Methylotenera mobilis gen. nov., sp. nov., an obligately methylamine-utilizing bacterium within the family Methylophilaceae.

A novel obligate methylamine utilizer (strain JLW8(T)), isolated from Lake Washington sediment, was characterized taxonomically. The isolate was an aerobic, Gram-negative bacterium. Cells were rod-shaped and motile by means of a single flagellum. Reproduction was by binary fission and no resting bodies were formed. Growth was observed within a pH range of 5-8.5, with optimum growth at pH 7.5. It utilized methylamine as a single source of energy, carbon and nitrogen. Methylamine was oxidized via methylamine dehydrogenase and formaldehyde was assimilated via the ribulose monophosphate cycle. The cellular fatty acid profile was dominated by C(16 : 0)omega7c and C(16 : 0) and the major phospholipid was phosphatidylethanolamine. The DNA G+C content was 54 mol%. 16S rRNA gene sequence analysis indicated that the new isolate was closely related (97-98 % similarity) to a broad group of sequences from uncultured or uncharacterized Betaproteobacteria, but only distantly related (93-96 % similarity) to known methylotrophs of the family Methylophilaceae. Strain JLW8(T) (=ATCC BAA-1282(T)=DSM 17540(T)) is proposed as the type strain of a novel species in a new genus within the family Methylophilaceae, Methylotenera mobilis gen. nov., sp. nov.

Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates.

The taxonomic positions and phylogenetic relationships of two new methylotrophic isolates from Lake Washington (USA) sediment, FAM5T and 500, and the previously described methylotrophic strain EHg5 isolated from contaminated soil in Estarreja (Portugal) were investigated. All three strains were facultative methylotrophs capable of growth on a variety of C1 and multicarbon compounds. Optimal growth occurred at pH 7.5-8 and 30-37 degrees C. The major fatty acids were C16:1omega7c and C16:0. The major quinone was ubiquinone Q8. Neither methanol dehydrogenase nor methanol oxidase activities were detectable in cells grown on methanol, suggesting an alternative, as-yet unknown, mechanism for methanol oxidation. The isolates assimilated C1 units at the level of formaldehyde, via the serine cycle. The DNA G+C content of the strains ranged between 64 and 65 mol%. 16S rRNA gene sequence similarity between the three new isolates was 99.85-100%, but was below 94% with other members of the Betaproteobacteria, indicating that the isolates represent a novel taxon. Based on physiological, phenotypic and genomic characteristics of the three isolates, a new genus, Methyloversatilis gen. nov., is proposed within the family Rhodocyclaceae. The type strain of Methyloversatilis universalis gen. nov., sp. nov. is FAM5T (=CCUG 52030T=JCM 13912T).

Fluorescence in situ hybridization-flow cytometry-cell sorting-based method for separation and enrichment of type I and type II methanotroph populations.

A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10(4) or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

Highly divergent genes for methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection.

The origins and the evolutionary history of tetrahydromethanopterin-linked C1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.