Leucothrix sargassi sp. nov., isolated from a marine alga [Sargassum natans (L.) Gaillon].

A novel bacterial strain, C3212T, was isolated from a marine alga collected from the sea shore of Yantai, China. The strain was Gram-stain-negative, rod-shaped, aerobic, non-motile, and oxidase- and catalase-positive. Growth was observed at 8-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 1.0-7.0 % (w/v) NaCl (optimum, 4.0 %). The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain C3212T consisted of diphosphatidylglycerol (cardiolipin), phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified polar lipid. The major fatty acids were C16 : 1ω6c and/or C16 : 1ω7c, and C18 : 1ω6c and/or C18 : 1ω7c. The DNA G+C content of strain C3212T was 44.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Leucothrix pacifica XH122T, Leucothrix arctica IMCC 9719T and Leucothrix mucor DSM 2157T with similarities of 98.0, 97.5 and 94.3 %, respectively. Estimated DNA-DNA hybridization values were 14.2, 20.7 and 13.9 % between strain C3212T and L. pacifica XH122T, L. arctica IMCC 9719T and L. mucor DSM 2157T, respectively. Phenotypic, phylogenetic and genomic analyses revealed that strain C3212T represents a novel species of the genus Leucothrix, for which the name Leucothrix sargassi sp. nov. is proposed. The type strain is C3212T (=MCCC 1K03600T=KCTC 72121T).

Thioflexithrix psekupsensis gen. nov., sp. nov., a filamentous gliding sulfur bacterium from the family Beggiatoaceae.

A sulfur-oxidizing, filamentous, gliding micro-organism, strain D3T, was isolated from a sulfidic spring in Goryachy Klyuch, Krasnodar, Russia. The cell walls were Gram-negative. The new isolate was a microaerophilic facultative anaerobe and an obligate chemolithoautotroph. The pH range for growth was pH 6.8-7.6, with an optimum at pH 7.2. The temperature range for growth was 10-46 °C, with an optimum at 32 °C. The G+C content of DNA was 42.1 mol%. Phylogenetic analysis of the 16S rRNA gene showed that strain D3T belongs to the family Beggiatoaceae, order Thiotrichales and was distantly related to the genera of the family Beggiatoaceae(86-88 % sequence similarity). The major respiratory quinone was ubiquinone-6. Major fatty acids were C18:1 ω7 (37.6 %), C16 : 0 (34.7 %) and C16: 1 ω7 (27.7 %). On the basis of its physiological properties and the results of phylogenetic analysis, strain D3T is considered to represent a novel species of a new genus, for which the name Thioflexithrix psekupsensis gen. nov., sp. nov. is proposed. The type strain is D3T (=KCTC 62399=UNIQEM U981).

Leucothrix arctica sp. nov., isolated from Arctic seawater.

A Gram-stain-negative, non-motile, oxidase- and catalase-positive, rod-shaped bacterium was isolated from a coastal seawater sample from the Arctic Circle and designated strain IMCC9719T. On the basis of 16S rRNA gene sequence analysis, it was shown that strain IMCC9719T belonged to the genus Leucothrix and was closely related to the type strains of Leucothrix pacifica (97.6 % similarity) and Leucothrix mucor (95.1 %), while the strain shared <90.6 % sequence similarity with other bacterial species. The average nucleotide identity and genome-to-genome distance values between strain IMCC9719T and L. pacifica JCM 18388T were 71.7 and 16.9 %, respectively. The DNA G+C content of strain IMCC9719T was 43.5 mol%. Optimum growth of strain IMCC9719T was observed at 15 °C, at pH 7.5-8.5 and in the presence of 2.0-2.5 % (w/v) NaCl. The major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and C16 : 0. Cells of strain IMCC9719T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified polar lipid, an unidentified aminophospholipid and two unidentified phospholipids. The major respiratory quinone was ubiquinone-8 (Q-8). Based on the taxonomic data collected in this study, strain IMCC9719T represents a novel species of the genus Leucothrix, for which the name Leucothrix arctica sp. nov. is proposed. The type strain is IMCC9719T (=KACC 18010T=NBRC 110382T).

Description of a phototrophic bacterium, Thiorhodococcus alkaliphilus sp. nov.

Strain JA878T was purified from a photoheterotrophic enrichment obtained from a sediment sample of a brown pond near Nari Salt Pan, Bhavnagar, Gujarat, India. Cells of the isolate were coccoid, motile by means of single polar flagellum and Gram-stain-negative. The internal photosynthetic membrane architecture was vesicular. Strain JA878T contained bacteriochlorophyll a and spirilloxanthin series of carotenoids with rhodopin (>85 %) as the major component. Strain JA878T grew optimally at pH 10-11, and had no requirement for NaCl (tolerated up to 6 %, w/v) or vitamins for growth. C16 : 1ω7c/C16 : 1ω6c, C18 : 1ω7c/C18 : 1ω6c and C16 : 0 were identified as the major fatty acids (>10 %). Phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and an unknown polar lipid were identified. Q8 was the predominant quinone system in strain JA878T. The DNA G+C content was 62.4 mol%. Highest 16S rRNA gene sequence similarity through EzTaxon-based blast analysis of strain JA878T was found with the type strains of Thiorhodococcus fuscus (99 %), Thiorhodococcus kakinadensis (98.6 %), Thiohalobacter thiocyanaticus (98.4 %), Thiophaeococcus fuscus (97.3 %) and other members of the class Gammaproteobacteria (<97.3 %), revealing a close affiliation to the genera Thiorhodococcus, Thiohalobacter and Thiophaeococcus. However, the phylogenetic treeing firmly placed the strain in the genus Thiorhodococcus. Phenotypic and chemotaxonomic evidence supported the affiliation of strain JA878T to the genus Thiorhodococcus and not to Thiohalobacter, Thiophaeococcus or other known genera of Chromatiaceae. Distinct physiological, genotypic and chemotaxonomic differences indicate that strain JA878T represents a novel species of the genus Thiorhodococcus, for which the name Thiorhodococcus alkaliphilus sp. nov. is proposed. The type strain is JA878T (=KCTC 15531T=JCM 31245T).

Microbial Community Associated with Thioploca sp. Sheaths in the Area of the Posolski Bank Methane Seep, Southern Baikal.

Bacterial mats formed by a colorless sulfur bacterium Thioploca sp. in the area of the Posolski Bank cold methane seep (southern Baikal) were -studied using electron microscopy and phylogenetic analysis. Morphologically the bacteria were identified as Thioploca ingrica.- Confocal microscopy of DAPI-stained samples revealed numerous rod-shaped, filamentous, and spiral microorganisms in the sheaths, as well as in- side and between the trichomes. Transmission electron microscopy revealed nonvacuolated bacteria and small cells-without cell envelopes within the sheath. Bacteria with pronounced intracytoplasmic membranes characteristic; of type I methanotrophs were observed at the outer side of the sheath. Based on analysis of the 16S rRNA gene sequences, the following phyla were idenified in the sheath community: Bacteroidetes, Nitro- spira, Chloroflexi, Planctomycetes, Verrucomicrobia,'y-, and 6-Proteobacteria, Euryarchaeota, Crenarchaeota, and Thaumarchaeota, as well as anammox bacteria. A hypothetical scheme of matter flows in the Lake Baikal bacterial mats was proposed based on the data on metabolism of the cultured homologues.

Leucothrix pacifica sp. nov., isolated from seawater, and emended description of the genus Leucothrix.

A Gram-stain-negative, strictly aerobic, non-flagellated, non-gliding, oxidase- and catalase-positive, white-pigmented and rod-shaped bacterium, designated strain XH122T, was isolated from a surface seawater sample collected from the South Pacific Gyre (45° 58' E 163° 11' S) during Integrated Ocean Drilling Program Expedition 329.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain XH122T belonged to the genus Leucothrix and showed highest 16S rRNA gene sequence similarity to Leucothrix mucor DSM 2157T (94.3%). It showed lower sequence similarities ( < 90.7%) with all other representatives of the class Gammaproteobacteria. Optimal growth occurred in the presence of 2% (w/v) NaCl, at pH 8.0 and at 28 °C. The DNA G+C content of strain XH122T was 46.2 mol%. The major fatty acids were C16 : 0, C16 : 1ω9c and C18 : 1ω9c. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of data from this polyphasic study, strain XH122T is considered to represent a novel species of the genus Leucothrix, for which the name Leucothrix pacifica sp. nov. is proposed. The type strain is XH122T ( = DSM 25984T = JCM 18388T).

Nitrogen losses in anoxic marine sediments driven by Thioploca-anammox bacterial consortia.

Ninety per cent of marine organic matter burial occurs in continental margin sediments, where a substantial fraction of organic carbon escapes oxidation and enters long-term geologic storage within sedimentary rocks. In such environments, microbial metabolism is limited by the diffusive supply of electron acceptors. One strategy to optimize energy yields in a resource-limited habitat is symbiotic metabolite exchange among microbial associations. Thermodynamic and geochemical considerations indicate that microbial co-metabolisms are likely to play a critical part in sedimentary organic carbon cycling. Yet only one association, between methanotrophic archaea and sulphate-reducing bacteria, has been demonstrated in marine sediments in situ, and little is known of the role of microbial symbiotic interactions in other sedimentary biogeochemical cycles. Here we report in situ molecular and incubation-based evidence for a novel symbiotic consortium between two chemolithotrophic bacteria--anaerobic ammonium-oxidizing (anammox) bacteria and the nitrate-sequestering sulphur-oxidizing Thioploca species--in anoxic sediments of the Soledad basin at the Mexican Pacific margin. A mass balance of benthic solute fluxes and the corresponding nitrogen isotope composition of nitrate and ammonium fluxes indicate that anammox bacteria rely on Thioploca species for the supply of metabolic substrates and account for about 57 ± 21 per cent of the total benthic N2 production. We show that Thioploca-anammox symbiosis intensifies benthic fixed nitrogen losses in anoxic sediments, bypassing diffusion-imposed limitations by efficiently coupling the carbon, nitrogen and sulphur cycles.

Macplocimine A, a new 18-membered macrolide isolated from the filamentous sulfur bacteria Thioploca sp.

Macplocimine A (1), a rare naturally occurring 18-membered macrolide, was isolated from the marine-derived filamentous sulfur bacteria Thioploca sp. The structure was determined by a combination of spectroscopic techniques, including HRESIMS, 1D and 2D NMR analyses. 1 features a thymine group, which is attached to an aromatic fused 18-membered macrolide ring structure derived from a polyketide synthase biosynthetic pathway.

Phylogenetic and morphologic complexity of giant sulphur bacteria.

The large sulphur bacteria, first discovered in the early nineteenth century, include some of the largest bacteria identified to date. Individual cells are often visible to the unaided eye and can reach 750 µm in diameter. The cells usually feature light-refracting inclusions of elemental sulphur and a large internal aqueous vacuole, which restricts the cytoplasm to the outermost periphery. In some taxa, it has been demonstrated that the vacuole can also serve for the storage of high millimolar concentrations of nitrate. Over the course of the past two centuries, a wide range of morphological variation within the family Beggiatoaceae has been found. However, representatives of this clade are frequently recalcitrant to current standard microbiological techniques, including 16S rRNA gene sequencing and culturing, and a reliable classification of these bacteria is often complicated. Here we present a summary of the efforts made and achievements accomplished in the past years, and give perspectives for investigating the heterogeneity and possible evolutionary developments in this extraordinary group of bacteria.

Experimental taphonomy of giant sulphur bacteria: implications for the interpretation of the embryo-like Ediacaran Doushantuo fossils.

The Ediacaran Doushantuo biota has yielded fossils interpreted as eukaryotic organisms, either animal embryos or eukaryotes basal or distantly related to Metazoa. However, the fossils have been interpreted alternatively as giant sulphur bacteria similar to the extant Thiomargarita. To test this hypothesis, living and decayed Thiomargarita were compared with Doushantuo fossils and experimental taphonomic pathways were compared with modern embryos. In the fossils, as in eukaryotic cells, subcellular structures are distributed throughout cell volume; in Thiomargarita, a central vacuole encompasses approximately 98 per cent cell volume. Key features of the fossils, including putative lipid vesicles and nuclei, complex envelope ornament, and ornate outer vesicles are incompatible with living and decay morphologies observed in Thiomargarita. Microbial taphonomy of Thiomargarita also differed from that of embryos. Embryo tissues can be consumed and replaced by bacteria, forming a replica composed of a three-dimensional biofilm, a stable fabric for potential fossilization. Vacuolated Thiomargarita cells collapse easily and do not provide an internal substrate for bacteria. The findings do not support the hypothesis that giant sulphur bacteria are an appropriate interpretative model for the embryo-like Doushantuo fossils. However, sulphur bacteria may have mediated fossil mineralization and may provide a potential bacterial analogue for other macroscopic Precambrian remains.

Diversity of freshwater Thioploca species and their specific association with filamentous bacteria of the phylum Chloroflexi.

Phylogenetic diversity among filamentous sulfur-oxidizing bacteria of the genus Thioploca inhabiting freshwater/brackish environments was analyzed in detail. The 16S rRNA gene sequence of Thioploca found in a freshwater lake in Japan, Lake Okotanpe, was identical to that of Thioploca from Lake Ogawara, a brackish lake. The samples of the two lakes could be differentiated by the sequences of their 23S rRNA genes and 16S-23S rRNA internal transcribed spacer (ITS) regions. The 23S rRNA-based phylogenetic relationships between Thioploca samples from four lakes (Lake Okotanpe, Lake Ogawara, Lake Biwa, and Lake Constance) were similar to those based on the 16S rRNA gene sequences. In addition, multiple types of the ITS sequences were obtained from Thioploca inhabiting Lake Okotanpe and Lake Constance. Variations within respective Thioploca populations were also observed in the analysis of the soxB gene, involved in sulfur oxidation. As major members of the sheath-associated microbial community, bacteria of the phylum Chloroflexi were consistently detected in the samples from different lakes. Fluorescence in situ hybridization revealed that they were filamentous and abundantly distributed within the sheaths of Thioploca.

Vacuolated Beggiatoa-like filaments from different hypersaline environments form a novel genus.

In this study, members of a specific group of thin (6-14 µm filament diameter), vacuolated Beggiatoa-like filaments from six different hypersaline microbial mats were morphologically and phylogenetically characterized. Therefore, enrichment cultures were established, filaments were stained with fluorochromes to show intracellular structures and 16S rRNA genes were sequenced. Morphological characteristics of Beggiatoa-like filaments, in particular the presence of intracellular vacuoles, and the distribution of nucleic acids were visualized. In the intracellular vacuole nitrate reached concentrations of up to 650 mM. Fifteen of the retrieved 16S rRNA gene sequences formed a monophyletic cluster and were phylogenetically closely related (≥ 94.4% sequence identity). Sequences of known filamentous sulfide-oxidizing genera Beggiatoa and Thioploca that comprise non-vacuolated and vacuolated filaments from diverse habitats clearly delineated from this cluster. The novel monophyletic cluster was furthermore divided into two sub-clusters: one contained sequences originating from Guerrero Negro (Mexico) microbial mats and the other comprised sequences from five distinct Spanish hypersaline microbial mats from Ibiza, Formentera and Lake Chiprana. Our data suggest that Beggiatoa-like filaments from hypersaline environments displaying a thin filament diameter contain nitrate-storing vacuoles and are phylogenetically separate from known Beggiatoa. Therefore, we propose a novel genus for these organisms, which we suggest to name 'Candidatus Allobeggiatoa'.

A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria.

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.

A novel, mat-forming Thiomargarita population associated with a sulfidic fluid flow from a deep-sea mud volcano.

A mat-forming population of the giant sulfur bacterium Thiomargarita was discovered at the flank of the mud volcano Amon on the Nile Deep Sea Fan in the Eastern Mediterranean Sea. All cells were of a spherical and vacuolated phenotype and internally stored globules of elemental sulfur. With a diameter of 24-65 µm, Thiomargarita cells from the Eastern Mediterranean were substantially smaller than cells of previously described populations. A 16S rRNA gene fragment was amplified and could be assigned to the Thiomargarita-resembling cells by fluorescence in situ hybridization. This sequence is monophyletic with published Thiomargarita sequences but sequence similarities are only about 94%, indicating a distinct diversification. In the investigated habitat, highly dynamic conditions favour Thiomargarita species over other sulfur-oxidizing bacteria. In contrast to Thiomargarita namibiensis populations, which rely on periodic resuspension from sulfidic sediment into the oxygenated water column, Thiomargarita cells at the Amon mud volcano seem to remain stationary at the sediment surface while environmental conditions change around them due to periodic brine flow.

Cocleimonas flava gen. nov., sp. nov., a gammaproteobacterium isolated from sand snail (Umbonium costatum).

A Gram-stain-negative, aerobic, yellow-pigmented, heterotrophic, sulfur-oxidizing, non-motile, rod-shaped bacterium, designated KMM 3898(T), was isolated from an internal tissue of the sand snail Umbonium costatum, collected from the shallow sediments of the Sea of Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain KMM 3898(T) formed a distinct phylogenetic lineage within the class Gammaproteobacteria and was most closely related to Leucothrix mucor DSM 2157(T) (89.2 % 16S rRNA gene sequence similarity) and members of the genus Thiothrix (86.7-88.5 %). Chemotaxonomically, strain KMM 3898(T) contained the isoprenoid quinone Q-8, the polar lipids phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unknown phospholipid and the fatty acids C(18 : 1)ω7c, C(16 : 1)ω7c and C(16 : 0) as predominant components (>10 %). The DNA G+C content of strain KMM 3898(T) was 43.4 mol%. On the basis of phenotypic features and phylogenetic analysis, strain KMM 3898(T) represents a novel genus and species, for which the name Cocleimonas flava gen. nov., sp. nov. is proposed. The type strain is KMM 3898(T) (=NRIC 0757(T) =JCM 16494(T)).

Distribution, ecology and molecular identification of Thioploca from Danish brackish water sediments.

The distribution of Thioploca populations was investigated in Danish fjords, brackish lakes and coastal waters. Thioploca was found in three geographically distinct populations, where biomasses reached 33.8+/-14.3 g wet weight m(-2) (mean+/-SD). Mats or lawns were not formed at the sediment surfaces and Thioploca biomasses peaked 4-7 cm into the sediment and extended down to 18 cm depth. Morphology and 16S rRNA gene sequences classified all populations as Thioploca ingrica. A sequence divergence of 1.7-2.2% indicated that T. ingrica comprise at least two genotypes. Physiological analysis showed that T. ingrica accumulate nitrate in concentrations of approximately 3 mM and that bicarbonate and acetate are used as a carbon source. The presence of oxygen promoted carbon incorporation, but T. ingrica could survive up to 3 months without an external supply of nitrate or oxygen. Thioploca ingrica populations were exclusively found close to river outlets in a bioturbated sediment with separate sulphidic spots and worm burrow walls containing nitrate and oxygen. It is hypothesized that the subsurface T. ingrica have a special advantage in this heterogeneous environment using their sheath surrounding the bacterial trichomes when navigating between electron donor and acceptor.

Novel observations of Thiobacterium, a sulfur-storing Gammaproteobacterium producing gelatinous mats.

The genus Thiobacterium includes uncultivated rod-shaped microbes containing several spherical grains of elemental sulfur and forming conspicuous gelatinous mats. Owing to the fragility of mats and cells, their 16S ribosomal RNA genes have not been phylogenetically classified. This study examined the occurrence of Thiobacterium mats in three different sulfidic marine habitats: a submerged whale bone, deep-water seafloor and a submarine cave. All three mats contained massive amounts of Thiobacterium cells and were highly enriched in sulfur. Microsensor measurements and other biogeochemistry data suggest chemoautotrophic growth of Thiobacterium. Sulfide and oxygen microprofiles confirmed the dependence of Thiobacterium on hydrogen sulfide as energy source. Fluorescence in situ hybridization indicated that Thiobacterium spp. belong to the Gammaproteobacteria, a class that harbors many mat-forming sulfide-oxidizing bacteria. Further phylogenetic characterization of the mats led to the discovery of an unexpected microbial diversity associated with Thiobacterium.

[Colorless sulfur bacteria Thioploca from different sites in Lake Baikal].

The colorless sulfur bacteria Thioploca spp. found in Lake Baikal are probably a marker for the influx of subterranean mineralized fluids. Bacteria act as a biological filter; by consuming sulfide in their metabolism, they detoxicate it and maintain the purity of Lake Baikal's water. The bacteria were investigated by various techniques. According to analysis of the 16S rRNA gene fragment, Thioploca sp. from Frolikha Bay, Baikal belongs to the clade of freshwater species found in Lake Biwa and Lake Constance; it is most closely related to Thioploca ingrica.

The biogeographical distribution of closely related freshwater sediment bacteria is determined by environmental selection.

The role of environmental selection in governing the structure of communities of freshwater sulfur bacteria (Achromatium spp) was experimentally tested by mixing sediments from two geographically separated lakes (Rydal Water (RY) and Hell Kettles (HK)) that harboured Achromatium spp. Community profiles of Achromatium spp in sediment microcosms at day 0 and after 60 days were compared to determine whether initial Achromatium community composition or subsequent selection by the sediment environment had greater influence in dictating the final Achromatium community structure. It was found that Achromatium spp from the HK community became established in mixed sediments at the expense of members of the RY community. This selection for the HK Achromatium community was more pronounced when sediment composition was manipulated to resemble HK sediments. Our findings definitively demonstrate that environmental selection is the primary determinant of Achromatium community structure in these sediments.

Impact of septic compounds and operational conditions on the microbiology of an activated sludge system.

In activated sludge (AS) biotreatment, septic compounds such as volatile organic acids and reduced sulphur compounds have been frequently cited as a major cause of Thiothrix and Type 021N filamentous bulking. These filaments are common in Canadian pulp and paper biotreatment systems, where they cause settling problems in secondary clarifiers. We conducted a 14-week study of a TMP/newsprint mill effluent to characterize the septic compounds entering the biotreatment, and to determine correlations with AS biomass characteristics and biotreatment operating parameters. A significant correlation was found between the sludge volume index, the abundance of Type 021N, and the propionic acid (PA) concentration in the primary clarified effluent. PA also induced a significant change in the flocculating bacteria size distribution determined by digital imaging. Consequently, the correlation observed between PA and Type 021N bulking is an indirect effect of inhibition of floc-forming microorganisms, giving a competitive advantage to filaments.