E. I. Katsy

The effect of mutations affecting synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by Azospirillum brasilense

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.

[Analysis of DNA, lipopolysaccharide structure, and some cultural and morphological properties in closely related strains of Azospirillum brasilense].

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85 MDa (p85) and 120 MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-MDa (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-MDa plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-MDa replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to the cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.

Wheat root colonization by Azospirillum brasilense strains with different motility

Migration of associative bacteria Azospirillum brasilense in semisolid media is performed mainly by swarming (Swa+ phenotype), which depends on the flagellar functioning and intercellular contacts. Non-swarming mutants of A. brasilense Sp245 lacking a polar flagellum migrate in semisolid media with microcolony formation using a unrevealed mechanism (Gri+ phenotype). The study of wheat root colonization dynamics demonstrated that A. brasilense Sp245 Gri+ mutants exhibited lower capacity for wheat root adsorption. However, after “anchoring” has occurred, both A. brasilense Sp245 and its Swa-Gri+ mutants colonized the growing roots with virtually the same efficiency. All strains under study formed microcolonies on the surface of roots, stimulated root branching, and exhibited changes in the composition of protein antigens exposed on the bacterial cell surface. Indirect evidence was obtained for enhanced production of genus-specific protein antigens in the process of A. brasilense Sp245 adaptation to growth on plant roots.

Mobile elements of an Azospirillum brasilense Sp245 85-MDa plasmid involved in replicon fusions.

Sequence analysis of approximately 25kb of an Azospirillum brasilense Sp245 85-MDa ( approximately 142kb) plasmid, p85, identified two novel IS elements mediating p85 fusions with a suicide plasmid vector, pJFF350. These IS elements, 1465-bp ISAzba1 and 1112-bp ISAzba3, belong to the IS256 family and to the IS5 family/IS903 group, respectively. Truncated ISAzba2 from the ISL3 family was found near one of the copies of ISAzba1 that flank pJFF350 in p85::pJFF350. As another factor potentially contributing to the known genetic plasticity of p85, a phage integrase gene was identified in this plasmid.

Hemagglutinating activity and motility of the bacterium Azospirillum brasilense in the presence of various nitrogen sources

Hemagglutinating activity of the Azospirillum brasilense strain Sp245 grown in liquid media and the swarming motility of those bacteria grown in semisolid media vary significantly depending on the nitrogen source. In media with nitrate or nitrite, an increase in the hemagglutinating activity and a decrease in the swarming circles’ diameter of Sp245 were observed, compared to bacteria grown in the presence of ammonium or N2. A ∼67-kDa hemagglutinin exhibiting affinity to the O-specific polysaccharide, an acidic D-rhamnan (OPS-I), was isolated from the surface of Sp245 cells. Introduction of the hemagglutinin into the media resulted in a decrease in the Sp245 cell motility while not affecting its mutants lacking the acidic D-rhamnan or the Sp245.5 mutant with a different OPS structure. Cells of strain Sp245.5 demonstrated hemagglutinating activity two times higher than that of the parent Sp245 strain and formed “diffuse” colonies, rather than distinct swarming circles Sp245 formed when grown in a semisolid medium. The data obtained demonstrate that intercellular contacts mediated by the interaction between the surface hemagglutinin and OPS-I, which is sensitive to environmental factors, affect the collective motility of cells.

Detection of a sheath on Azospirillum brasilense polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.

Determination of the Structure of the Repeated Unit of the Azospirillum brasilense SR75 O-Specific Polysaccharide and Homology of the lps Loci in the Plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the previously studied OPSs of A. brasilense strain Sp245, isolated from surfacesterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains were localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects.

Effect of Congo red on the motility of the bacterium Azospirillum brasilense

In semiliquid laboratory media, the bacterium Azospirillum brasilense migrates with the formation of swarming rings. It is demonstrated that adsorption of the sulfonated azodye Congo Red confers on A. brasilense the ability to consistently spread in a semiliquid agar with formation of microcolonies. Spontaneous variants of A. brasilense with increased swarming rate are described, as well as variants that swarm in the presence of Congo Red. It is assumed that at least two types of compounds are formed, which are necessary for swarming and/or spreading with the formation of microcolonies and are capable of interacting with Congo Red.

Plasmid Rearrangements in Azospirillum brasilense

1 Bacteria of the genus Azospirillum live in various econiches in association with plants. Azospirillum cells contain many plasmids. The 90-MDa plasmid (pRhico) of type strain A. brasilense Sp7 and the 120and 85-MDa plasmids (p120 and p85, respectively) of A. brasilense Sp245 have been found to be involved in the regulation of motility (Mot phenotype) and swarming (Swa phenotype), as well as in the formation of polar (Fla phenotype) and lateral flagella (Laf phenotype) and the synthesis of lipopolysaccharides (LPS phenotype) and calcofluor-binding exopolysaccharides (Cal phenotype) [1, 2]. The Rhico plasmid has regions that are homologous to the lps/cal and fla/swa loci of p120, whereas the 115-MDa plasmid (p115) of strain Sp7 has regions that are homologous to the fla/laf and mot/swa loci of p85 [1]. According to their electrophoretic profiles, p85 and p115 have a smaller copy number than p120 and pRhico [1].

The structure of the O-specific polysaccharide from a mutant of nitrogen-fixing rhizobacterium Azospirillum brasilense Sp245 with an altered plasmid content

AbstractThe rhizobacteria Azospirillum brasilense Sp245 produce immunochemically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeating units of O-specific polysaccharides (OPS). In this study, we report the structure of the OPS from A. brasilense LPSI−LPSII− mutant Sp245.5, which spontaneously lost the p85 and p120 plasmids upon the formation of a new 300-MDa megaplasmid after the long-term storage of the bacteria in a rich medium. The repeating unit of the OPS of A. brasilense Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid: