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.

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.

Methylopila carotae sp. nov., a facultative methylotroph, isolated from a root of Daucus carota L.

An aerobic facultatively methylotrophic bacterium, designated strain Das4.1, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission.

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 .

Individual stages of bacterial dichloromethane degradation mapped by carbon and chlorine stable isotope analysis.

The fractionation of carbon and chlorine stable isotopes of dichloromethane (CHCl, DCM) upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed. Isotope effects for individual steps of the multi-stage DCM degradation process, including transfer across the cell wall from the aqueous medium to the cell cytoplasm, dehalogenase binding, and catalytic reaction, were considered. The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of CCl bonds, and not by inflow of DCM into cells.

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.

Distribution of 1-aminocyclopropane-1-carboxylate deaminase and D-cysteine desulfhydrase genes among type species of the genus Methylobacterium.

The presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase determines the ability of bacteria to increase the resistance of plants to various types of stress. The genes of ACC deaminase (acdS) and the closely related enzyme D-cysteine desulfhydrase (dcyD) were searched in type strains of various representatives of the genus Methylobacterium. Using PCR screening and in silico searching in the available complete genome sequences of type strains, the genes were found in 28 of 48 species of the genus.

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.

Ancylobacter sonchi sp. nov., a novel methylotrophic bacterium frоm roots of Sonchus arvensis L.

An aerobic facultatively methylotrophic bacterium was isolated from roots of Sonchus arvensis L. and designated strain Osot The cells of this strain were Gram-stain-negative, asporogenous, motile short rods multiplying by binary fisson. They utilized methanol, methylamines and a variety of polycarbon compounds as the carbon and energy sources.

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.

[Functionality of Metdi5511gene in Methylobacterium dichloromethanicum DM4].

A knockout mutant of Methylobacterium dichloromethanicum DM4 with an inactivated gene of a putative transcription regulator METDI5511 (ΔMETDI5511) has been obtained. The expression of this gene increases many times when the strain is grown on dichloromethane compared to methanol. The mutant had a low growth rate on dichloromethane as compared with the original strain and was found to be more sensitive to influences of various types of stress (oxidative, osmotic stress, heat, and drying).

[Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow Oblast].

During the summer period (15–25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus.

[A novel Delftia plant symbiont capable of autotrophic methylotrophy].

A facultative methylotrophic bacterium, strain Lp-1, which was isolated from root nodules of lupine (Lupinus polyphyllus L.) on the medium with methanol as a carbon and energy source, exhibited high similarity of the 16S rRNA gene sequences to Delftia strains (94‒99.9%).

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.

Methylobacillus methanolivorans sp. nov., a novel non-pigmented obligately methylotrophic bacterium.

Three strains of obligately methylotrophic Betaproteobacteria (ZT, SP and M3) with the ribulose monophosphate pathway of C1 assimilation are described. The isolates were strictly aerobic, Gram-stain-negative, asporogenous, motile (strains ZT and M3) or non-motile (strain SP) rods that multiplied by binary fisson, and were mesophilic and neutrophilic. All three strains utilized methanol but only strains SP and M3 utilized methylamine as carbon and energy sources.

Draft Genome Sequence of Methylophaga muralis Bur 1, a Haloalkaliphilic (Non-Methane-Utilizing) Methylotroph Isolated from a Soda Lake.

The draft genome sequence of Methylophaga muralis strain Bur 1 (VKM B-3046T), a non-methane-utilizing methylotroph isolated from a soda lake, is reported here. Strain Bur 1 possesses genes for methanol and methylamine (methylamine dehydrogenase and N-methylglutamate pathway) oxidation. Genes for the biosynthesis of ectoine were also found.

Draft Genome Sequence of Methyloligella halotolerans С2T, a New Halotolerant Methylotroph, Accumulating Poly-3-Hydroxybutyrate and Ectoine.

Methyloligella halotolerans С2 is a moderate halophilic obligate methylotroph, accumulating ultra-high-molecular-weight poly-3-hydroxybutyrate (up to 8 to 10 MDa) from methanol. Here we report a draft genome and annotation of Methyloligella halotolerans C2 (VKM B-2706 = CCUG 61687 = DSM 25045).

Emended Description of Methylovorus glucosotrophus Govorukhina and Trotsenko 1991.

Phylogeneticanalysis based,on comparison of the 16S rRNA gene sequences in combination with comparative analysis of physiological, biochemical, and chemotaxonomic characteristics and DNA-DNA hybridization revealed that "Methylobacillusfructoseoxidans" 34 (VKM B-1609 = DSM 5897 and-Methylov- orus glucosotrophus 6B 1T (ATCC 49758T = DSM 6874T = VKM B- 1745T = NCIMB 13222 ) belong to the same Methylovorus species. Extended description of the limited facultative methylotroph Methylovorus gluco- sotrophus is proposed, which includes the fructose-utilizing strain 34. Emended description of Methylovorus glucosotrophus is provided.

[Enhanced Resistance of Pea Plants to Oxidative: Stress Caused by Paraquat during Colonization by Aerobic Methylobacteria].

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Sidel) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H2O2, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls a, b and carotenoids).

[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.

[Functionality of the xoxF Gene in Methylobacterium dichloromethanicum DM4].

Activation of expression of the xoxFgene encoding PQQ-dependent methanol/ethanol dehydrogenase (METDI2492) in dichloromethane- (DCM) -grown Methylobacterium dichloromethanicum DM4 was first demonstrated. The sequence of the only XoxF homolog found in the genome of strain DM4 exhibited 50% identity to that of the protein (MxaF) of the large subunit of methanol dehydrogenase (MDH). A knockout mutant with the inactivate xoxF gene (ΔxoxF) was found to be unable to grow on methanol due to the absence of the expression of the gene cluster of the classical MDH, as was confirmed by the GFP test.

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.