Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

Pseudomethylobacillus aquaticus gen. nov., sp. nov., a new member of the family Methylophilaceae isolated from an artificial reservoir.

A bacterial strain, designated H-5T, was isolated from an artificial reservoir in Taiwan and characterized using a polyphasic taxonomic approach. Cells of strain H-5T were Gram-stain-negative, aerobic, motile by means of a single polar flagellum, rod-shaped, covered by large capsules and formed white colonies. Growth occurred at 15-30 °C (optimum, 25 °C), at pH 6-8 (optimum, pH 7) and with 0-0.5 % NaCl (optimum, 0 %). Phylogenetic analyses based on the 16S rRNA gene, the methanol dehydrogenase gene and the coding sequences of 92 protein clusters indicated that strain H-5T was affiliated with genera in the family Methylophilaceae in the class Betaproteobacteria. Strain H-5T was most closely related to Methylobacillus methanolivorans ZT with a 95.0 % 16S rRNA gene sequence similarity. Strain H-5T showed less than 73.7 % average nucleotide identity and less than 23.6 % digital DNA-DNA hybridization identity compared to the strains of related genera within the family Methylophilaceae. The predominant fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The major isoprenoid quinone was Q-8 and the DNA G+C content was 58.3 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, one uncharacterized aminophospholipid, one uncharacterized phospholipid and one uncharacterized lipid. On the basis of the genotypic and phenotypic data presented here, strain H-5T represents a novel species of a new genus in the family Methylophilaceae, for which the name Pseudomethylobacillus aquaticus gen. nov., sp. nov. is proposed. The type strain is H-5T (=BCRC 81154T=KCTC 62865T).

Evolution in action: habitat transition from sediment to the pelagial leads to genome streamlining in Methylophilaceae.

The most abundant aquatic microbes are small in cell and genome size. Genome-streamlining theory predicts gene loss caused by evolutionary selection driven by environmental factors, favouring superior competitors for limiting resources. However, evolutionary histories of such abundant, genome-streamlined microbes remain largely unknown. Here we reconstruct the series of steps in the evolution of some of the most abundant genome-streamlined microbes in freshwaters ("Ca. Methylopumilus") and oceans (marine lineage OM43). A broad genomic spectrum is visible in the family Methylophilaceae (Betaproteobacteria), from sediment microbes with medium-sized genomes (2-3 Mbp genome size), an occasionally blooming pelagic intermediate (1.7 Mbp), and the most reduced pelagic forms (1.3 Mbp). We show that a habitat transition from freshwater sediment to the relatively oligotrophic pelagial was accompanied by progressive gene loss and adaptive gains. Gene loss has mainly affected functions not necessarily required or advantageous in the pelagial or is encoded by redundant pathways. Likewise, we identified genes providing adaptations to oligotrophic conditions that have been transmitted horizontally from pelagic freshwater microbes. Remarkably, the secondary transition from the pelagial of lakes to the oceans required only slight modifications, i.e., adaptations to higher salinity, gained via horizontal gene transfer from indigenous microbes. Our study provides first genomic evidence of genome reduction taking place during habitat transitions. In this regard, the family Methylophilaceae is an exceptional model for tracing the evolutionary history of genome streamlining as such a collection of evolutionarily related microbes from different habitats is rare in the microbial world.

Isolation and genomic characterization of Novimethylophilus kurashikiensis gen. nov. sp. nov., a new lanthanide-dependent methylotrophic species of Methylophilaceae.

Recently, it has been found that two types of methanol dehydrogenases (MDHs) exist in Gram-negative bacterial methylotrophs, calcium-dependent MxaFI-MDH and lanthanide-dependent XoxF-MDH and the latter is more widespread in bacterial genomes. We aimed to isolate and characterize lanthanide-dependent methylotrophs. The growth of strain La2-4T on methanol, which was isolated from rice rhizosphere soil, was strictly lanthanide dependent. Its 16S rRNA gene sequence showed only 93.4% identity to that of Methylophilus luteus MimT , and the name Novimethylophilus kurashikiensis gen. nov. sp. nov. is proposed. Its draft genome (ca. 3.69 Mbp, G + C content 56.1 mol%) encodes 3579 putative CDSs and 84 tRNAs. The genome harbors five xoxFs but no mxaFI. XoxF4 was the major MDH in the cells grown on methanol and methylamine, evidenced by protein identification and quantitative PCR analysis. Methylamine dehydrogenase gene was absent in the La2-4T genome, while genes for the glutamate-mediated methylamine utilization pathway were detected. The genome also harbors those for the tetrahydromethanopterin and ribulose monophosphate pathways. Additionally, as known species, isolates of Burkholderia ambifaria, Cupriavidus necator and Dyadobacter endophyticus exhibited lanthanide dependent growth on methanol. Thus, lanthanide can be used as an essential growth factor for methylotrophic bacteria that do not harbor MxaFI-MDH.

Microbial megacities fueled by methane oxidation in a mineral spring cave.

Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000 p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales (Gammaproteobacteria) and methylotrophic Methylophilaceae (Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed.

[Culturable psychrotolerant methanotrophic bacteria in landfill cover soil].

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10@C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20 degrees C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40 degrees C from a sample collected in May (the temperature of the cover soil was 11.5-12.5 degrees C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S r-RNA genes with the type strain SV96(T)) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15 degrees C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.

Novel methylotrophic isolates from lake sediment, description of Methylotenera versatilis sp. nov. and emended description of the genus Methylotenera.

Phylogenetic positions, and genotypic and phenotypic characteristics of three novel methylotrophic isolates, strains 301(T), 30S and SIP3-4, from sediment of Lake Washington, Seattle, USA, are described. The strains were restricted facultative methylotrophs capable of growth on single carbon compounds (methylamine and methanol) in addition to a limited range of multicarbon compounds. All strains used the N-methylglutamate pathway for methylamine oxidation. Strain SIP3-4 possessed the canonical (MxaFI) methanol dehydrogenase, but strains 301(T) and 30S did not. All three strains used the ribulose monophosphate pathway for C1 assimilation. The major fatty acids in the three strains were C(16:0) and C(16:1)ω7c. The DNA G+C contents of strains 301(T) and SIP3-4 were 42.6 and 54.6 mol%, respectively. Based on 16S rRNA gene sequence phylogeny and the relevant phenotypic characteristics, strain SIP3-4 was assigned to the previously defined species Methylovorus glucosotrophus. Strains 301(T) and 30S were closely related to each other (100% 16S rRNA gene sequence similarity) and shared 96.6% 16S rRNA gene sequence similarity with a previously described isolate, Methylotenera mobilis JLW8(T). Based on significant genomic and phenotypic divergence with the latter, strains 301(T) and 30S represent a novel species within the genus Methylotenera, for which the name Methylotenera versatilis sp. nov. is proposed; the type strain is 301(T) (=VKM B-2679(T)=JCM 17579(T)). An emended description of the genus Methylotenera is provided.

Genomes of three methylotrophs from a single niche reveal the genetic and metabolic divergence of the methylophilaceae.

The genomes of three representatives of the family Methylophilaceae, Methylotenera mobilis JLW8, Methylotenera versatilis 301, and Methylovorus glucosetrophus SIP3-4, all isolated from a single study site, Lake Washington in Seattle, WA, were completely sequenced. These were compared to each other and to the previously published genomes of Methylobacillus flagellatus KT and an unclassified Methylophilales strain, HTCC2181. Comparative analysis revealed that the core genome of Methylophilaceae may be as small as approximately 600 genes, while the pangenome may be as large as approximately 6,000 genes. Significant divergence between the genomes in terms of both gene content and gene and protein conservation was uncovered, including the varied presence of certain genes involved in methylotrophy. Overall, our data demonstrate that metabolic potentials can vary significantly between different species of Methylophilaceae, including organisms inhabiting the very same environment. These data suggest that genetic divergence among the members of this family may be responsible for their specialized and nonredundant functions in C1 cycling, which in turn suggests means for their successful coexistence in their specific ecological niches.

Complete genome sequence of the bacterium Methylovorus sp. strain MP688, a high-level producer of pyrroloquinolone quinone.

Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.

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.

Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil.

Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.