Surface layers of methanotrophic bacteria

Structural and functional characteristics of the regular glycoprotein layers in prokaryotes are analyzed with a special emphasis on aerobic methanotrophic bacteria. S-Layers are present at the surfaces of Methylococcus, Methylothermus, and Methylomicrobium cells. Different Methylomicrobium species either synthesize S-layers with planar (p2, p4) symmetry or form cup-shaped or conical structures with hexagonal (p6) symmetry. A unique, copper-binding polypeptide ‘CorA’/MopE (27/45 kDa), which is coexpressed with the diheme periplasmic cytochrome c peroxidase ‘CorB’/Mca (80 kDa) was found in Methylomicrobium album BG8, Methylomicrobium alcaliphilum 20Z, and Methylococcus capsulatus Bath. This tandem of the surface proteins is functionally analogous to a new siderophore: methanobactin. Importantly, no ‘CorA’/MopE homologue was found in methanotrophs not forming S-layers. The role of surface proteins in copper metabolism and initial methane oxidation is discussed.     

Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs

The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline. M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

Genome sequence of the haloalkaliphilic methanotrophic bacterium Methylomicrobium alcaliphilum 20Z

Methylomicrobium strains are widespread in saline environments. Here, we report the complete genome sequence of Methylomicrobium alcaliphilum 20Z, a haloalkaliphilic methanotrophic bacterium, which will provide the basis for detailed characterization of the core pathways of both single-carbon metabolism and responses to osmotic and high-pH stresses. Final assembly of the genome sequence revealed that this bacterium contains a 128-kb plasmid, making M. alcaliphilum 20Z the first methanotrophic bacterium of known genome sequence for which a plasmid has been reported.     

Primary characterization of dominant cell surface proteins of halotolerant methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Cell surface-associated proteins with molecular masses of 27 and 80 kDa found in Methylomicrobium alcaliphilum were studied. The MEALZv2_1030034 and MEALZv2_1030035 genes encoding these proteins were identified in the partially annotated genome of the methanotroph. Polypeptides MEALZv2_1030034 and MEALZv2_1030035 showed high homology (>50% identity) with the surface proteins CorA and CorB of Methylomicrobium album BG8 expressed in conditions of low copper content in the growth medium. Electron microscopic analysis with antibodies revealed localization of the 27-kDa protein in the base of the cup-shaped structures of S-layers. The mutant with an insertion in the MEALZv2_1030034 gene lost the ability to grow on the medium with methane, but grew in the presence of 0.2% methanol. In this case, the cup-shaped structures of S-layers were not bound to the cell wall but occurred as regular aggregates in the intercellular space. The 80-kDa protein was found mainly in the periplasm, had a peroxide-degrading activity, and was classified as a di-heme cytochrome c peroxidase. The mutant deficient in the MEALZv2_1030035 gene grew on methane at a high rate and had higher activities of C₁ compound oxidation enzymes than did the parent strain. The role of the proteins MEALZv2_1030034 and MEALZv2_1030035 and distribution of their homologues in other methanotrophs are discussed.