E. N. Kaparullina

Halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes

Seven strains of moderately halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes (Perm krai, Russia) were isolated in pure cultures and characterized. The isolates were represented by gram-negative and gram-positive (strain 2395B) cells. All the cells were shown to multiply by binary fission without formation of spores or prosthecae. All isolates except strain 2395B were able to oxidize methanol by a classical methanol dehydrogenase. The ribulose monophosphate (RMP) (strain LS), serine (strains S12, S3, 2395A), or ribulose bisphosphate (strains SK15 and S3270) pathways of C1-assimilation were used. In strain 2395B, the key enzymes of the RMP and serine metabolic pathways were determined. Using polyphasic taxonomy, three strains were identified as representatives of the known species: Arthrobacter protophormiae 2395B, Methylophaga thalassica LS, and Ancylobacter rudongensis S3270. Three more strains were identified as members of new species: Methylopila oligotropha sp. nov. (strain 2395AT; VKM B-2788T = CCUG 63805T), Ancylobacter defluvii sp. nov. (strain SK15T; VKM B-2789T = CCUG 63806T), and Paracoccus communis sp. nov. (strain S3T; VKM B-2787T = CCUG 63804T). According to the results of 16S rRNA gene sequencing, the obligately methylotrophic strain S12 had less than 94% similarity with the known genera of the Proteobacteria and was probably a representative of a novel genus.

Phosphate-solubilizing activity of aerobic methylobacteria

Phosphate-solubilizing activity was found in 14 strains of plant-associated aerobic methylobacteria belonging to the genera Methylophilus, Methylobacillus, Methylovorus, Methylopila, Methylobacterium, Delftia, and Ancyclobacter. The growth of methylobacteria on medium with methanol as the carbon and energy source and insoluble tricalcium phosphate as the phosphorus source was accompanied by a decrease in pH due to the accumulation of up to 7 mM formic acid as a methanol oxidation intermediate and by release of 120–280 μM phosphate ions, which can be used by both bacteria and plants. Phosphate-solubilizing activity is a newly revealed role of methylobacteria in phytosymbiosis.

Methylovorus menthalis, a novel species of aerobic obligate methylobacteria associated with plants

A bacterial strain (MM) utilizing methanol as the only carbon and energy source was isolated from corn mint rhizoplane. The cells of the strain were gram-negative colorless motile rods. Spores and prosthecae were not formed, reproduced by binary fission, and did not require vitamins and growth factors. The organism was strictly aerobic, urease-, oxidase-, and catalase-positive. Used the KDPG variant of the ribulose monophosphate pathway. Possessed NAD+ dependent 6-phosphogluconate dehydrogenase activity and enzymes of the glutamate cycle. The activities of α-ketoglutarate dehydrogenase and of the glyoxylate bypass enzymes (isocitrate lyase and malate synthase) were absent. Palmitic (C16:0) and palmitoleic (C16:1) acids were predominant in the cell fatty-acid composition. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. The dominant ubiquinone was Q8. The strain formed indole from tryptophan. The DNA G + C content was 54.5 mol % (Tm). According to the data of the 16S rRNA gene sequencing, strain MM showed high similarity (98–99%) to Methylovorus glucosotrophus VKM B-1745T and Methylovorus mays VKM B-2221T, but the level of DNA-DNA homology with these cultures was only 40 and 58%, respectively. The strain was classified as a new species, Methylovorus menthalis sp. nov. (VKM B-2663T).

Aerobic degradation of ethylenediaminetetraacetate (review)

Literature data on the influence of complexing compound ethylenediaminetetraacetate (EDTA) on environmental and ecological risks related with its application were analyzed and summarized. Methods of abiotic and biotic degradation of EDTA were systemized. Special attention was paid to microbiological degradation of EDTA was paid. Data on EDTA transport and metabolism pathways in aerobic bacteria are represented. The practical aspects of application of aerobic bacteria-destructors of EDTA in ecobiotechnology were discussed.

Phylogenetic Position and Phosphate Solubilization Activity of Lactic Acid Bacteria Associated with Different Plants

Lactic acid bacteria (LAB) are grampositive, non� sporeforming (except for some members of the genus Sporolactobacillus), catalasepositive bacteria, devoid of cytochromes, aeroand acidtolerant, which form lactic acid as a final metabolite in the lactic acid fer� mentation of sugars. LABs have been found in the nutrientrich foods (dairy products, meat, and vegetables) and inhabit the digestive system of humans and animals; some LAB species were found on plants (1). We hypothesize that LABs are imported into human and animal intestines with plants, to which they are probably associated in a form of phytosymbiosis. In view of the above, the goal of the present work was to isolate pure LAB cultures from different plant species in winter and in summer and to determine their phosphate solubilization activity and phyloge� netic position. Enrichment LAB cultures were obtained as fol� lows: the plant material (leaves and buds) was washed twice with sterile distilled water, ground, and placed into hermetically sealed screwcapped test tubes, fill� ing them to about 80% of their volume. The residual volume was filled with sterile water; the test tubes were hermetically closed and incubated at 30°С in a ther� mostat for 24 h. The tubes were manually shaken two times during the incubation. After the incubation, the samples from the liquid phase were plated on an agar� ized nutrient medium 2T5 and incubated in a thermo� stat at 30°С for 20-40 h. The 2T5 medium developed in our laboratory con� tained (per 1 L of Н2О) yeast extract, 5 g; tryptone, 1.5 g; soy peptone, 0.7 g; aminopeptide from animal blood plasma, 20 mL; biomass autolysate (12 g/L by dry substance weight) of the fungus Cephaliophora trop� ica D3, 50 mL; pumpkin juice, 60 mL; tomato juice, 20 mL; pH 6.0.

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].

[Degradation of the EDTA and EDTA complexes with metals by immobilized cells of Chelativorans oligotrophicus LPM-4 bacteria].

Enzymatic oxidative degradation of EDTA and EDTA complexes with metals has been investigated using immobilized cells of Chelativorans oligotrophicus LPM-4. A polarographic method, which makes it possible to register oxygen consumption by cells, has been used. For the first time, it has been indicated that the Cd-EDTA and Ni-EDTA complexes undergo degradation by the bacteria under study.

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow region

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. 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.

Erratum: “Methylopila turkiensis sp. nov., a new aerobic facultatively methylotrophic phytosymbiont”

A new facultative methylotroph, strain Side1T, was isolated from the phyllosphere of Bougainvillea sp. L. The isolate is represented by rod-shaped, aerobic gram-negative asporogenous bacteria which divide by binary fission. Methanol and mono- and trimethylamine were utilized, as well as a limited spectrum of polycarbon substrates, while methane and dichloromethane were not used. Growth occurred at pH 6.0–9.0 with the optimum at pH 7.0 within the temperature range from 20 to 40°C (optimum at 28–30°C) and 0–2.5% NaCl in the medium. The predominant fatty acids were cis-11-octadecenoic (C18:1ω7c), 11-methyl-octadecenoic (C18:ω7c11Me), and stearic (C18:0) acids. Phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, and diphosphatidylglycerol were the dominant phospholipids. Q10 was the dominant ubiquinone. The isolate oxidized methanol and methylamine by the appropriate dehydrogenases. The isocitrate lyase-negative variant of the serine pathway was used. Ammonium assimilation involved glutamate dehydrogenase and the glutamate cycle (glutamate synthase and glutamine synthetase). The strain synthesized indole and siderophores; it solubilized insoluble phosphates. The DNA G+C content (Tm) was 65.4 mol %. While the nucleotide sequence of the 16S rRNA gene of strain Side1 exhibited high similarity to those of Methylopila species (M. musalis MUSAT and M. capsulata IM1T), DNA-DNA homology with these cultures was 32–37%. The results obtained supported classification of strain Side1T as a new species Methylopila turkiensis sp. nov. (VKM B-2748T= DSM 27566T).