Mannitol as a Growth Substrate for Facultative Methylotroph Methylobrevis pamukkalensis PK2.

Biochemistry of carbon assimilation in aerobic methylotrophs growing on reduced C1 compounds has been intensively studied due to the vital role of these microorganisms in nature. The biochemical pathways of carbon assimilation in methylotrophs growing on multi-carbon substrates are insufficiently explored. Here we elucidated the metabolic route of mannitol assimilation in the alphaproteobacterial facultative methylotroph Methylobrevis pamukkalensis PK2.

Genomic Insights into Moderately Thermophilic Methanotrophs of the Genus .

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture.

Interchangeability of class I and II fumarases in an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z is an industrially promising candidate for bioconversion of methane into value-added chemicals. Here, we have study the metabolic consequences of the breaking in the tricarboxylic acid (TCA) cycle by fumarase knockout. Two fumarases belonging to non-homologous class I and II fumarases were obtained from the bacterium by heterologous expression in Escherichia coli.

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation.

Methylomonas rapida sp. nov., a novel species of fast-growing, carotenoid-producing obligate methanotrophs with high biotechnological potential.

The genus Methylomonas accommodates strictly aerobic, obligate methanotrophs, with their sole carbon and energy sources restricted to methane and methanol. These bacteria inhabit oxic-anoxic interfaces of various freshwater habitats and have attracted considerable attention as potential producers of a single-cell protein. Here, we characterize two fast-growing representatives of this genus, strains 12 and MP1, which are phylogenetically distinct from the currently described Methylomonas species (94.

Complete Genome Sequence of Methylococcus capsulatus MIR, a Methanotroph Capable of Growth on Methanol.

Methylococcus capsulatus MIR is an aerobic methanotroph that was isolated from an activated sludge sample and is capable of growth on methanol. The finished genome of strain MIR is 3.2 Mb in size.

AcdR protein is an activator of transcription of 1-aminocyclopropane-1-carboxylate deaminase in Methylobacterium radiotolerans JCM 2831.

It has been previously shown that a number of plant associated methylotrophic bacteria contain an enzyme aminocyclopropane carboxylate (ACC) deaminase (AcdS) hydrolyzing ACC, the immediate precursor of ethylene in plants. The genome of the epiphytic methylotroph Methylobacterium radiotolerans JCM2831 contains an open reading frame encoding a protein homologous to transcriptional regulatory protein AcdR of the Lrp (leucine-responsive regulatory protein) family. The acdR gene of M.

Expanding Characterized Diversity and the Pool of Complete Genome Sequences of Species, the Bacteria of High Environmental and Biotechnological Relevance.

The bacterial genus , which comprises aerobic thermotolerant methanotrophic cocci, was described half-a-century ago. Over the years, a member of this genus, Bath, has become a major model organism to study genomic and metabolic basis of obligate methanotrophy. High biotechnological potential of fast-growing species, mainly as a promising source of feed protein, has also been recognized.

Enzymes of an alternative pathway of glucose metabolism in obligate methanotrophs.

Aerobic methanotrophic bacteria utilize methane as a growth substrate but are unable to grow on any sugars. In this study we have shown that two obligate methanotrophs, Methylotuvimicrobium alcaliphilum 20Z and Methylobacter luteus IMV-B-3098, possess functional glucose dehydrogenase (GDH) and gluconate kinase (GntK). The recombinant GDHs from both methanotrophs were homotetrameric and strongly specific for glucose preferring NAD over NADP.

Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli.

Objectives: Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments.

Results: Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as His-tagged proteins were characterized. The simultaneous substitutions of arginine-180, arginine-198 and serine-178 residues by alanine in the enzyme pocket sequence as well as other amino acid changes decreased both NADPH- and NADH-dependent activities in comparison to the wild-type enzyme.

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.

Ectoine degradation pathway in halotolerant methylotrophs.

Background: Microorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acid ectoine, the major osmoprotector in halotolerant methane-utilizing bacteria.

Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification.

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 k/K ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction.

Metabolic Features of Aerobic Methanotrophs: News and Views.

This review is focused on recent studies of carbon metabolism in aerobic methanotrophs that specifically addressed the properties, distribution and phylogeny of some of the key enzymes involved in assimilation of carbon from methane. These include enzymes involved in sugar sythesis and cleavage, conversion of intermediates of the tricarboxylic acid cycle, as well as in osmoadaptation in halotolerant methanotrophs.

ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs.

The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph sp. 12. The recombinant proteins were obtained by the heterologous expression of the genes in .

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

The indicator enzyme of the serine pathway of assimilation of reduced C 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.

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12.

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C-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 C-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 C-compounds via the serine cycle.

Draft Genome Sequences of Two Gammaproteobacterial Methanotrophs Isolated from Rice Ecosystems.

The genomes of the aerobic methanotrophs "" strain 73a and strain 175 were sequenced. Both strains were isolated from rice plants. strain 73a represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus .

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-His, PHI-His, and the two-domain protein HPS-PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His, HPS-His (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.

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z.

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks.

[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.

Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine Ecosystems.

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained.

Acetate kinase-an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z.

Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His6-tagged AcK were determined. The M.