Features of psychrophilic and thermophilic methanotrophic microorganisms

Обзор посвящен описанию структурно-функциональных и физиолого-биохимических особенностей метанотрофов, обитающих в экотопах с низкими и высокими температурами. Показано, что в холодных экосистемах обитают метанотрофы I типа, для которых характерны ассимиляция углерода по рибулозомонофосфатному пути, преобладание жирных кислот с 16 атомами углерода, мембранная форма метанмонооксигеназы (ММО). Среди термофилов доминируют метанотрофы II типа, которым свойственны наличие как растворимой, так и мембранной формы ММО, реализация, наряду с РМФ циклом, минорных серинового и РБФ путей Сі-ассимиляции. This paper considers some structural-functional and physiological-biochemical features of methanotrophs living in ecotopes with low and high temperatures. It has been established that cold ecosystems are characterized by type I methanotrophs, with the assimilation of carbon along the ribulose monophosphate pathway, the predominance of fatty acids with 16 carbon atoms, and the presence of membrane methane monooxygenase (MMO). Representatives of both types are detected among thermophilic methanotrophs, but type II with the presence of soluble and membrane forms of MMO is dominant, where, along with the RMF cycle, minor serine and RBF Ci-assimilation pathways are realized.

Methylophaga nitratireducenticrescens sp. nov. and Methylophaga frappieri sp. nov., isolated from the biofilm of the methanol-fed denitrification system treating the seawater at the Montreal Biodome

Two bacterial strains, designated JAM1T and JAM7T, were isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. They were affiliated within the genus Methylophaga of the Gammaproteobacteria by analysis of the 16S rRNA gene sequences. Strain JAM1T had the capacity to grow under denitrifying conditions by reducing nitrate into nitrite which is unique among the species of the genus Methylophaga . Major fatty acids were C16 : 1ω7c or ω6c, C16 : 0 and C18 : 1ω7c or ω6c. The major ubiquinone was Q8. Both strains required vitamin B12 and Na+ ions for growth. The genomes of strains JAM1T and JAM7T have been completely sequenced and showed a DNA G+C content of 44.7 mol% and 47.8 mol%, respectively. Growth occurred at pH 6–11 and at 0.5–8 % NaCl. Both genomes contained predicted ORFs encoding the key enzymes of the ribulose monophosphate pathway. Also, operons encoding two nitrate reductases (Nar), two nitric oxide reductases (Nor), one nitrous oxide reductase (Nos) and one truncated nitrite reductase (NirK) were clustered in a 67 kb chromosomal region in strain JAM1T. No such operons were found in strain JAM7T. These results supported the affiliation of the two strains as novel species within the genus Methylophaga . The names Methylophaga nitratireducenticrescens sp. nov. for type strain JAM1T ( = DSM 25689T = ATCC BAA-2433T) and Methylophaga frappieri sp. nov. for type strain JAM7T ( = DSM 25690T = ATCC BAA-2434T) are proposed.

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

A novel methanotroph, designated strain HT12T, was isolated from forest soil in Japan. Cells of strain HT12T 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 C16 : 0 (46.9 %) and C14 : 0 (34.2 %), whereas unsaturated C16 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 2T (93.1 % sequence similarity) and Methylobacter tundripaludum SV96T (92.6 % similarity). Phylogenetic analysis based on the pmoA gene indicated that strain HT12T formed a distinct lineage within the type I methanotrophs and analysis of the deduced pmoA amino acid sequence of strain HT12T 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 HT12T 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 HT12T ( = NBRC 106162T = DSM 23269T = ATCC BAA-2070T).

Methylophaga aminisulfidivorans sp. nov., a restricted facultatively methylotrophic marine bacterium

A novel restricted facultatively methylotrophic marine strain, MPT, possessing the ribulose monophosphate pathway of C1-carbon compound assimilation was isolated from a seawater sample obtained from Mokpo, South Korea. The novel isolate is aerobic, Gram-negative, asporogenous and a non-motile short rod. It grows well on methanol, methylated amines, dimethylsulfide and DMSO. Optimal growth occurs with 3 % NaCl at 30 °C and pH 7.0. Fructose is utilized as a multicarbon source. Growth factors are not required and vitamin B12 does not stimulate growth. The cellular fatty acid profile of the novel strain consists primarily of straight-chain saturated C16 : 0 and unsaturated C16 : 1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content is 44.9 mol% (T m). Based on 16S rRNA gene sequence analysis and DNA–DNA relatedness (25–41 %) with the type strains of marine methylotrophs belonging to the genus Methylophaga, it is suggested that isolate MPT represents a novel species, Methylophaga aminisulfidivorans sp. nov. (type strain MPT=KCTC 12909T=VKM B-2441T=JCM 14647T).

An Extremely Oligotrophic Bacterium, Rhodococcus erythropolis N9T-4, Isolated from Crude Oil

ABSTRACT Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO2-limiting conditions but could grow on a medium containing NaHCO3 under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO2. Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N′-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO2 fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4.

The Ribulose Monophosphate Pathway Substitutes for the Missing Pentose Phosphate Pathway in the Archaeon Thermococcus kodakaraensis

ABSTRACT The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Δhps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.