D. N. Fedorov

Phytosymbiosis of aerobic methylobacteria: New facts and views

This review highlightsrecent findings on the phytosymbiosis of aerobic methylobacteria, including their biodiversity, occurrence, and their role in associations with plants, as well as the capacity for biosynthesis of bioactive compounds (auxins, cytokinins, and vitamin Bl2) and nitrogen fixation. Future research directions in phytosymbiosis of aerobic methylobacteria during the postgenomics era are discussed.

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 kcat value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (kcat/Km). 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.

A new system of degenerate oligonucleotide primers for detection and amplification of nifHD genes

247 Biological nitrogen fixation is a process of dinitrogen reduction to assimilable ammonium [1, 2]. Many prokaryotes fix nitrogen by means of nitrogenase, a highly conservative enzyme complex consisting of dinitrogenase (MoFe-protein) and dinitrogenase reductase (Fe-protein) encoded by the nifDK and nifH genes, respectively [1]. The most convenient method of assessing the nitrogen-fixing ability in different prokaryotes is amplification of nitrogenase structural genes ( nif genes) using degenerate oligonucleotide primers in polymerase chain reaction (PCR). Nitrogen fixation can be assessed by other methods, such as acetylene reduction, hybridization of genome DNA with labeled probes, or methods using 15 N. However, these methods require expensive equipment and reagents, and their results are not always reliable.

Aerobic methylobacteria as promising objects of modern biotechnology

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

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 σ70-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.

Analysis of the 3′-region of the dcmA gene of dichloromethane dehalogenase of Methylobacterium dichloromethanicum DM4

Two hypothetical genes of a dichloromethane (DCM) degrader Methylobacterium dichloromethanicum DM4, METDI2657 and METDI2658, located in the 3′-region of the dcmA gene of DCM dehalogenase, have been studied. The method of reverse transcription polymerase chain reaction (RT-PCR) was used to show that the cells of M. dichloromethanicum DM4 grown on both DCM and methanol contained the transcripts of all three of the above genes. RT-PCR amplification of the intergenic regions showed that the genes dcmA, METDI2657, and METDI2658 formed an operon. Orthologs of the METDI2657 and METDI2658 genes were also found in the DCM destructors Methylopila helvetica DM9, Methylorhabdus multivorans DM13 and DM15, Ancylobacter dichloromethanicus DM16, and Methylobacterium extorquens DM17. A mobilized suicide vector pK18mob was used to obtain a knock-out mutant of M. dichloromethanicum DM4 (NOK353) with the “turned-off” METDI2657 gene, the nucleotide sequence of which was interrupted by insertion of a gentamycin cassette. After cultivation on methanol, the NOK353 mutant had a lower rate of growth on DCM than the wild type strain DM4.

Methanol metabolism of the rhizosphere phytosymbiont Methylobacterium nodulans

854 Methanol, a natural plant metabolite, is the carbon and energy source for aerobic methylotrophic bacteria (methylobacteria). It was demonstrated that the plant phyllosphere is inhabited by methylobacteria of differ ent taxonomic groups, phytosymbiotic organisms which supply various bioactive compounds to the plant [1, 2]. The methylobacteria living in plant rhizo sphere remain poorly understood. The nitrogen fixing and root nodule forming facultative methylotroph Methylobacterium nodulans was isolated from root nodules of the African legume Crotalaria podocarpa [3, 4]. The genome of this methylotrophic bacterium was sequenced (Refseq/GenBank NC_011894/ CP001349); however, the pathways of C1 metabolism were not investigated. The goal of the present work was to carry out a comparative genomic and enzymatic analysis of the pathways of methanol metabolism in M. nodulans. Strain M. nodulans ORS2060T (CNCM I 2342T=LMG 21967T) was grown at 29°C on a shaker (180 rpm) in flasks with the K medium (pH 7.2) con taining the following: (g/l): KH2PO4, 2; (NH4)2SO4, 2; NaCl, 0.5; MgSO4 · 7H2O, 0.025; and FeSO4 · 7H2O, 0.002. Methanol (0.5% vol/vol) was added as a carbon and energy source. Exponentially grown cells were sonicated using an MSE sonicator (United King dom) at 150 W and 20 kHz (4 × 30 s, 4°C) and centri fuged (10000 g, 45 min); the supernatant was used for enzymatic analysis [5]. The protein content was deter mined by the Lowry method. The table shows that M. nodulans has all the enzymes involved in the process of methanol oxida tion to CO2, i.e. methanol, formaldehyde, and for mate dehydrogenase. Methanol is oxidized by the “classical” methanol dehydrogenase (MDH) (Mnod_8040 localized in the plasmid pMnod02), which exhibits its maximum activity at pH 9.0 and is stimulated by ammonium ions. It was recently estab lished that, along with this MDH, the pink pigmented phyllosphere methylotroph Methylobacterium extorquens AM1 has an MDH paralog, XoxF (MexAM1_META1p2757) with the amino acid sequence showing a 50% similarity with the sequence of the MxaF subunit of the periplasmic MDH (MexAM1_META1p4538) [6]. In addition to MDH, an amino acid sequence showing 88% similarity with that of the XoxF of M. extorquens AM1 was detected in the M. nodulans genome. It is likely that, in the case of M. nodulans, both the “classical” MDH and its para log (XoxF) are involved in methanol oxidation. The activity of formaldehyde dehydrogenase with phena zine methosulfate (PMS) is higher than that of the NAD+ dependent GSH stimulated form of this enzyme. Formate dehydrogenases exhibiting activity both with PMS and NAD were detected in the cell extracts. Moreover, the M. nodulans genome contains the genes which encode the enzymes of the tetrahy drofolate and tetrahydro methanopterin pathways of formaldehyde oxidation (Mnod_6007, 7114, 6004, 5997–6000, 6002, 7115, and 4459), indicating that the pathways of C1 assimilation of M. nodulans are similar to those of M. extorquens AM1 [7].

Genetic modification of Methylobacterium extorquens G10 producer strain of polyhydroxybutyrate

The effect of the increased copy number of polyhydroxybutyrate (PHB) biosynthesis genes in pink-pigmented methylobacterium Methylobacterium extorquens G10 on properties of the biopolymer was studied. The activity of poly-3-hydroxybutyrate-synthase (PHB-synthase) was shown to increase and the molecular weight of synthesized PHB decreases twofold (150 → 79 kDa) after insertion of extra copies of phaC and phaCAB genes into cells of the producer strain, whereas the physicochemical properties of the plastic changed insignificantly. White mutant M. extorquens G10-W with disrupted synthesis of the carotenoid pigment (defect by the crtI gene, which codes for phytoene desaturase) was established to have the same rate of growth and level of PHB accumulation as the initial strain G10. The G10-W strain is a promising producer of PHB, with decreased expenses for purification and PHB biosynthesis.

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.

1-aminocyclopropane-1-carboxylate deaminase of the aerobic facultative methylotrophic actinomycete Amycolatopsis methanolica 239

584 One of the key mechanisms of the effect of bacteria on plant growth and development is their ability to reduce the level of ethylene due to the activity of 1 aminocyclopropane 1 carboxylate deaminase (ACCD) (EC 3.5.99.7) [1]. This enzyme catalyzes the hydrolysis of 1 aminocyclopropane 1 carboxylate (ACC), which is an immediate precursor in ethylene biosynthesis, to α ketobutyrate and ammonium ions. Ethylene is one of the main phytohormones; it regu lates the aging process, induces fruit ripening and flower withering, and plays a key role in stress signal transduction [2]. Increased ethylene concentration in plant roots as a part of stress response inhibits root elongation, nodulation and transport of auxins, and accelerates tissue aging and exfoliation [3, 4]. It has been shown that ACCD possessing bacteria contrib ute to the enhancement of plant resistance to such negative impacts as drought, soil salinity, heavy metal pollution, and the presence of phytopathogens [1].