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.

[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). The cells were stained with Fluorescent Brightener 28 (Calcofluor white), and the intensity of their fluorescence showed that the ΔMETDI5511 mutant had significantly increased numbers of surface polysaccharides with ß-1,3 and ß-1,4-glycoside bonds. The results indicate that the METDI5511 gene is involved in the regulation of surface polysaccharides that play an important role in adaptation of cells to growth on dichloromethane.

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

During the summer period (15­25°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. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 µg/mL) and are probably plant symbionts.

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 cells of Delftia sp. Lp-1 were motile gram-negative rods dividing by binary fission. Predominant fatty acids were C16:0 (34.2%), C16:1ω9 (14.5%), and C18:1ω7c (17.3%). Phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol were the dominant phospholipids. Q8 was the major ubiquinone. Optimal growth occurred at 24‒26°C and pH 7.1‒7.3; growth was inhibited by 1% NaCl. The organism oxidized methanol with the classical methanol dehydrogenase and used the ribulose bisphosphate pathway of C1 metabolism. Analysis of translated amino acid sequence of the large subunit of the MxaF methanol dehydrogenase revealed 85.5‒94% similarity to the sequences of such autotrophic methylotrophs of the class Alphaproteobacteria as Angulomicrobium, Starkeya, and Ancylobacter, indicating the possible acquisition of the mxaF gene via horizontal gene transfer. Delftia sp. Lp-1 (VKM B-3039, DSM 24446), the first methylotrophic member of the genus Delftia, was shown to be a plant symbiont, stimulating plant growth and morphogenesis, increasing the level of photosynthetic pigments and specific leaf weight. It possesses the nifH gene of nitrogen fixation, is capable of phosphate solubilization, synthesis of auxins and siderophores, and is antagonistic to plant pathogenic fungi and bacilli.

[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). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.

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.

[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.

[Aerobic methylobacteria as promising objects of modern biotechnology].

The experimental data of the past decade concerning the metabolic peculiarities of aerobic meth ylobacteria and the prospects for their use in different fields of modern biotechnology, including genetic engineering techniques, have been summarized.

[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. When grown of succinate, the ΔxoxF mutant exhibited a lower growth rate on DCM than the original strain and was more sensitive to various stress factors (oxidative, osmotic, and heat shock). Based on these data, the xoxF gene was hypothesized to belong to a group of genes affecting expression of the proteins of general stress response.

Cloning and characterization of indolepyruvate decarboxylase from Methylobacterium extorquens AM1.

For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k(cat) value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k(cat)/K(m)). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.

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.