicated in the activation process. The hupT and hupUV genes were isolated, and show similarity to the histidine kinase element of the H2-driven signal transduction system and to the regulatory hydrogenases of Ralstonia eutropha and Rhodobacter capsulatus, respectively. Although the genes of the entire H2 sensing and regulation system were present, the expression of the hupSL genes was not affected by the presence or absence of H2. Using reverse transcription PCR, we could not detect any mRNA specific to the hupTUV genes in cells grown under diverse conditions. The hupT and hupUV mutant strains had the same phenotype as the wild-type strains. The hupT gene product, expressed from a plasmid, repressed HupSL synthesis as expected while introduction of actively expressed hupTUV genes together derepressed the HupSL activity in T. roseopersicina. The gene product of hupUV behaves similarly to other regulatory hydrogenases and shows H-D exchange activity.

The hydrogenases of Thiocapsa roseopersicina

The purple sulphur phototrophic bacterium, Thiocapsa roseopersicina BBS, contains several NiFe hydrogenases. One of these enzymes (HynSL) is membrane associated, remarkably stable and can be used for practical applications. HupSL is also located in the photosynthetic membrane, its properties are similar to other known Hup-type NiFe hydrogenases. A third hydrogenase activity was located in the soluble fraction and was analogous to the NAD-reducing hydrogenases of cyanobacteria. The hoxEFUYH genes are transcribed together. HoxE is needed for the in vivo electron flow to and from the soluble hydrogenase. Some of the accessory genes were identified using random mutagenesis, and sequencing of the T. roseopersicina genome is in progress. The HupD, HynD and HoxW gene products corresponded to the proteases processing the C-termini of the three NiFe hydrogenases respectively. HypF and HupK mutants displayed significant in vivo H(2) evolution, which could be linked to the nitrogenase activity for the DeltahypF and to the bidirectional Hox activity in the DeltahupK strain. Both HypC proteins are needed for the biosynthesis of each NiFe hydrogenase. The hydrogenase expression is regulated at the transcriptional level through distinct mechanisms. The expression of hynSL is up-regulated under anaerobic conditions with the participation of an FNR (fumarate and nitrate reduction regulator)-type protein, FnrT. Although the genes encoding a typical H(2) sensor (hupUV) and a two-component regulator (hupR and hupT) are present in T. roseopersicina, the system is cryptic in the wild-type BBS strain. The hupR gene was identified in the gene cluster downstream from hupSL. Introduction of actively expressed hupT repressed the hupSL gene expression as expected by analogy with other bacteria.

Improvement of biohydrogen production and intensification of biogas formation

H2 is considered as the ultimate cleanest energy carrier to be generated from renewable sources. This minireview intends to point out that in addition to this function, biologically produced hydrogen is important for environmental biotechnological applications. The purple sulphur phototrophic bacterium, Thiocapsa roseopersicina BBS contains several NiFe hydrogenases. These enzymes can be used e.g., as fuel cell H2 splitting catalyst or in photoheterotrophic H2 production. Microorganisms that supply H2 in situ facilitate the biodegradation of organic material and concomitant biogas production. Fast, efficient, and economic treatment of organic waste, sludge, manure is achieved and generation of significant amount of renewable fuel from waste is intensified. The technology has been field tested under mesophilic and thermophilic conditions with positive results.

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.

Hydrogenases, accessory genes and the regulation of [NiFe] hydrogenase biosynthesis in Thiocapsa roseopersicina

The purple (Sulphur) phototrophic bacterium, Thiocapsa roseopersicina BBS contains several [NiFe] hydrogenases, of which two are membrane bound. Mutant T. roseopersicina cells, carrying deletions in both gene clusters showed hydrogenase activity. This activity was located in the cytoplasm. The structural gene cluster hoxEFUYH was identified and sequenced. In addition, genes homologous to hupUV/hoxBC, the hydrogen sensing hydrogenase have been identified and sequenced. Regulation of hydrogenase biosynthesis was studied in detail for HydSL (renamed HynSL). A random mutagenesis system was optimised for T. roseopersicina. One of the mutations was in a gene similar to that coding for the HypF proteins in other organisms. Inactivation of the hypF gene resulted in a 60-fold increase in hydrogen evolution under nitrogen fixing conditions. In addition to hypF, the following accessory genes were identified: hydD, hupK, hypC1, hypC2, hypDE. Characterisation of the corresponding gene products and search for additional accessory genes are in progress.

Unusual organization of the genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS.

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947-951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567-3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25-31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.

Hydrogenase of purple bacteria: properties and regulation of synthesis

Physico-chemical properties of homogeneous preparations of soluble and membrane-bound hydrogenases from the purple sulfur bacterium Thiocapsa roseopersicina BBS and membrane-bound hydrogenase of Rhodopseudomonas capsulata, strain B10 have been studied. Compared to the enzymes from other sources, the hydrogenase of Thiocapsa roseopersicina is more stable to O2 and products of its reduction (O 2 - , H2O2), temperature and a number of other factors of the medium. A natural electron donor for T. roseopersicina hydrogenase is a low-potential cytochrome C′3, while the natural electron acceptors for hydrogenases of R. capsulata, T. roseopersicina, Ectothiorhodospira shaposhnikovii and Anabaena cylindrica are cytochromes of groups “c” and “b”.