Cloning and Characterization of Polyhydroxybutyrate Synthase from Methylobacterium extorquens AM1

В результате поиска генов, кодирующих вероятные ПГБ-синтазы в геномах бактерий рода Methylobacterium, выявлены множественные (до пяти у одного штамма) гены ПГБ-синтаз. Филогенетическим анализом показано, что белки PhaC1, PhaC2, PhaC3 относятся к I классу ПГБ-синтаз, белки PhaC4 – к ПГБ-синтазам III класса, тогда как PhaC5, по-видимому, представляет неохарактеризованный класс ПГБ-синтаз. Впервые выделена и охарактеризована рекомбинантная ПГБ-синтаза I класса (КФ 2.3.1.B2) из Methylobacterium extorquens AM1, кодируемая геном phaC1. Молекулярная масса мономера фермента составила 78 кДа. Константа Михаэлиса (Km) для PhaC1 из штамма AM1 составила 1,3 мМ, а максимальная скорость реакции (Vmax) – 0,1 мкмоль∙мин-1∙мг-1. Получен делеционный мутант Methylobacterium extorquens по гену phaC, перспективный для дальнейшего исследования особенностей биосинтеза ПГБ метилобактериямиMultiple genes encoding putative PHB synthases (up to 5 in single strain) were found in Methylobacterium genomes. As a result of phylogenetic analysis proteins PhaC1, PhaC2, PhaC3 were identified as class I PHB synthases, PhaC4 proteins were identified as class III PHB synthases, while PhaC5 apparently belongs to uncharacterized class of PHB synthases. Firstly, the recombinant class I PBH synthase (EC 2.3.1.B2) encoded by phaC1 gene from Methylobacterium extorquens AM1 was purified and characterized. Molecular mass of enzyme monomer was 78 kDa. Michaelis constant (Km) for PhaC1 was 1,3 mM and maximal reaction rate (Vmax) was 0,1 μmol∙min–1∙mg–1. The deletion mutant of Methylobacterium extorquens in the phaC gene was obtained which is promising for further study of peculiarities of methylobacteria’s PHB biosynthesi

Methylophaga lonarensis sp. nov., a novel moderately haloalkaliphilic methylotroph isolated from the soda lake sediments of a meteorite impact crater

A novel moderately haloalkaliphilic methylotrophic bacterium (strain MPL(T)), possessing the ribulose monophosphate pathway for carbon assimilation, was isolated from two week old Lonar Lake sediment microcosms that were actively oxidizing methane. The isolate, which utilizes methanol, is an aerobic, Gram-negative, asporogenous, motile short rod that multiplies by binary fission. It requires NaHCO(3) or NaCl for growth and although not auxotrophic for vitamin B12, the addition of vitamin B12 enhances its growth. Optimal growth occurs with 0.5-2% (w/v) NaCl at 28-30 °C and pH 9.0-10.0. The cellular fatty acid profile of the strain consists primarily of straight-chain saturated C(16:0) and unsaturated C(16:1)?7c and C(18:1)?7c acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Cells accumulate the cyclic imino acid ectoine as the main compatible solute. The DNA G+C content is 50.0 mol%. Based on 16S rRNA gene sequence similarity (94.0-95.4 %) with the type strains of methylotrophs belonging to the genus Methylophaga and DNA-DNA relatedness (31%) with the closest described species Methylophaga alcalica M39(T), it is proposed that isolate MPL(T) represents a novel species, Methylophaga lonarensis sp. nov. (type strain MPL(T) =VKM B-2684(T) =MCC 1002(T))