Single-cell genomics-based analysis reveals a vital ecological role of Thiocapsa sp. LSW in the meromictic Lake Shunet, Siberia.

Meromictic lakes usually harbour certain prevailing anoxygenic phototrophic bacteria in their anoxic zone, such as the purple sulfur bacterium (PSB) Thiocapsa sp. LSW (hereafter LSW) in Lake Shunet, Siberia. PSBs have been suggested to play a vital role in carbon, nitrogen and sulfur cycling at the oxic-anoxic interface of stratified lakes; however, the ecological significance of PSBs in the lake remains poorly understood. In this study, we explored the potential ecological role of LSW using a deep-sequencing analysis of single-cell genomics associated with flow cytometry. An approximately 2.7 Mb draft genome was obtained based on the co-assembly of five single-cell genomes. LSW might grow photolithoautotrophically and could play putative roles not only as a carbon fixer and diazotroph, but also as a sulfate reducer/oxidizer in the lake. This study provides insights into the potential ecological role of Thiocapsa sp. in meromictic lakes.

The HydS C-terminal domain of the Thiocapsa bogorovii HydSL hydrogenase is involved in membrane anchoring and electron transfer.

Thiocapsa bogorovii BBS (former name Thiocapsa roseopersicina) contains HydSL hydrogenase belonging to 1e subgroup of NiFe hydrogenases (isp-type). The operon of these hydrogenases contains gene for small subunit (hydS), gene for large subunit (hupL), and genes isp1 and isp2 between them. It is predicted that last two genes code electron transport careers for electron transfer from/to HydSL hydrogenase. However, the interaction between them is unclear. The aim of this study was to determine structural and functional role of T. bogorovii HydS C-terminal end. For this purpose, we modelled all subunits of the complex HydS-HydL-Isp1-Isp2. Hydrophobicity surface analysis of the Isp1 model revealed highly hydrophobic helices suggesting potential membrane localization, as well as the hydrophilic C-terminus, which is likely localized outside of membrane. Isp1 model was docked with models of full length and C-terminal truncated HydSL hydrogenases and results illustrate the possibility of HydSL membrane anchoring via transmembrane Isp1 with essential participation of C-terminal end of HydS in the interaction. C-terminal end of HydS subunit was deleted and our studies revealed that the truncated HydSL hydrogenase detached from cellular membranes in contrast to native hydrogenase. It is known that HydSL hydrogenase in T. bogorovii performs the reaction of elemental sulfur reduction (S0 + H2 = ≥H2S). Cells with truncated HydS produced much less H2S in the presence of H2 and S0. Thus, our data support the conclusion that C-terminal end of HydS subunit participates in interaction of HydSL hydrogenase with Isp1 protein for membrane anchoring and electron transfer.

[Dynamics of Purple Sulfur Bacteria in a Meromictic Saline Lake Shunet (Khakassia, Siberia) in 2007-2013].

According to the results of seasonal monitoring, in 2007-2013 purple sulfur bacteria morphologically similar to Thiocapsa sp. Shira_1 (AJ633676 in EMBL/GenBank) predominated in the anoxygenic phototrophic community of the water column of the meromictic Lake Shira (Khakassia, Siberia). No pronounced seasonal periodicity in the total cell number in the water column was revealed during the period of observation. In some years cell number during the period when the lake was covered with ice was reliably higher than in summer. The absence ofseasonal periodicity was probably due to the low amplitude of seasonal variations in temperature and illumination in the redox zone, resulting from its relatively deep location (12-16 m). The year-to-year dynamics was characterized by a reliable decrease of the total cell number in 2009-2010 and maxima in 2007 and 2011-2012. Canonical correlation analysis revealed that water temperature in the redox zone was the best predictor of the PSB abundance in Lake Shira. Water temperature, in turn, depended on the depth of mixing of the water column. Intense mixing in 2009-2011 was probably responsible for decreased PSB abundance in the lake. On the other hand, the absence of deep winter mixing, resulting in stable conditions in the chemocline, favored the preservation of relatively high PSB biomass. Prediction of circulation depth, which.depends mainly on the weather conditions and dynamics of the water level, is required for prediction of PSB abundance in Lake Shira. These results may be useful for paleolimnological reconstructions of the history of the lake based on the remnants of purple sulfur bacteria in bottom sediments.

Anaerobic phototrophic nitrite oxidation by Thiocapsa sp. strain KS1 and Rhodopseudomonas sp. strain LQ17.

In anaerobic enrichment cultures for phototrophic nitrite-oxidizing bacteria from different freshwater sites, two different cell types, i.e. non-motile cocci and motile, rod-shaped bacteria, always outnumbered all other bacteria. Most-probable-number (MPN) dilution series with samples from two freshwater sites yielded only low numbers (

De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry.

We have determined the primary structure of cytochrome c(4) from Thiocapsa roseopersicina by de novo protein sequencing using the 'bottom up' approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination.

Thiocapsa imhoffii, sp. nov., an alkaliphilic purple sulfur bacterium of the family Chromatiaceae from Soap Lake, Washington (USA).

An alkaliphilic purple sulfur bacterium, strain SC5, was isolated from Soap Lake, a soda lake located in east central Washington state (USA). Cells of strain SC5 were gram-negative, non-motile, and non-gas vesiculate cocci, often observed in pairs or tetrads. In the presence of sulfide, elemental sulfur was deposited internally. Liquid cultures were pink to rose red in color. Cells contained bacteriochlorophyll a and spirilloxanthin as major photosynthetic pigments. Internal photosynthetic membranes were of the vesicular type. Optimal growth of strain SC5 occurred in the absence of NaCl (range 0-4%), pH 8.5 (range pH 7.5-9.5), and 32 degrees C. Photoheterotrophic growth occurred in the presence of sulfide or thiosulfate with only a limited number of organic carbon sources. Growth factors were not required, and cells could fix N2. Dark, microaerobic growth occurred in the presence of both an organic carbon source and thiosulfate. Sulfide and thiosulfate served as electron donors for photoautotrophy, which required elevated levels of CO2. Phylogenetic analysis placed strain SC5 basal to the clade of the genus Thiocapsa in the family Chromatiaceae with a 96.7% sequence similarity to its closest relative, Thiocapsa roseopersicina strain 1711T (DSM217T). The unique assemblage of physiological and phylogenetic properties of strain SC5 defines it as a new species of the genus Thiocapsa, and we describe strain SC5 herein as Tca. imhoffii, sp. nov.

Cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochrome c4.

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.

Characterization of purple sulfur bacteria from the South Andros Black Hole cave system: highlights taxonomic problems for ecological studies among the genera Allochromatium and Thiocapsa.

A dense 1 m thick layer of phototrophic purple sulfur bacteria is present at the pycnocline (17.8 m depth) in the meromictic South Andros Black Hole cave system (Bahamas). Two purple sulfur bacteria present in samples collected from this layer have been identified as belonging to the family Chromatiaceae. One isolate (BH-1), pink coloured, is non-motile, non-gas vacuolated, 2-3 microm in diameter and surrounded by a capsule. The other isolates (BH-2 and BH-2.4), reddish-brown coloured, are small celled (4 microm x 2 microm), motile by means of a single polar flagellum. In both isolates (BH-1 and BH-2), the intracellular photosynthetic membranes are of the vesicular type and bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series are present. Both isolates grow well in the presence of sulfide and carbon dioxide in the light. During photoautotrophic growth sulfur globules are stored intracellularly as intermediate oxidation products. According to the 16S rRNA gene sequence data the isolates belong to the genera Thiocapsa and Allochromatium. However, at the species level a number of inconsistencies exist between the phenotypic and phylogenetic data, highlighting taxonomic problems within these genera. These inconsistencies may have implications for microbiologists studying the ecology of anoxygenic phototrophs. For ecologists studying the functioning of an ecosystem it may not be particularly important to know whether a specific isolate belongs to one species or another. However, if one wants to study the role of different populations within a particular functional group then the species concept is important. This study demonstrates that further work is still required on the taxonomy of purple sulfur bacteria in order that microbial ecologists are able to accurately identify a population/species isolated from hitherto undescribed aquatic ecosystems.

Thiocapsa marina sp. nov., a novel, okenone-containing, purple sulfur bacterium isolated from brackish coastal and marine environments.

Four marine, phototrophic, purple sulfur bacteria (strains 5811T, 5812, BM-3 and BS-1) were isolated in pure culture from different brackish to marine sediments in the Mediterranean Sea, the White Sea and the Black Sea. Single cells of these strains were coccus-shaped, non-motile and did not contain gas vesicles. The colour of cell suspensions that were grown in the light was purple-red. Bacteriochlorophyll a and carotenoids of the okenone series were present as photosynthetic pigments. Photosynthetic membrane systems were of the vesicular type. Hydrogen sulfide, thiosulfate, elemental sulfur and molecular hydrogen were used as electron donors during photolithotrophic growth under anoxic conditions; carbon dioxide was utilized as the carbon source. During growth on sulfide, elemental sulfur globules were stored inside the cells. In the presence of hydrogen sulfide, several organic substances could be photoassimilated. Comparative 16S rDNA sequence analysis revealed an affiliation of these four strains to the genus Thiocapsa. Both phylogenetic analysis and the results of DNA-DNA hybridization studies revealed that these strains formed a separate cluster within the genus Thiocapsa. Thus, according to phenotypic characteristics and mainly the carotenoid composition, 16S rDNA sequence analysis and DNA-DNA hybridization data, it is proposed that these strains should be classified as a novel species, Thiocapsa marina sp. nov., with strain 5811T (=DSM 5653T=ATCC 43172T) as the type strain.

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.

Is there a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases?

The question of the existence of a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases was taken up by using the sets of data available in the case of two specific enzymes: the hydrogenase from Thiocapsa roseopercisina, in which isotope effects have been systematically investigated over a wide pH range, and the enzyme from Desulfovibrio fructosovorans, for which the activities and the redox properties have been studied in two different forms, the wild type and the P238C mutant. When these data are analyzed in the light of appropriate kinetic models, it is concluded that electron transfer and proton transfer are rate limiting in the H2 uptake and H2 evolution reactions, respectively. This proposal is consistent with the data available from other Ni-Fe enzymes.

Influence of metal ions on hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina.

The effects of some metal ions on the activity and activation of Thiocapsa roseopersicina hydrogenase have been studied. Inhibitory effects of Ni2+ and Cd2+ on the catalytic activity of the enzyme were reversible and competitive with respect to methyl viologen (MV) in the reaction of hydrogen oxidation. The affinity of these metal ions to the enzyme increased significantly with increasing pH, suggesting that their interactions are determined by electrostatic forces. Cu2+ and Hg2+ irreversibly inhibited the hydrogenase activity. A decrease in absorption of hydrogenase at 400 nm in the presence of these metal ions is indicative of the destruction of the FeS cluster in the enzyme.

Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme.

The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.

Thiocapsa litoralis sp. nov., a new purple sulfur bacterium from microbial mats from the White Sea.

A new phototrophic purple sulfur bacterium, isolated from benthic microbial mats from the White Sea littoral zone, is described. Individual cells were spherical, non-motile and lacked gas vesicles. In pure cultures cells appeared in regular platelet-like arrangements of four, eight or sixteen cells. Cell division occurred inside a common envelope, surrounded by a thick capsule. Internal photosynthetic membranes were of the vesicular type. The colour of cell suspensions was pink to rose-red. Bacteriochlorophyll a and carotenoids of the spirilloxanthin series were found as photosynthetic pigments. Under anoxic conditions in the light, photolithoautotrophic growth occurred with sulfide, thiosulfate, sulfite and elemental sulfur as electron donors. Sulfur globules were stored as an intermediary oxidation product and were visible microscopically inside the cells. In the presence of sulfide and bicarbonate, photomixotrophic growth occurred with a number of organic substrates. Sulfate could serve as sole assimilatory source of sulfur. Chemolithoautotrophic growth in the dark was possible with sulfide and thiosulfate as electron donors. Optimum growth occurred in the presence of 1% NaCl, at pH 6.5 and at 30 degrees C. The DNA base composition of the type strain, BM5T, was 64.0 mol% G+C. According to 16S rDNA sequence information and DNA-DNA hybridization, the new isolate clearly belongs to the genus Thiocapsa, but is sufficiently different from other recognized Thiocapsa species to be described as a new species of this genus for which the name Thiocapsa litoralis sp. nov. is proposed. The type strain is BM5T (= ATCC 700894).

Fabrication of an electrode-viologen-hydrogenase heterogeneous system and the electrochemical hydrogen evolution.

An indium tin oxide (ITO) electrode was chemically modified by one layer of viologen (VIO) derivative, which possessed a persistent and reproducible electrochemical response. A monolayer of a thermal stable hydrogenase from Thiocapsa roseopersicina was stabilized on a synthesized poly-L-lysine subphase surface and transferred onto the electrode for fabrication of an ITO-VIO-hydrogenase heterogeneous system. Electrochemical properties of both the ITO-VIO monolayer and the heterogeneous ITO-VIO-hydrogenase system have been investigated. Hydrogen evolution could be measured by potentiostating the VIO-hydrogenase-covered ITO electrode to "electroplate" [(VIO+)n]surf, and a large increase in hydrogen evolution was observed when using an electrolyte solution containing sodium dithionite. We discuss the possible electron transfer process.

Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli.

An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E. coli led to the purification of the enzyme to homogeneity. The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction. However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E. coli strain DH5alpha. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) .

A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli.

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.