Hiroko Makita

Allying with armored snails: the complete genome of gammaproteobacterial endosymbiont

Deep-sea vents harbor dense populations of various animals that have their specific symbiotic bacteria. Scaly-foot gastropods, which are snails with mineralized scales covering the sides of its foot, have a gammaproteobacterial endosymbiont in their enlarged esophageal glands and diverse epibionts on the surface of their scales. In this study, we report the complete genome sequencing of gammaproteobacterial endosymbiont. The endosymbiont genome displays features consistent with ongoing genome reduction such as large proportions of pseudogenes and insertion elements. The genome encodes functions commonly found in deep-sea vent chemoautotrophs such as sulfur oxidation and carbon fixation. Stable carbon isotope (13C)-labeling experiments confirmed the endosymbiont chemoautotrophy. The genome also includes an intact hydrogenase gene cluster that potentially has been horizontally transferred from phylogenetically distant bacteria. Notable findings include the presence and transcription of genes for flagellar assembly, through which proteins are potentially exported from bacterium to the host. Symbionts of snail individuals exhibited extreme genetic homogeneity, showing only two synonymous changes in 19 different genes (13 810 positions in total) determined for 32 individual gastropods collected from a single colony at one time. The extremely low genetic individuality in endosymbionts probably reflects that the stringent symbiont selection by host prevents the random genetic drift in the small population of horizontally transmitted symbiont. This study is the first complete genome analysis of gastropod endosymbiont and offers an opportunity to study genome evolution in a recently evolved endosymbiont.

Methylothermus subterraneus sp. nov., a moderately thermophilic methanotroph isolated from a terrestrial subsurface hot aquifer

A novel methane-oxidizing bacterium, strain HTM55(T), was isolated from subsurface hot aquifer water from a Japanese gold mine. Strain HTM55(T) was a Gram-negative, aerobic, motile, coccoid bacterium with a single polar flagellum and the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain HTM55(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 37-65 °C (optimum 55-60 °C). The isolate grew at pH 5.2-7.5 (optimum 5.8-6.3) and with 0-1 % NaCl (optimum 0-0.3 %). The ribulose monophosphate pathway was operative for carbon assimilation. The DNA G+C content was 54.4 mol% and the major fatty acids were C(16 : 0) (52.0 %) and C(18 : 1)ω7c (34.8 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HTM55(T) was closely related to Methylothermus thermalis MYHT(T) (99.2 % 16S rRNA gene sequence similarity), which is within the class Gammaproteobacteria. However, DNA-DNA relatedness between strain HTM55(T) and Methylothermus thermalis MYHT(T) was ≤ 39 %. On the basis of distinct phylogenetic, chemotaxonomic and physiological characteristics, strain HTM55(T) represents a novel species of the genus Methylothermus, for which the name Methylothermus subterraneus sp. nov. is proposed. The type strain is HTM55(T) ( = JCM 13664(T) = DSM 19750(T)).

Bacteriogenic Fe(III) (Oxyhydr)oxides Characterized by Synchrotron Microprobe Coupled with Spatially Resolved Phylogenetic Analysis

Ubiquitous presence of microbes in aquatic systems and their inherent ability of biomineralization make them extremely important agents in the geochemical cycling of inorganic elements. However, the detailed mechanisms of environmental biomineralization (e.g., the actual reaction rates, the temporal and spatial dynamics of these processes) are largely unknown, because there are few adequate analytical techniques to observe the biogenic oxidation/reduction reactions in situ. Here, we report a novel technical approach to characterize specific biominerals associated with a target microbe on high spatial resolution. The technique was developed by combining directly in situ phylogenetic analysis, fluorescence in situ hybridization (FISH), with a synchrotron microprobe method, micro X-ray absorption fine structure spectroscopy (μ-XAFS), and was applied to iron mineral deposition by iron(II)-oxidizing bacteria (IOB) in environmental samples. In situ visualization of microbes revealed that in natural iron mats, Betaproteobacteria dominated by IOB were dominantly localized within 10 μm of the surface. Furthermore, in situ chemical speciation by the synchrotron microprobe suggested that the Fe local structure at the IOB accumulating parts was dominantly composed of short-ordered Fe-O(6) linkage, which is not observed in bulk iron mat samples. The present study indicates that coupled XAFS-FISH could be a potential technique to provide direct information on specific biogenic reaction mediated by target microorganism.

Isolation and characterization of a thermophilic, obligately anaerobic and heterotrophic marine Chloroflexi bacterium from a Chloroflexi-dominated microbial community associated with a Japanese shallow hydrothermal system, and proposal for Thermomarinilinea lacunofontalis gen. nov., sp. nov.

A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7T, was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7T was isolated from an enrichment culture at a high dilution rate. Strain SW7T was an obligately anaerobic heterotroph that grew with fermentation and had non-motile thin rods 3.5–16.5 μm in length and 0.2 μm in width constituting multicellular filaments. Growth was observed between 37–65°C (optimum 60°C), pH 5.5–7.3 (optimum pH 6.0), and 0.5–3.5% (w/v) NaCl concentration (optimum 1.0%). Based on the physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7T (=JCM15506T=KCTC5908T).

Biogeography of Persephonella in deep-sea hydrothermal vents of the Western Pacific

Deep-sea hydrothermal vent fields are areas on the seafloor with high biological productivity fueled by microbial chemosynthesis. Members of the Aquificales genus Persephonella are obligately chemosynthetic bacteria, and appear to be key players in carbon, sulfur, and nitrogen cycles in high temperature habitats at deep-sea vents. Although this group of bacteria has cosmopolitan distribution in deep-sea hydrothermal ecosystem around the world, little is known about their population structure such as intraspecific genomic diversity, distribution pattern, and phenotypic diversity. We developed the multi-locus sequence analysis (MLSA) scheme for their genomic characterization. Sequence variation was determined in five housekeeping genes and one functional gene of 36 Persephonella hydrogeniphila strains originated from the Okinawa Trough and the South Mariana Trough (SNT). Although the strains share >98.7% similarities in 16S rRNA gene sequences, MLSA revealed 35 different sequence types (ST), indicating their extensive genomic diversity. A phylogenetic tree inferred from all concatenated gene sequences revealed the clustering of isolates according to the geographic origin. In addition, the phenotypic clustering pattern inferred from whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis can be correlated to their MLSA clustering pattern. This study represents the first MLSA combined with phenotypic analysis indicative of allopatric speciation of deep-sea hydrothermal vent bacteria.

Physiological and genomic features of a novel sulfur-oxidizing gammaproteobacterium belonging to a previously uncultivated symbiotic lineage isolated from a hydrothermal vent.

Strain Hiromi 1, a sulfur-oxidizing gammaproteobacterium was isolated from a hydrothermal vent chimney in the Okinawa Trough and represents a novel genus that may include a phylogenetic group found as endosymbionts of deep-sea gastropods. The SSU rRNA gene sequence similarity between strain Hiromi 1 and the gastropod endosymbionts was approximately 97%. The strain was shown to grow both chemolithoautotrophically and chemolithoheterotrophically with an energy metabolism of sulfur oxidation and O2 or nitrate reduction. Under chemolithoheterotrophic growth conditions, the strain utilized organic acids and proteinaceous compounds as the carbon and/or nitrogen sources but not the energy source. Various sugars did not support growth as a sole carbon source. The observation of chemolithoheterotrophy in this strain is in line with metagenomic analyses of endosymbionts suggesting the occurrence of chemolithoheterotrophy in gammaproteobacterial symbionts. Chemolithoheterotrophy and the presence of homologous genes for virulence- and quorum sensing-related functions suggest that the sulfur-oxidizing chomolithotrophic microbes seek animal bodies and microbial biofilm formation to obtain supplemental organic carbons in hydrothermal ecosystems.

Direct Detection of Fe(II) in Extracellular Polymeric Substances (EPS) at the Mineral-Microbe Interface in Bacterial Pyrite Leaching

We herein investigated the mechanisms underlying the contact leaching process in pyrite bioleaching by Acidithiobacillus ferrooxidans using scanning transmission X-ray microscopy (STXM)-based C and Fe near edge X-ray absorption fine structure (NEXAFS) analyses. The C NEXAFS analysis directly showed that attached A. ferrooxidans produces polysaccharide-abundant extracellular polymeric substances (EPS) at the cell-pyrite interface. Furthermore, by combining the C and Fe NEXAFS results, we detected significant amounts of Fe(II), in addition to Fe(III), in the interfacial EPS at the cell-pyrite interface. A probable explanation for the Fe(II) in detected EPS is the leaching of Fe(II) from the pyrite. The detection of Fe(II) also indicates that Fe(III) resulting from pyrite oxidation may effectively function as an oxidizing agent for pyrite at the cell-pyrite interface. Thus, our results imply that a key role of Fe(III) in EPS, in addition to its previously described role in the electrostatic attachment of the cell to pyrite, is enhancing pyrite dissolution.

Distribution and Niche Separation of Planktonic Microbial Communities in the Water Columns from the Surface to the Hadal Waters of the Japan Trench under the Eutrophic Ocean.

The Japan Trench is located under the eutrophic Northwestern Pacific while the Mariana Trench that harbors the unique hadal planktonic biosphere is located under the oligotrophic Pacific. Water samples from the sea surface to just above the seafloor at a total of 11 stations including a trench axis station, were investigated several months after the Tohoku Earthquake in March 2011. High turbidity zones in deep waters were observed at most of the sampling stations. The small subunit (SSU) rRNA gene community structures in the hadal waters (water depths below 6000 m) at the trench axis station were distinct from those in the overlying meso-, bathy and abyssopelagic waters (water depths between 200 and 1000 m, 1000 and 4000 m, and 4000 and 6000 m, respectively), although the SSU rRNA gene sequences suggested that potential heterotrophic bacteria dominated in all of the waters. Potential niche separation of nitrifiers, including ammonia-oxidizing archaea (AOA), was revealed by quantitative PCR analyses. It seems likely that Nitrosopumilus-like AOAs respond to a high flux of electron donors and dominate in several zones of water columns including shallow and very deep waters. This study highlights the effects of suspended organic matter, as induced by seafloor deformation, on microbial communities in deep waters and confirm the occurrence of the distinctive hadal biosphere in global trench environments hypothesized in the previous study.

Thalassobius abyssi sp. nov., a marine bacterium isolated from the cold-seep sediment.

A novel marine bacterial strain, designated JAMH 043T, was isolated from cold-seep sediment in Sagami Bay, Japan. Cells were Gram-stain-negative, rod-shaped, non-motile and aerobic chemo-organotrophs. The isolate grew optimally at 25 °C, at pH 7.0-7.5 and with 3 % (w/v) NaCl. The major respiratory quinone was ubiquinone-10 (Q-10). The predominant fatty acid was C18 : 1ω7c. On the basis of 16S rRNA gene sequence analysis, the isolated strain was closely affiliated to members of the genus Thalassobius in the class Alphaproteobacteria, and 16S rRNA gene sequence similarity of the novel isolate with the type strain of its closest related species, Thalassobius aestuarii JC2049T, was 98.4 %. The DNA G+C content of the novel strain was 58.0 mol%. The hybridization values for DNA-DNA relatedness between strain JAMH 043T and reference strains belonging to the genus Thalassobius were less than 14.1 ± 2.2 %. Based on differences in taxonomic characteristics, the isolated strain represents a novel species of the genus Thalassobius, for which the name Thalassobius abyssi sp. nov. is proposed. The type strain is JAMH 043T ( = JCM 30900T = DSM 100673T).

Comparative Analysis of Microbial Communities in Iron-Dominated Flocculent Mats in Deep Sea Hydrothermal Environments

ABSTRACT It has been suggested that iron is one of the most important energy sources for photosynthesis-independent microbial ecosystems in the ocean crust. Iron-metabolizing chemolithoautotrophs play a key role as primary producers, but little is known about their distribution and diversity and their ecological role as submarine iron-metabolizing chemolithotrophs, particularly the iron oxidizers. In this study, we investigated the microbial communities in several iron-dominated flocculent mats found in deep-sea hydrothermal fields in the Mariana Volcanic Arc and Trough and the Okinawa Trough by culture-independent molecular techniques and X-ray mineralogical analyses. The abundance and composition of the 16S rRNA gene phylotypes demonstrated the ubiquity of zetaproteobacterial phylotypes in iron-dominated mat communities affected by hydrothermal fluid input. Electron microscopy with energy-dispersive X-ray microanalysis and X-ray absorption fine structure (XAFS) analysis revealed the chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species and the potential contribution of Zetaproteobacteria to the in situ generation. These results suggest that putative iron-oxidizing chemolithoautotrophs play a significant ecological role in producing iron-dominated flocculent mats and that they are important for iron and carbon cycles in deep-sea low-temperature hydrothermal environments. IMPORTANCE We report novel aspects of microbiology from iron-dominated flocculent mats in various deep-sea environments. In this study, we examined the relationship between Zetaproteobacteria and iron oxides across several hydrothermally influenced sites in the deep sea. We analyzed iron-dominated mats using culture-independent molecular techniques and X-ray mineralogical analyses. The scanning electron microscopy–energy-dispersive X-ray spectroscopy SEM-EDS analysis and X-ray absorption fine structure (XAFS) analysis revealed chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species as well as the potential contribution of the zetaproteobacterial population to the in situ production. These key findings provide important information for understanding the mechanisms of both geomicrobiological iron cycling and the formation of iron-dominated mats in deep-sea hydrothermal fields.