Ryosuke Nakai

Microflorae of aquatic moss pillars in a freshwater lake, East Antarctica, based on fatty acid and 16S rRNA gene analyses

Aquatic mosses in the genera Bryum and Leptobryum form unique tower-like “moss pillars” underwater in some Antarctic lakes, in association with algae and cyanobacteria. These are communities with a two-layer structure comprising an oxidative exterior and reductive interior. Although habitats and photosynthetic properties of moss pillars have been reported, microfloral composition of the two-layer structure has not been described. Here we report fatty acid analysis of one moss pillar and molecular phylogenetic analysis, based on the 16S rRNA gene, of this and one other moss pillar. Cluster analysis of the phospholipid fatty acid composition showed three groups corresponding to the exterior, upper interior, and lower interior of the pillar. This suggested that species composition differed by section, with the exterior dominated by photosynthetic organisms such as mosses, algae, and cyanobacteria, the upper interior primarily containing gram-positive bacteria and anaerobic sulfate-reducing bacteria, and the lower interior dominated by gram-negative bacteria. Molecular phylogenetic analysis revealed that Proteobacteria dominate the moss pillar as a whole; cyanobacteria were found on the exterior and the gram-positive obligate anaerobe Clostridium in the interior, while gram-positive sulfate-reducing bacteria were present in the lowest part of the interior. Nitrogen-fixing bacteria and denitrifying bacteria were found in all sections. Thus, fatty acid analysis and genetic analysis showed similar patterns. These findings suggest that microorganisms of different phylogenetic groups inhabit different sections of a single moss pillar and form a microbial community that performs biogeochemical cycling to establish and maintain a structure in an oxidation–reduction gradient between exterior and interior.

Metagenomic Analysis of 0.2-μm-Passable Microorganisms in Deep-Sea Hydrothermal Fluid

We pyrosequenced the bulk DNA extracted from microorganisms that passed through 0.2-μm-pore-size filters and trapped by 0.1-μm-pore-size filters in the hydrothermal fluid of the Mariana Trough. Using the 454-FLX sequencer, we generated 202,648 sequences with an average length of 173.8 bases. Functional profiles were assigned by the SEED Annotation Engine. In the metagenome of the 0.2-μm-passable microorganisms, genes related to membrane function, including potassium homeostasis classified as membrane transport, and multidrug-resistance efflux pumps classified as virulence, were dominant. There was a higher proportion of genes pertinent to the subsystem of membrane transport in our metagenomic library than in other oceanic and hydrothermal vent metagenomes. Genes associated with a RND-type efflux transporter for exogenous substances were specifically identified in the present study. After a comparative analysis with the genome of the known ultramicrobacterium Sphingopyxis alaskensis RB2256, we discovered 1,542 cases of significant hits (E < 1 × 10−2) in our metagenome, and 1,172 of those were related to the DNA repair protein RadA. In this way, the microbial functional profile of 0.2-μm-passable fraction in the present study differs from oceanic metagenomes in the 0.2-μm-trapped fractions and hydrothermal vent metagenomes reported in previous research.

Oligoflexus tunisiensis gen. nov., sp. nov., a Gram-negative, aerobic, filamentous bacterium of a novel proteobacterial lineage, and description of Oligoflexaceae fam. nov., Oligoflexales ord. nov. and Oligoflexia classis nov.

A phylogenetically novel proteobacterium, strain Shr3T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria. The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.

Phylogeographic analysis of filterable bacteria with special reference to Rhizobiales strains that occur in cryospheric habitats

Abstract Although the lower size limit of microorganisms was previously believed to be c. 0.2 μm, there is evidence for the existence of microorganisms that can pass through 0.2 μm-pore-size filters called ultramicrobacteria or nanobacteria. However, information on the phylogeny and biogeography of these bacteria is limited. We obtained 53 isolates of 0.2 μm-passable bacteria from 31 samples collected at 26 locations worldwide, including the Arctic Svalbard Islands, deserts, and Maritime Antarctica. Phylogenetic analysis of near full-length 16S rRNA gene sequences revealed that 18 of the 53 isolates were < 97% homologous with previously cultured isolates, representing potentially novel species. Two isolates (order Rhizobiales) (100% identical) collected from Byers Peninsula, Livingston Island in Maritime Antarctica, were closely related (99.8% similarity) to an isolate collected from intertidal sediments in East Antarctica. In addition, the sequence of this Antarctic isolate showed ≥ 97% similarity to 901 sequences derived from known isolates and samples collected at geographically disparate locations under various environmental conditions. Interestingly, among 13 sequences showing ≥ 99% similarity, ten were isolated from cryospheric habitats such as Arctic, Antarctic, and alpine environments. This implies that such Rhizobiales strains occur in the cryospheric regions, however, their abundance and biomass may be scarce depending on the geographic location.

Diversity and Ecology of Thraustochytrid Protists in the Marine Environment

Thraustochytrids are heterotrophic estuarine/marine protists belonging to the class Labyrinthulomycetes within the stramenopile lineage. Thraustochytrid protists have been a neglected agent of the microbial food chain; however, they occur in detectable amounts in seawater, sediment, and algal and animal tissues. They have the ability to degrade a wide variety of organic substrates, including refractory substrates, by means of extracellular enzymes. Their wide distribution and degradation capability exhibit their ecological significance as decomposers. In particular, thraustochytrids may grow on terrestrial refractory matter in riverine input, and play a role in enhancing carbon cycling in estuarine and coastal areas. Additionally, they produce high amounts of long-chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), which are essential fatty acids for marine animals. Furthermore, some members are known to be pathogens of marine mollusks. These distinctive characteristics mean that thraustochytrid protists play a number of important roles in marine environments.

Oligoflexia, the Newest Class of the Phylum Proteobacteria, Consisting of only One Cultured Species and Uncultured Bacterial Phylotypes from Diverse Habitats

The phylum Proteobacteria has recently gained a new taxon Oligoflexia that represents the seventh or eighth (if yet-to-be-validated “Zetaproteobacteria” is included) class, described by the only cultured species (as of December 2014), Oligoflexus tunisiensis, as the type species. This bacterium exhibits cellular polymorphism and presence of the uncommon fatty acid C16: 1ω5c as high as >65% of total fatty acids, besides its unique 16S rRNA gene sequence. The class Oligoflexia is characterized by the distinct phylogenetic cluster within the greater proteobacterial cluster, and certain environmentally-derived 16S rRNA gene sequences, a.k.a. environmental clones or phylotypes, of uncultured bacteria are now grouped into the Oligoflexia cluster; however, the content and extent of the cluster has not been clearly depicted. This mini-review illustrates that the Oligoflexia cluster hosts a variety of environmental clones from diverse sources. Currently 20 phylotypes (or clones) are affiliated with the Oligoflexia cluster, and the sources were ranging from soils to cyanobacterial mat, bio-filter, human skin, ant colony, desert, glacial ice, earthworm intestine, and seawater. However, their frequencies in respective clone libraries were generally as low as 85% similarities (class-level affiliation) to the 16S rRNA gene sequence of O. tunisiensis, which accounts for merely 0.04% of those registered in Meta-Metagenomic Data Base (MetaMeta DB). On the other hand, >97%-similarities sequences were found in rhizosphere, and <95%-similar sequences were found from hydrocarbon-rich habitats such as petroleum reservoir. Thus, it is suggested that members of Oligoflexia may display cosmopolite distribution in general as well as endemism in certain geochemical settings.

Aurantimicrobium minutum gen. nov., sp. nov., a novel ultramicrobacterium of the family Microbacteriaceae, isolated from river water.

A Gram-stain-positive, aerobic, non-motile, curved (selenoid), rod-shaped actinobacterium, designated KNCT, was isolated from the 0.2 μm-filtrate of river water in western Japan. Cells of strain KNCT were ultramicrosized (0.04-0.05 μm3). The strain grew at 15-37 °C, with no observable growth at 10 °C or 40 °C. The pH range for growth was 7-9, with weaker growth at pH 10. Growth was impeded by the presence of NaCl at concentrations greater than 1 %. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KNCT showed relatively high sequence similarity (97.2 %) to Alpinimonas psychrophila Cr8-25T in the family Microbacteriaceae. However, strain KNCT formed an independent cluster with cultured, but as-yet-unidentified, species and environmental clones on the phylogenetic tree. The major cellular fatty acids were anteiso-C15 : 0 (41.0 %), iso-C16 : 0 (21.8 %), C16 : 0 (18.0 %) and anteiso-C17 : 0 (12.9 %), and the major menaquinones were MK-11 (71.3 %) and MK-12 (13.6 %). The major polar lipids were phosphatidylglycerol and two unknown glycolipids. The cell-wall muramic acid acyl type was acetyl. The peptidoglycan was B-type, and contained 3-hydroxyglutamic acid, glutamic acid, aspartic acid, glycine, alanine and lysine, with the latter being the diagnostic diamino acid. The G+C content of the genome was unusually low for actinobacteria (52.1 mol%), compared with other genera in the family Microbacteriaceae. Based on the phenotypic characteristics and phylogenetic evidence, strain KNCT represents a novel species of a new genus within the family Microbacteriaceae, for which the name Aurantimicrobium minutum gen. nov., sp. nov. is proposed. The type strain of the type species is KNCT ( = NBRC 105389T = NCIMB 14875T).

Diversity of RuBisCO gene responsible for CO2 fixation in an Antarctic moss pillar

Antarctic “moss pillars” are lake-bottom biocenoses that are primarily comprised of aquatic mosses. The pillars consist of distinct redox-affected sections: oxidative exteriors and reductive interiors. Batteries of SSU rRNA genotypes of eukaryotes, eubacteria, and cyanobacteria, but no archaea, have been identified in these pillars. However, rRNA-based phylogenetic analysis provides limited information on metabolic capabilities. To investigate the microorganisms that have the potential for CO2 fixation in the pillars, we studied the genetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39)—an enzyme involved in CO2 fixation. PCR clone libraries targeting all forms of the RuBisCO large subunit-encoding gene were constructed and 1,092 clones were randomly sequenced. Phylogenetic analysis indicated that proteobacterial form IA operational RuBisCO units (ORUs) were detected at the same frequency as the cyanobacterial form IB ORUs. Surprisingly, the form IA ORU, which was closely related to the sequences from deep-sea environments, was detected from all moss pillar sections. The form IB ORU related to Bryophyta, considered to be derived from moss, was identical to the sequence of Leptobryum sp. isolated from Lake Hotoke-Ike where the pillars were found. Moreover, certain cyanobacterial ORUs were found exclusively in the exterior of the pillar, whereas form II ORUs related to chemolithoautotrophic sulfur oxidizers and purple sulfur bacteria were found exclusively in the interior. No forms IC, ID, or III RuBisCO genes were detected. This is the first report demonstrating that bacteria with the potential for CO2 fixation and chemoautotrophy are present in the Antarctic moss pillar ecosystem.

Complete Genome Sequence of Aurantimicrobium minutum Type Strain KNCT, a Planktonic Ultramicrobacterium Isolated from River Water

ABSTRACT Aurantimicrobium minutum type strain KNCT is a planktonic ultramicrobacterium isolated from river water in western Japan. Strain KNCT has an extremely small, streamlined genome of 1,622,386 bp comprising 1,575 protein-coding sequences. The genome annotation suggests that strain KNCT has an actinorhodopsin-based photometabolism.