Methylobrevis pamukkalensis gen. nov., sp. nov., a halotolerant restricted facultative methylotroph isolated from saline water

An aerobic halotolerant restricted facultatively methylotrophic bacterium was isolated from a saline hot spring in Pamukkale, Turkey, and designated strain PK2T. The cells of this strain were Gram-stain-negative, asporogenous, motile short rods multiplying by binary fission. They utilized methanol, methylamine and mannitol as carbon and energy sources. The organism grew optimally at 30 °C in media containing 85 mM NaCl and at pH 7.5–8.0. C1 compounds were assimilated via the isocitrate-lyase-positive variant of the serine pathway. Poly-β-hydroxybutyrate and the compatible solute ectoine were found in the cells. The dominant phospholipids were phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major cellular fatty acids of methanol-grown cells were C18 : 1ω7 and C16 : 1ω7c. The main ubiquinone was Q-10. The DNA G+C content was 67.9 mol% (T m). The 16S rRNA gene sequence suggests that strain PK2T is affiliated with the order Rhizobiales within the class Alphaproteobacteria , being most closely related to Mesorhizobium gobiense CCBAU 83330T (94 % similarity). A novel genus and species, Methylobrevis pamukkalensis gen. nov., sp. nov., is proposed on the basis of phenotypic and genotypic data, with PK2T (VKM B-2849T = JCM 30229T) as the type strain.

Methylophilus glucosoxydans sp. nov., a restricted facultative methylotroph from rice rhizosphere

Two restricted facultatively methylotrophic strains, designed BT and P, were isolated from rice roots. The isolates were strictly aerobic, Gram-negative, asporogenous, mesophilic, neutrophilic, motile rods that multiplied by binary fission and were able to synthesize indole-3-acetate. The cellular fatty acid profiles of the two strains were dominated by C16 : 0, C16 : 1ω7c and C16 : 0 2-OH. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol. Cardiolipin (diphosphatidylglycerol) was absent. The two strains assimilated methanol carbon at the level of formaldehyde via the ribulose monophosphate cycle (2-keto-3-deoxy-6-phosphogluconate variant). They lacked α-ketoglutarate dehydrogenase and glutamate dehydrogenase. They assimilated ammonium via the glutamate cycle enzymes glutamine synthetase and glutamate synthase. The DNA G+C contents of strains BT and P were 52.5 and 51.5 mol% (T m), respectively. The level of DNA–DNA reassociation between these strains was 78 %, indicating that they belong to one species. Phylogenetic analysis of strain BT based on 16S rRNA and methanol dehydrogenase (mxaF) gene sequences showed a high level of similarity to members of the genus Methylophilus. As the two isolates were clearly distinct from all recognized members of the genus Methylophilus based on phenotypic data and levels of DNA–DNA relatedness (30–46 %), they are considered to represent a novel species, for which the name Methylophilus glucosoxydans sp. nov. is proposed; the type strain is BT ( = VKM B-1607T = CCUG 59685T = DSM 5898T).

Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522

Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N′ -nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine–methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10−6 to 10−8 for each marker. Key words: methylotrophs, Hyphomicrobium, N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis, transposon mutagenesis, promiscuous plasmids, chromosome mobilization.