Phylogenetic and pangenomic analyses of members of the family Micrococcaceae related to a plant-growth-promoting rhizobacterium isolated from the rhizosphere of potato (Solanum tuberosum L.).

We report the results of taxonomic studies on members of the family Micrococcaceae that, according to the 16S rRNA, internal transcribed spacer 1 (ITS1), average nucleotide identity (ANI), and average amino acid identity (AAI) tests, are related to Kocuria rosea strain RCAM04488, a plant-growth-promoting rhizobacterium (PGPR) isolated from the rhizosphere of potato (Solanum tuberosum L.). In these studies, we used whole-genome phylogenetic tests and pangenomic analysis. According to the ANI > 95 % criterion, several known members of K. salina, K. polaris, and K. rosea (including K. rosea type strain ATCC 186T) that are related most closely to isolate RCAM04488 in the ITS1 test should be assigned to the same species with appropriate strain verification. However, these strains were isolated from strongly contrasting ecological and geographical habitats, which could not but affect their genotypes and phenotypes and which should be taken into account in evaluation of their systematic position. This contradiction was resolved by a pangenomic analysis, which showed that the strains differed strongly in the number of accessory and strain-specific genes determining their individuality and possibly their potential for adaptation to different ecological niches. Similar results were obtained in a full-scale AAI test against the UniProt database (about 250 million records), by using the AAI-profiler program and the proteome of K. rosea strain ATCC 186T as a query. According to the AAI > 65 % criterion, members of the genus Arthrobacter and several other genera belonging to the class Actinomycetes, with a very wide geographical and ecological range of sources of isolation, should be placed into the same genus as Kocuria. Within the paradigm with vertically inherited phylogenetic markers, this could be regarded as a signal for their following taxonomic reclassification. An important factor in this case may be the detailing of the gene composition of the strains and the taxonomic ratios resulting from analysis of the pangenomes of the corresponding clades.

Improving the efficacy of potato clonal micropropagation by inoculation with the rhizosphere bacteria Azospirillum baldaniorum Sp245 and Ochrobactrum cytisi IPA7.2.

Sustainable development of agriculture depends on the provision of quality seeds to the market. Inoculation with plant-growth-promoting rhizobacteria in in vitro culture can be used to improve the growth eff icacy and performance of microplants. We examined the effect of in vitro inoculation of microplants of the cultivars Nevsky and Kondor with the strains Azospirillum baldaniorum Sp245 and Ochrobactrum cytisi IPA7.2 separately and in combination. We examined the morphological variables of plant growth in in vitro culture and under ex vitro adaptation conditions; we also investigated the growth and performance of the plants in the greenhouse. The dependence of the inoculation eff icacy on potato genotype, growth stage, and inoculum composition was ascertained throughout the experiment. In vitro, A. baldaniorum Sp245 alone and in combination with O. cytisi IPA7.2 promoted the formation of roots on the microplants of both cultivars and the growth of Nevsky shoots. During plant growth ex vitro, all growth variables of the Nevsky microplants were promoted by O. cytisi IPA7.2 alone and in combination with A. baldaniorum Sp245. In both cultivars grown in the greenhouse, shoot growth was promoted in most inoculation treatments. The survival ability of the Nevsky microplants in the greenhouse increased 1.7-fold under the effect of simultaneous inoculation. Inoculation of microplants with a combination of A. baldaniorum Sp245 and O. cytisi IPA7.2 increased the number of Nevsky minitubers 1.5-fold and the number of Kondor minitubers 3.5-fold. Inoculation with the tested strains can be used to promote the growth of microplants and increase the yield of minitubers in potato seed breeding for the production of healthy planting material.

Analysis of the microbial cell-Ab binding in buffer solution by the piezoelectric resonator.

The possibility of the registration of the interaction of the cells Azospirillum lipoferum Sp59b with the specific antibodies directly in the conducting suspensions by using an acoustic sensor was shown. The main element of the sensor is a piezoelectric resonator with a lateral electric field. The analysis is based on a comparison of the resonator's electrical impedance before and after the specific biological interaction between the cells and antibodies. By using this sensor one can detect and identify the bacterial cells directly in the buffer solution with the conductivity between 2.4 and 20 µS/cm. The minimum detectable concentration of the bacterial cells turned out to be ∼103 cells/ml and for a short time (less than 10 min). Also the possibility of the detection of the cells in the presence of the extraneous microflora was shown. The results provide the opportunities for the development of a new class of the methods for the analysis of the microbial cells in real-time directly in the buffer solution.

[Immunochemical Detection of Azospirilla in Soil with Genus-Specific Antibodies].

Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

Capsular polysaccharide of the bacterium Azospirillum lipoferum Sp59b: structure and antigenic specificity.

Antigenic differences were revealed between the cell wall outer membrane lipopolysaccharides and the capsular high molecular weight bioglycans for a typical strain of the nitrogen-fixing rhizobacterium Azospirillum lipoferum Sp59b using antibodies prepared against the homologous lipopolysaccharide and lipopolysaccharide-protein complex. From the capsular lipopolysaccharide-protein and polysaccharide-lipid complexes of A. lipoferum Sp59b, polysaccharides were isolated and their structure was for the first time established in Azospirillum by monosaccharide analysis which included determination of the absolute configurations, methylation, O-deacetylation, and one- and two-dimensional NMR spectroscopy. The polysaccharides of the capsular complexes were shown to have identical structure of the branched tetrasaccharide repeating unit, which differs from the structure of the O-specific polysaccharide within the outer membrane lipopolysaccharide of this strain.

[Electrooptical properties of the microbial suspensions during a cell's interaction with the antibodies of a different specificity].

The electrooptical abilities of the microbial suspensions during a cells interaction with antibodies (ABs) of a different specificity have been studied on the example of the Azospirillum brasilense Sp245 cells and their interaction with the polyclonal monospecific and polyspecific antibodies. Measuring of the orientational spectra of the cells has been performed using the ELUS electrooptical analyzer. A discrete frequency set of an orienting electric field (740, 1000, 1450, 2000, and 2800 kHz) was used. It has been shown that an interaction of the polyspecific AB with the investigated cells redoubles the value of an electrooptical signal of the cells' suspension as compared with the monospecific antibodies. These findings can be used for a development a new method of microorganism detection.

On the Enhanced Antibacterial Activity of Antibiotics Mixed with Gold Nanoparticles.

The bacterial action of gentamicin and that of a mixture of gentamicin and 15-nm colloidal-gold particles on Escherichia coli K12 was examined by the agar-well-diffusion method, enumeration of colony-forming units, and turbidimetry. Addition of gentamicin to colloidal gold changed the gold color and extinction spectrum. Within the experimental errors, there were no significant differences in antibacterial activity between pure gentamicin and its mixture with gold nanoparticles (NPs). Atomic absorption spectroscopy showed that upon application of the gentamicin-particle mixture, there were no gold NPs in the zone of bacterial-growth suppression in agar. Yet, free NPs diffused into the agar. These facts are in conflict with the earlier findings indicating an enhancement of the bacterial activity of similar gentamicin-gold nanoparticle mixtures. The possible causes for these discrepancies are discussed, and the suggestion is made that a necessary condition for enhancement of antibacterial activity is the preparation of stable conjugates of NPs coated with the antibiotic molecules.

[Composition and immunochemical characteristics of exopolysaccharides from the rhizobacterium Paenibacillus polymyxa 1465].

Exopolysaccharides (EPS) synthesized by Paenibacillus polymyxa 1465 in the course of batch cultivation were proven to contain neutral and acidic fractions. EPS are heterogeneous polysaccharides, represented by a complex of macromolecules with molecular mass of 7 x 10(4) to 2 x 10(6) Da. The acidic component was shown to be predominant in EPS preparations isolated from bacteria cultivated on glucose, which corresponds to a higher viscosity of EPS water solutions. The exoglycans were shown to contain glucose, mannose, galactose, and uronic acids. Polyclonal rabbit antibodies against the isolated P. polymyxa 1465 EPS preparations were used in a comparative immunodiffusion analysis of a number of P. polymyxa strains.

[Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum].

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab(Sp59b) and Ab(Cd) were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.