Serine-glyoxylate aminotranferases from methanotrophs using different C1-assimilation pathways.

The indicator enzyme of the serine pathway of assimilation of reduced C1 compounds, serine-glyoxylate aminotransferase (Sga), has been purified from three methane-oxidizing bacteria, Methylomicrobium alcaliphilum 20Z, Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. The native enzymes were shown to be dimeric (80 kDa, strain 20Z), tetrameric (~ 170 kDa, strain OB3b) or trimeric (~ 120 kDa, strain Bath). Sga from the three methanotrophs catalyse the pyridoxal phosphate-dependent transfer of an amino group from serine to glyoxylate and pyruvate; the enzymes from strains 20Z and Bath also transfer an amino group from serine to α-ketoglutarate and from alanine to glyoxylate. No other significant differences between the Sga from the three methanotrophs were found. The three methanotrophic Sga have their highest catalytic efficiencies in the reaction between glyoxylate and serine, which is in agreement with their function to provide circulation of the serine assimilation pathway.The disruption of the sga gene in Mm. alcaliphilum resulted in retardation of growth rate of the mutant cells and in a prolonged lag-phase after passaging from methane to methanol. In addition, the growth of the mutant strain is accompanied by formaldehyde accumulation in the culture liquid. Hence, Sga is important in the serine cycle of type I methanotrophs and this pathway could be related to the removal of excess formaldehyde and/or energy regulation.

Properties of Malic Enzyme from the Aerobic Methanotroph Methylosinus trichosporium.

Recombinant malic enzyme from the aerobic methanotroph Methylosinus trichosporium was obtained by heterologous expression in Escherichia coli and purified by affinity metal-chelating chromatography. The homohexameric enzyme of 6×80 kDa catalyzed the reversible reaction of oxidative decarboxylation of malate to pyruvate in the presence of mono- and divalent cations and NADP+ as a cofactor. The kcat/Km ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction. Analysis of the protein sequence revealed that the C-region of the enzyme contains a large domain homologous to phosphoacetyltransferase, but no phosphoacetyltransferase activity was detected either for a full chimeric malic enzyme or for the C-end fragment obtained as a separate protein. This C-end domain promoted activity of the malic enzyme.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

Characterization of Two Recombinant 3-Hexulose-6-Phosphate Synthases from the Halotolerant Obligate Methanotroph Methylomicrobium alcaliphilum 20Z.

Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His6, PHI-His6, and the two-domain protein HPS-PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His6, HPS-His6 (2 × 20 kDa), and the fused protein HPS-PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose-5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the kcat/Km ratio, HPS-His6 had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS-PHI. AMP and ADP were powerful inhibitors of both HPS and HPS-PHI activities. The two-domain HPS-PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.

[Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z].

A number of vectors were constructed based on the plasmid from the broad range of pMHA200 hosts. Also, the expression of some key genes of the haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z was studied. The activities of the promoter regions of genes for hexulose phosphate synthase, glutamine synthetase, and glucokinase, as well as the promoter of the ectABC-ask operon, which encodes enzymes for osmoprotectant ectoine biosynthesis, were evaluated with the use of the gfp gene; the evaluation was proven to be ineffective. Conversely, glucokinase and a heterologous enzyme of chloramphenicol acetyltransferase were useful for the evaluation of promoter activity. In M. alcaliphilum 20Z cells, the expression level of chloramphenicol acetyltransferase transcribed from the methanol dehydrogenase promoter was higher as compared with that of glucokinase. This seems to be due to a regulatory mechanism for homologous protein expression. The introduction of a synthetic nucleotide sequence forming the secondary structure in the 5' untranslated region of the glucokinase mRNA resulted in an increase of this enzyme level. This is the first attempt to use M. alcaliphilum 20Z for homo- and heterologous protein expression.

[Biosynthesis of secondary metabolites in methanotrophs: biochemical and genetic aspects (review)].

The review summarizes the data on the metabolic potential of methanotrophs as producers of biopolymers, alternative biofuel, bioprotectants, and other secondary metabolites. The work provides the examples of modern 'omic' technologies used for genetic engineering of efficient methanotrophic producers.

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

Properties of recombinant ATP-dependent fructokinase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

In the cluster of genes for sucrose biosynthesis and cleavage in Methylomicrobium alcaliphilum 20Z, a gene whose encoded sequence showed high similarity to sugar kinases of the ribokinase family was found. By heterologous expression of this gene in Escherichia coli cells and following metal chelate affinity chromatography, the electrophoretically homogenous recombinant enzyme with six histidine residues on the C-end was obtained. The enzyme catalyzes ATP-dependent phosphorylation of fructose into fructose-6-phosphate but is not active with other sugars as phosphoryl acceptors. The fructokinase of M. alcaliphilum 20Z is most active in the presence of Mn(2+) at pH 9.0 and 60°C, being inhibited by ADP (K(i) = 2.50 ± 0.03 mM). The apparent K(m) values for fructose and ATP are 0.26 and 1.3 mM, respectively; the maximal activity is 141 U/mg protein. The enzyme shows the highest similarity of translated amino acid sequence with putative fructokinases of methylotrophic and autotrophic proteobacteria whose fruK gene is located in the gene cluster of sucrose biosynthesis. The involvement of fructokinase in sucrose metabolism in M. alcaliphilum 20Z and other methanotrophs and autotrophs is discussed.

Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica.

In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ(70)-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.

Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath.

The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co2+ and inhibited by EDTA. The enzyme displays low K(m) to fructose-1,6-bisphosphate (FBP) and higher K(m) to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co2+ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C(1) assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.

Malyl-CoA lyase provides glycine/glyoxylate synthesis in type I methanotrophs.

The biochemical routes for assimilation of one-carbon compounds in bacteria require many clarifications. In this study, the role of malyl-CoA lyase in the metabolism of the aerobic type I methanotroph Methylotuvimicrobium alcaliphilum 20Z has been investigated by gene inactivation and biochemical studies. The functionality of the enzyme has been confirmed by heterologous expression in Escherichia coli. The mutant strain lacking Mcl activity demonstrated the phenotype of glycine auxotrophy. The genes encoding malyl-CoA lyase are present in the genomes of all methanotrophs, except for representatives of the phylum Verrucomicrobium. We suppose that malyl-CoA lyase is the enzyme that provides glyoxylate and glycine synthesis in the type I methanotrophs supporting carbon assimilation via the serine cycle in addition to the major ribulose monophosphate cycle.

[Detection of osmoprotector ectoine content in methylotrophic bacteria using of normal-phase high performance liquid chromatography].

Detection and quantitative analysis of ectoine in bacterial biomass were performed by normal-phase high performance liquid chromatography with ultraviolet detection at 230 nm. Quantitative analysis was not hindered by glutamate and sucrose accumulation in bacteria. Measurement of ectoine concentration in haloalkaliphilic methanotrophs Methylobacter marinus 7C and Methylomicrobium alcaliphilum 5S showed that ectoine accumulation reached maximum (5 and 12% of dry cell weight) in the presence of NaCl at concentrations of 4 and 6%, respectively.

[Biotechnological potential of methylotrophic bacteria: a review of current status and future prospects].

Major results of the authors' findings on the implementation of biotechnological potential of aerobic methylobacteria and methanotrophs for obtaining forage proteins, biopolymers (polybutyrate and polysaccharides), enzymes (oxidoreductases), and bioprotectors (ectoin), as well as for degrading toxic C1 and Cn compounds have been reviewed. Unique features of the structural and functional organization of the metabolism of extremophilic (tolerant) methylotrophs are discussed, with a view for their prospective use in various fields of modern biotechnology, including biocatalysis and nanotechnology.

Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden.

Methane-utilizing bacteria were enriched from deep igneous rock environments and affiliated by amplification of functional and phylogenetic gene probes. Type I methanotrophs belonging to the genera Methylomonas and Methylobacter dominated in enrichment cultures from depths below 400 m. A pure culture of an obligate methanotroph (strain SR5) was isolated and characterized. Pink-pigmented motile rods of the new isolate contained intracytoplasmic membranes as stacks of vesicles, assimilated methane via the ribulose monophosphate pathway and had an incomplete tricarboxylic acid cycle. Phosphatidyl glycerol, methylene ubiquinone and cytochrome c552 were prevailing. The DNA G+C content is 53.3 mol %. Strain SR5 grew at temperatures between 5 and 30 degrees C with optimum at 15 degrees C, close to its in situ temperature. Analyses of 16S rRNA gene, whole cell protein, enzymatic and physiological analyses of strain SR-5 revealed significant differences compared to the other representatives of Type I methanotrophs. Based on pheno- and genotypic characteristics we propose to refer the strain SR5 as to a new species, Methylomonas scandinavica.

[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 conicalstructures 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.

Highly efficient methane biocatalysis revealed in a methanotrophic bacterium.

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

Cloning, purification, and characterization of diaminobutyrate acetyltransferase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

L-2,4-Diaminobutyrate (DAB) acetyltransferase (DABAcT) catalyzes one of the key reactions of biosynthesis of the bacterial osmoprotectant ectoine--acetylation of L-2,4-DAB yielding Ngamma-acetyl-2,4-DAB. Gene ectA encoding DABAcT was cloned from DNA of the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z and expressed in Escherichia coli with an additional six His residues at the C-terminus. Homogeneous enzyme preparation with specific activity 200 U/mg was obtained by affinity metal-chelating chromatography. DABAcT was found to be a homodimer with molecular mass 40 kD. The enzyme is most active at pH 9.5 and 20 degrees C, and its activity increased threefold in the presence of 0.1-0.2 M NaCl or 0.2 M KCl. The Km values of recombinant DABAcT measured at the optimal pH and temperature in the presence of 0.2 M KCl were 460 and 36.6 microM for L-2,4-DAB and acetyl-CoA, respectively. The enzyme is specific for L-2,4-DAB and acetyl-CoA and is also active against propionyl-CoA (20%). Zn2+ and Cd2+ at 1 mM concentration completely inhibit the recombinant enzyme; 10 mM ATP inhibits 26% of the enzyme activity, whereas EDTA, o-phenanthroline, ADP, NAD(P), and NAD(P)H do not significantly effect the enzyme activity. The possible participation of DABAcT in regulation of ectoine biosynthesis in M. alcaliphilum 20Z is discussed.

[Aerobic methanotrophic communities in the bottom sediments of Lake Baikal].

The results of the first methodical investigation into the aerobic methanotrophic communities inhabiting the bottom sediments of Lake Baikal are reported. Use of the radioisotopic method revealed methane consumption in 12 10- to 50-cm-long sediment cores. The maximum methane consumption rates (495-737 microl/(dm3 day) were recorded in sediments in the regions of hydrothermal vents and oil and gas occurrence. Methane consumption was most active in the surface layers of the sediments (0-4 cm); it decreased with the sediment depth and became negligible or absent at depths below 20 cm. The number of methanotrophic bacteria usually ranged from 100 to 1000 cells/cm3 of sediment and reached 1 million cells/cm3 in the regions of oil and gas occurrence. The 17 enrichment cultures obtained were represented mainly by morphotype II methanotrophs. Phylogenetic analysis of the enrichment cultures in terms of the amino acid sequence of the alpha subunit of the membrane-bound methane monooxygenase revealed the predominance of methanotrophs of the genus Methylocystis. The results obtained suggest the presence of an active aerobic methanotrophic community in Lake Baikal.