Methylovulum miyakonense gen. nov., sp. nov., a type I methanotroph isolated from forest soil.

A novel methanotroph, designated strain HT12(T), was isolated from forest soil in Japan. Cells of strain HT12(T) were Gram-reaction-negative, aerobic, non-motile, coccoid and formed pale-brown colonies. The strain grew only with methane and methanol as sole carbon and energy sources. Cells grew at 5-34 °C (optimum 24-32 °C). The strain possessed both particulate and soluble methane monooxygenases and assimilated formaldehyde using the ribulose monophosphate pathway. The major cellular fatty acids were C(16 : 0) (46.9 %) and C(14 : 0) (34.2 %), whereas unsaturated C(16) fatty acids, typical of type I methanotrophs, were absent. Comparative 16S rRNA gene sequence analysis showed that the most closely related strains were Methylosoma difficile LC 2(T) (93.1 % sequence similarity) and Methylobacter tundripaludum SV96(T) (92.6 % similarity). Phylogenetic analysis based on the pmoA gene indicated that strain HT12(T) formed a distinct lineage within the type I methanotrophs and analysis of the deduced pmoA amino acid sequence of strain HT12(T) showed that it had a 7 % divergence from that of its most closely related species. The DNA G+C content was 49.3 mol%. Based on this evidence, strain HT12(T) represents a novel species and genus of the family Methylococcaceae, for which the name Methylovulum miyakonense gen. nov., sp. nov. is proposed. The type strain of the type species is HT12(T) ( = NBRC 106162(T)  = DSM 23269(T)  = ATCC BAA-2070(T))

Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis.

Eukaryotic methylotrophs, which are able to obtain all the carbon and energy needed for growth from methanol, are restricted to a limited number of yeast species. When these yeasts are grown on methanol as the sole carbon and energy source, the enzymes involved in methanol metabolism are strongly induced, and the membrane-bound organelles, peroxisomes, which contain key enzymes of methanol metabolism, proliferate massively. These features have made methylotrophic yeasts attractive hosts for the production of heterologous proteins and useful model organisms for the study of peroxisome biogenesis and degradation. In this paper, we describe recent insights into the molecular basis of yeast methylotrophy

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii

The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256–292 or Δ107–237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1–20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S.

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere

Yeast Hog1 proteins are sequestered in stress granules during high-temperature stress

The yeast high-osmolarity glycerol (HOG) pathway plays a central role in stress responses. It is activated by various stresses, including hyperosmotic stress, oxidative stress, high-temperature stress and exposure to arsenite. Hog1, the crucial MAP kinase of the pathway, localizes to the nucleus in response to high osmotic concentrations, i.e. high osmolarity; but, otherwise, little is known about its intracellular dynamics and regulation. By using the methylotrophic yeast Candida boidinii, we found that CbHog1-Venus formed intracellular dot structures after high-temperature stress in a reversible manner. Microscopic observation revealed that CbHog1-mCherry colocalized with CbPab1-Venus, a marker protein of stress granules. Hog1 homologs in Pichia pastoris and Schizosaccharomyces pombe also exhibited similar dot formation under high-temperature stress, whereas Saccharomyces cerevisiae Hog1 (ScHog1)-GFP did not. Analysis of CbHog1-Venus in C. boidinii revealed that a β-sheet structure in the N-terminal region was necessary and sufficient for its localization to stress granules. Physiological studies revealed that sequestration of activated Hog1 proteins in stress granules was responsible for downregulation of Hog1 activity under high-temperature stress

Community composition and methane oxidation activity of methanotrophs associated with duckweeds in a fresh water lake

Methanotrophs are the only biological sink of the greenhouse gas methane. To understand the ecological features of methanotrophs in association with plants in the methane emitting environments, we investigated the community composition and methane oxidation of methanotrophs associated with duckweeds in a fresh water lake. Duckweeds collected from Lake Biwa, Japan over three summers showed methane consumption activity between 0.0067 and 0.89 μmol h⁻¹ g⁻¹ (wet weight), with the highest values occurring from the end of July to August. The methanotrophic community on duckweeds consisted primarily of γ-proteobacterial groups including the genera Methylomonas and Methylocaldum. Further analysis of co-cultures of a methanotroph isolate with sterilized duckweed revealed that the duckweed plant as well as the duckweed spent culture supernatant exerted an enhancing effect on methane oxidation. These results indicate that duckweeds not only provide a habitat for methanotrophs but also stimulate methanotrophic growth

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore-iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species

Methanol bioeconomy: promotion of rice crop yield in paddy fields with microbial cells prepared from natural gas‐derived C 1 compound

微生物やその細胞壁成分の葉面散布による酒米の増収に成功 --メタノールを原料に生産した微生物製剤を出穂後1度の散布で--. 京都大学プレスリリース. 2020-12-11.Methylotrophs, which can utilize methanol as a sole carbon source, are promising microorganisms to be exploited in a methanol‐based bioeconomy, in which a variety of useful compounds are biotechnologically produced from natural gas‐derived methanol. Pink‐pigmented facultative methylotrophs (PPFMs) are common plant phyllospheric bacteria and are known to enhance seedling growth and total biomass of various plants. However, improvement of crop yield by inoculation of PPFMs at the field level has not been well investigated. We herein describe improvement of crop yield of several rice cultivars by foliar spraying of PPFMs. After selection of PPFM strains and rice cultivars by the in vitro seedling growth test, we further conducted paddy field experiments. The crop yield of the sake‐brewing rice Oryza sativa cultivar Hakutsurunishiki was reproducibly improved in a commercial paddy field for over a 5‐year period. A one‐time foliar spray of PPFM cells (living or killed) or a cell wall polysaccharide fraction, after the heading date, acted in the phyllosphere and effectively improved crop yield. Our results show that the established process with PPFMs is feasible for improvement of food production in the methanol bioeconomy

Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels

Draft genomes of gammaproteobacterial methanotrophs isolated from terrestrial ecosystems

Genome sequences of Methylobacter luteus, Methylobacter whittenburyi, Methylosarcina fibrata, Methylomicrobium agile, and Methylovulum miyakonense were generated. The strains represent aerobic methanotrophs typically isolated from various terrestrial ecosystems