Metagenomics Revealed a New Genus 'Candidatus Thiocaldithrix dubininis' gen. nov., sp. nov. and a New Species 'Candidatus Thiothrix putei' sp. nov. in the Family Thiotrichaceae, Some Members of Which Have Traits of Both Na+- and H+-Motive Energetics.

Two metagenome-assembled genomes (MAGs), GKL-01 and GKL-02, related to the family Thiotrichaceae have been assembled from the metagenome of bacterial mat obtained from a sulfide-rich thermal spring in the North Caucasus. Based on average amino acid identity (AAI) values and genome-based phylogeny, MAG GKL-01 represented a new genus within the Thiotrichaceae family. The GC content of the GKL-01 DNA (44%) differed significantly from that of other known members of the genus Thiothrix (50.1-55.6%). We proposed to assign GKL-01 to a new species and genus 'Candidatus Thiocaldithrix dubininis' gen. nov., sp. nov. GKL-01. The phylogenetic analysis and estimated distances between MAG GKL-02 and the genomes of the previously described species of the genus Thiothrix allowed assigning GKL-02 to a new species with the proposed name 'Candidatus Thiothrix putei' sp. nov. GKL-02 within the genus Thiothrix. Genome data first revealed the presence of both Na+-ATPases and H+-ATPases in several Thiothrix species. According to genomic analysis, bacteria GKL-01 and GKL-02 are metabolically versatile facultative aerobes capable of growing either chemolithoautotrophically or chemolithoheterotrophically in the presence of hydrogen sulfide and/or thiosulfate or chemoorganoheterotrophically.

A bacterium that is not a microbe.

A new discovery challenges the prevailing view of the boundaries of bacterial cell size.

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles.

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Candidatus (Ca.) Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells. These features, along with compartmentalization of genomic material and ribosomes in translationally active organelles bound by bioenergetic membranes, indicate gain of complexity in the Thiomargarita lineage and challenge traditional concepts of bacterial cells.

Mammoth mangrove bacterium has complex cell.

"Eye-opening" discovery challenges evolutionary thinking on microbes.

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).

Potential effects of suspended TiO2 nanoparticles on activated sludge floc properties in membrane bioreactors.

With the rapid development and application of consumer products containing nanoparticles (NPs), especially titanium dioxide (TiO2) NPs, the potential effects of suspended NPs on wastewater treatment has been a concern over the recent years. This study investigated the potential effects of suspended TiO2 NPs on activated sludge flocculation properties in a membrane bioreactor (MBR). Results showed that suspended TiO2 NPs inhibited the viability of activated sludge flocs, and led to bacterial protein secretion for bacterial protection, causing an overall protein increase of soluble microbial products. Suspended TiO2 NPs also destabilized the activated sludge floc structure and reduced flocculation capacity of flocs, causing an over production of organic matter and resulting in a floc size decrease of over 50%. Suspended TiO2 NPs also caused a change in the phylogenetic distribution of bacterial community. Whereby, the dominant species in activated sludge was replaced from Comamonadaceae to Thiotrichaceae in 50 mg/L suspended TiO2 NPs.

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).

Imaging of Cellular Oxidoreductase Activity Suggests Mixotrophic Metabolisms in Thiomargarita spp.

The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be monitored in time course experiments or to be individually sorted for omics-based investigations. Here we investigated the metabolism of individual cells of Thiomargarita spp. by using a novel application of a tetrazolium-based dye that measures oxidoreductase activity. When coupled with microscopy, staining of the cells with a tetrazolium-formazan dye allows metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita sp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox dye-based assay suggest that Thiomargarita is the most metabolically versatile under anoxic conditions, where it appears to express cellular oxidoreductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Candidatus Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular oxidoreductase response of bacteria attached to the exterior of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.IMPORTANCE The metabolic potential of many microorganisms that cannot be grown in the laboratory is known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolism with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation-independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

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).

Ecology: Electrical Cable Bacteria Save Marine Life.

Animals at the bottom of the sea survive oxygen depletion surprisingly often, and a new study identifies cable bacteria in the sediment as the saviors. The bacterial electrical activity creates an iron 'carpet', trapping toxic hydrogen sulfide.

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.

Cable bacteria generate a firewall against euxinia in seasonally hypoxic basins.

Seasonal oxygen depletion (hypoxia) in coastal bottom waters can lead to the release and persistence of free sulfide (euxinia), which is highly detrimental to marine life. Although coastal hypoxia is relatively common, reports of euxinia are less frequent, which suggests that certain environmental controls can delay the onset of euxinia. However, these controls and their prevalence are poorly understood. Here we present field observations from a seasonally hypoxic marine basin (Grevelingen, The Netherlands), which suggest that the activity of cable bacteria, a recently discovered group of sulfur-oxidizing microorganisms inducing long-distance electron transport, can delay the onset of euxinia in coastal waters. Our results reveal a remarkable seasonal succession of sulfur cycling pathways, which was observed over multiple years. Cable bacteria dominate the sediment geochemistry in winter, whereas, after the summer hypoxia, Beggiatoaceae mats colonize the sediment. The specific electrogenic metabolism of cable bacteria generates a large buffer of sedimentary iron oxides before the onset of summer hypoxia, which captures free sulfide in the surface sediment, thus likely preventing the development of bottom water euxinia. As cable bacteria are present in many seasonally hypoxic systems, this euxinia-preventing firewall mechanism could be widely active, and may explain why euxinia is relatively infrequently observed in the coastal ocean.

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).

Metatranscriptomic analysis of diminutive Thiomargarita-like bacteria ("Candidatus Thiopilula" spp.) from abyssal cold seeps of the Barbados Accretionary Prism.

Large sulfur-oxidizing bacteria in the family Beggiatoaceae are important players in the global sulfur cycle. This group contains members of the well-known genera Beggiatoa, Thioploca, and Thiomargarita but also recently identified and relatively unknown candidate taxa, including "Candidatus Thiopilula" spp. and "Ca. Thiophysa" spp. We discovered a population of "Ca. Thiopilula" spp. colonizing cold seeps near Barbados at a ∼4.7-km water depth. The Barbados population consists of spherical cells that are morphologically similar to Thiomargarita spp., with elemental sulfur inclusions and a central vacuole, but have much smaller cell diameters (5 to 40 µm). Metatranscriptomic analysis revealed that when exposed to anoxic sulfidic conditions, Barbados "Ca. Thiopilula" organisms expressed genes for the oxidation of elemental sulfur and the reduction of nitrogenous compounds, consistent with their vacuolated morphology and intracellular sulfur storage capability. Metatranscriptomic analysis further revealed that anaerobic methane-oxidizing and sulfate-reducing organisms were active in the sediment, which likely provided reduced sulfur substrates for "Ca. Thiopilula" and other sulfur-oxidizing microorganisms in the community. The novel observations of "Ca. Thiopilula" and associated organisms reported here expand our knowledge of the globally distributed and ecologically successful Beggiatoaceae group and thus offer insight into the composition and ecology of deep cold seep microbial communities.

Ecophysiology of Thioploca ingrica as revealed by the complete genome sequence supplemented with proteomic evidence.

Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8 Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of T. ingrica in lake sediment.

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.

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.

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.

X-ray mosaic nanotomography of large microorganisms.

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state.