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.

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 (

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.

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.

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.

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.