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.

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.

[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.

[Advenella kashmirensis subsp. methylica PK1, a facultative methylotroph from carex rhizosphere].

A strain (PK1) of facultative methylobacteria growing on methanol as a carbon and energy source was isolated from carex rhizosphere (Pamukkale National Park, Turkey). The cells were nonmotile gram-negative rods propagating by binary fission. The organism was a strict anaerobe, oxidase- and catalase-positive. Optimal growth occurred at 29°C, pH 8.0-8.5, and 0.5% NaCl; no growth occurred at 2% NaCl. The organism used the ribulose bisphosphate pathway of C1 assimilation. Predominant fatty acids were 11-octodecenoic (18:1ω7) and cis-hexadecenoic (16:1ω7c). Phosphatidylethanolamine and diphosphatidylglycerol were the dominant phospholipids. Q8 was the main ubiquinone. DNA G+C content was 55.4 mol % (mp). Sequencing of the 16S rRNA gene revealed that strain PK1 belonged to the genus Advenella with 98.8 and 99.2% similarity to the type strains A. incenata CCUG 45225T and A. kashmirensis WT001T, respectively. DNA-DNA homology of strain PK1 and A. kashmirensis WT001T was 70%. While MALDI analysis confirmed their close clusterization, RAPD analysis revealed the differences between strain PKI and other Advenella strains. Based on its geno- and phenotypic properties, the isolate PK1 was classified as A. kashmirensis subsp. methylica PK1 (VKM-B 2850 = DSM 27514), the first known methylotroph of the genus Advenella.

[Effect of polyoxyalkanoate -based cover on properties of suture material].

The results of a surgical intervention and, in particular, reconstructive operations on arteries are largely influenced by suture material. Despite a wide range of choice, the market concerned lacks suture material which would make it possible to decrease the risk of thrombosis in the area of the vascular anastomosis. In order to increase bio- and haemocompatibility, the surface of a polypropylene thread was covered with a layer based on polyoxyalkanoates - polyhydroxybutyrate and copolymer polyhydroxybutyrate-oxyvaleriate (PHBV). The strongest and most uniformly distributed coating of the polypropylene thread was provided by PHBV. We assessed the bio- and haemocompatibility properties of suture material modified by PHBV, revealing that this modification does not exert negative effect on the main components of blood - thrombocytes and erythrocytes. Treatment of the thread's surface with PHBV promoted a significant (p<0.05) decrease in the amount of absorbed proteins in the area of the vascular anastomosis in experiment: the amount of IgM decreased by 26%, that of fibrinogen by 29%, and that of D-Dimer by 281%, being on the whole indicative of bio- and haemocompatibility of the modified suture material.

[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.

[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-hydroxybutyril-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.

[Comparative characteristics of biosynthesis of polyhydroxybutyrate from methanol by Methylobacteria extorquens G10 and Methyloligella halotolerans C2].

The biosynthesis of polyhydroxybutyrate by Methylobacteria extorquens G10 and Methyloligella halotolerans C2 via the serine pathway of C1 metabolism was comparatively studied. Nitrogen limitation stimulated synthesis of the biopolymer in both cultures. It was shown that, despite the similarity of the pathways of methanol metabolism and those of polyhydroxybutyrate biosynthesis, the methylobacteria synthesized polymers of different molecular weights. In the case of M. extorquens G10, an increase in the content of the residual nitrogen in the culture medium was found to result in a reduction of the molecular weight of the polymer from 250 to 85 kDa, whereas M. halotolerans C2 synthesized a polymer of high molecular weight (approximately 3000 kDa) regardless of the residual content of the nitrogen source. It was established that the examined methylobacteria can utilize not only pure methanol but also a crude one, a feature that made it possible to significantly reduce the cost of the resulting polyhydroxybutyrate.

[Biosynthesis of polyhydroxybutyrate/valerate with different molecular weights during the growth of Methylobacterium extorquens G-10 on a methanol-pentanol mixture].

The influence of the concentration and time of addition of cosubstrate (pentanol) on the molecular weight (MW) of the polyhydroxybutyrate/valerate (PHBV) copolymer synthesized by Methylobacterium extorquens G-10 during cultivation in a methanol-containing medium has been studied. It was shown that an increase in the pentanol concentration to 20% in a mixture with methanol stimulated the biosynthesis of PHBV with a MW of approximately 1500 kDa and increased the content of valerate up to 50%, especially when pentanol was added to the log phase culture. High pentanol concentrations are toxic for the producer and reduce the total yield of PHBV. An MW increase to 1500 kDa lowers the melting temperature (from 172 to 162 degrees C) and the crystallinity degree (from 63 to 8%) of the biopolymer but increases its elasticity. The revealed variability of PHBV properties extends considerably the potential application areas of synthetic bioplastics.

[Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection].

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6-8.8 mM) led to a change in the transistor's output signal, which was determined by the appearance of H+ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria--Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM 17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM 16--the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

[EDTA degradation by cells of Chelativorans oligotrophicus immobilized on a biofilter].

A biofilter based on light expanded clay aggregate (LECA) and cells of the obligate ethylenediamine tetraacetate (EDTA) destructor Chelativorans oligotrophicus LPM-4 has been developed. The culture steadily maintained a high level of EDTA monooxygenase activity of 180-200 nmol/min/mg of protein during three months. EDTA was converted completely or by 80% at initial concentrations of 0.5-0.7 or 2.0 g/l, respectively, in a 2-dm2 biofilter at a flow rate of 20 ml/h.

[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.

[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.

[Effect of plant-associated methanolic bacteria on methane and methanol concentration dynamics in atmosphere of pressurized chamber].

Stability of Chinese cabbage crop colonization by methanolic bacteria Methylovorus mays, Methylomonas methanica and Methylosinus trichosporium inoculated using a space-applicable method was evaluated. Besides, trends of methane and methanol concentrations in the pressurized chamber with inoculated and uninoculated crops were calculated. Methylovorus mays and Methylosinus trichosporium were shown to establish more stable colonization as compared to Methylomonas methanica. Also, stable association of methanolic bacteria with plants reduced airborne methanol 75% faster owing to its uptake by bacteria. Therefore, inoculation of these microorganisms can be viewed as a promising method of controlling volatile pollutants in space vehicle atmosphere. Methane drop after 6-hour exposure to inoculated control and test crops was not significant.