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Azospirillum brasilense forms biofilms on various surfaces. It was previously shown that inactivation of the ahpC gene of alkyl hydroperoxide reductase in the SK586 mutant of A. brasilense Sp245 led to an increased sensitivity to peroxides and to changes in flagellation, motility, and cell surface properties. This work revealed that under nitrogen limitation, Sp245 switches predominantly to the biofilm mode of life, whereas this tendency is not characteristic of the mutant SK586. Inactivation of the ahpC gene negatively affects the viability of SK586 in biofilms, which is most noticeable under nitrogen limitation. While no significant morphological differences were found between cyst-like forms of Sp245 and SK586, the dormant mutant forms grown in a nitrogen-free medium were more sensitive to hydrogen peroxide than the dormant forms of Sp245. In Sp245 biofilms, viable forms resistant to drying were found even after 9 months of storage, while in SK586 biofilms, such bacteria remained viable for less than a month. In the course of adaptation to the plant root system, the number of Azospirillum cells resistant to drying increased, as compared to the number of such cells from mature biofilms on abiotic surfaces. Unlike Sp245, SK586 did not promote the development of the root system and the growth of the aerial parts of 10-day-old plants, possibly owing to its lower ability to colonize the roots of wheat seedlings incubated in a liquid medium. Alkyl hydroperoxide reductase is probably essential for better resistance of azospirilla to various stresses and for manifestation of their plant-growth-promoting activity.

The bacterium Azospirillum brasilense can swim and swarm owing to the work of polar and lateral flagella. Its major surface glycopolymers consist of lipopolysaccharides (LPS) and Calcofluor-binding polysaccharides (Cal+ phenotype). Motility and surface glycopolymers are important for the interactions of plant-associated bacteria with plants. The facultative plant endophyte A. brasilense Sp245 produces two antigenically different LPS, LpsI, and LpsII, containing identical O-polysaccharides. Previously, using vector pJFF350 for random Omegon-Km mutagenesis, we constructed a mutant of Sp245 named KM018 that still possessed flagella, although paralyzed. The mutant was no longer able to produce Calcofluor-binding polysaccharides and LpsII. Because of the limited experimental data on the genetic aspects of surface glycopolymer production and flagellar motility in azospirilla, the aim of this study was to identify and examine in more detail the coding sequence of strain Sp245, inactivated in the mutant. We found that pJFF350 was integrated into a coding sequence for a putative integral membrane protein of unknown function (AZOBR_p60025) located in the sixth plasmid of Sp245. To clarify the role of the putative protein, we cloned AZOBR_p60025 in the expression vector pRK415 and used it for the genetic complementation of mutant KM018. The SDS–PAGE, immunodiffusion, and linear immunoelectrophoresis analyses showed that in strain KM018 (pRK415–p60025), the wild-type LpsI+ LpsII+ profile was restored. The complemented mutant had a Cal+ phenotype and it was capable of swimming and swarming motility. Thus, the AZOBR_p60025-encoded protein significantly affects the composition of the major cell-surface glycopolymers and the single-cell and social motility of azospirilla.

Due to the primary localization of both epiphytic and endophytic plant growth-promoting rhizobacteria on the surface of the plant root system, biofilm formation is an adaptive trait for these microorganisms. Under conditions of nitrogen limitation in liquid media, nitrogen-fixing Azospirillum brasilense strains switch mainly to the biofilm mode of growth. Overall ultrastructural similarities of the cells within A. brasilense biofilms were revealed, and their resistance to desiccation and oxidative stress was characterized. In strains Sp7, Cd, and Sp245, several types of single and undivided cells were revealed, as well as cystlike cells with pronounced morphological diversity. Resistance to desiccation and to oxidative stress was higher in the biofilm populations of these strains than in planktonic cultures. Dormant forms remained viable in dry biofilms of strains Sp245, Sp7, and Cd formed in a nitrogen-free medium after storage for 120 days. Viability of the biofilms of the same strains formed in the presence of nitrogen was retained for 120, 90, and 60 days, respectively. The minimal inhibitory concentration of H2O2 for biofilms was 1.0% for strains Sp7 and Cd and 0.1% for strain Sp245. Both the dormant forms and biofilms of strain Sp245 were more sensitive to H2O2 than those of strains Sp7 and Cd. Peroxidase activity was not previously reported in Azospirillum biofilms.

The bacterium Azospirillum brasilense can swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella, respectively. They also form biofilms on various interfaces. Experimental data on flagellar assembly and social behaviours in these bacteria are scarce. Here, for the first time, the chromosomal coding sequence mmsB1 for a homologue of 3-hydroxyisobutyrate dehydrogenase (protein accession Nos. ADT80774 and E7CWE2) was shown to play a role in the assembly of motile Fla and in biofilm biomass accumulation. In the previously obtained mutant SK039 of A. brasilense Sp245, an Omegon-Km insertion in mmsB1 was concurrent with changes in cell-surface properties and with suppression of Fla assembly (partial) and Fla-dependent motility (complete). Here, the immotile leaky Fla− mutant SK039 was complemented with the expression vector pRK415-borne mmsB1 gene of Sp245. In the complemented mutant, the elevated relative cell hydrophobicity and changed relative membrane fluidity of SK039 returned to the wild-type levels; also, biofilm biomass accumulation increased and even reached Sp245’s levels under nutritionally rich conditions. In strain SK039 (pRK415-mmsB1), the percentage of cells with Fla became significantly higher than that in mutant SK039, and the Fla-driven swimming velocity was equal to that in strain Sp245.

The polar flagellum significantly affects the morphological and behavioral responses of the bacterium Azospirillum brasilense Sp245 to changes in the density of the medium and the maintenance of its biofilms at the interface between solid and liquid media.

The facultative plant endophyte Azospirillum brasilense Sp245 synthesizes two high-molecular-weight lipopolysaccharides, LPSI and LPSII, which comprise identical d-rhamnan O-polysaccharides and, presumably different core oligosaccharides. Previously, using random insertion mutagenesis, we constructed the LpsII- mutant KM139 of strain Sp245 that possessed an Omegon-Km insertion in plasmid AZOBR_p6. Here, we found that in KM139, Omegon-Km disrupted the coding sequence AZOBR_p60126 for a putative glycosyltransferase related to mannosyltransferases and rhamnosyltransferases. To verify its function, we cloned the AZOBR_p60126 gene of strain Sp245 in the expression vector plasmid pRK415 and transferred the construct pRK415-p60126 into KM139. In the complemented mutant KM139 (pRK415-p60126), the wild-type LPSI+ LPSII+ profile was recovered. We also compared the swimming and swarming motilities of strains Sp245, Sp245 (pRK415), KM139, KM139 (pRK415), and KM139 (pRK415-p60126). All these strains had the same flagellar-dependent swimming speeds, but on soft media, the LpsI+ LpsII- strains KM139 and KM139 (pRK415) swarmed significantly faster than the other LpsI+ LpsII+ strains. Such interstrain differences in swarming motility were more pronounced on 0.4% than on 0.5% soft agar plates. These data show that the AZOBR_p60126-encoded putative glycosyltransferase significantly affects the lipopolysaccharide profile and, as a consequence, the social motility of azospirilla.

Herein, we reveal the alteration in phenol oxidase enzymes complex production from Azospirillum brasilense Sp245 omegon mutants with polar and lateral flagella dysfunction and from A. brasilense Sp7 phase variants with different plasmid composition. The enzymatic activities for various laccases, tyrosinases, Mnperoxidases, and lignin peroxidases as well as the isomorphic composition of intracellular laccases and tyrosinases were estimated for the studied variants and the parent strains. It was noted that various genetic events correlating with phenotypic heterogeneity in A. brasilense populations affect their phenol oxidase activity level.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla− Laf− fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

Bacteria Azospirillum brasilense may swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf). They also construct sessile biofilms on various interfaces. As compared to the wild-type strain Sp245, the previously characterized Fla− Laf− flhB1 mutant Sp245.1063 accumulated less biomass in mature biofilms, which also were susceptible to the forces of hydrodynamic shear. In this study, we compared biofilms formed by strain Sp245 and its previously constructed derivatives on the interfaces between a minimal (malate–salt medium, or MSM) or rich (LB) liquid growth medium and a hydrophilic (glass) or hydrophobic (polystyrene) solid surface under static or dynamic conditions. In all experimental settings, the alterations in Sp245.1063’s mature biofilm traits were partially (in MSM) or completely (in LB) rescued in the complemented mutant Sp245.1063 (pRK415–150177), which received the pRK415-borne coding sequence for the putative FlhB1 protein of the flagellar type III secretion system. Although Laf were not found in the biofilms of azospirilla, Fla was present on the biofilm cells of the complemented mutant Sp245.1063 (pRK415–150177) and other studied strains, which had normal flagellation on liquid and solid nutritional media. Accordingly, mature biofilms of these strains contained more biomass and were significantly more resistant to shaking at 140 rpm, as compared to the biofilms of the flagella-free mutant bacteria. These data proved that the polar flagellum of A. brasilense Sp245 plays a significant positive role in biofilm biomass increase and in biofilm stabilization.

The previously obtained insertion mutants ofAzospirillum brasilense Sp245 in the genes mmsBl and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense- Sp245 strains SK039 and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide prepa- rations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.

Alphaproteobacteria of the species Azospirillum brasilense have a multicomponent genome that undergoes frequent spontaneous rearrangements, yielding changes in the plasmid profiles of strains. Specifically, variants (Cd, Sp7.K2, Sp7.1, Sp7.4, Sp7.8, etc.) of the type strainA. brasilense Sp7 that had lost a 115-MDa plasmid were previously selected. In many of them, the molecular weight of a 90-MDa plasmid (p90 or pRhico), which is a kind of "depot" for glycopolymer biosynthesis genes, increased. In this study, a collection of primers was designed to the plasmid pRhico and to the DNA of prophage phiAb-Cd integrated in it. The use ofthese primers in polymerase chain reactions allowed the detection of the probable excision of phiAb-Cd phage from the DNA of A. brasilense variants Sp7.4 and Sp7.8 and other alterations of the pRhico structure in A. brasilense strains Cd, Sp7.K2, and Sp7.8. The developed primers and PCR conditions may be recoin mended for primary analysis of spontaneous plasmid rearrangements in A. brasilense Sp7 and related strains.

In the bacterium Azospirillum brasilense Sp245, extracellular calcofluor-binding polysaccharides (Cal+ phenotype) and two types of lipopolysaccharides, LPSI and LPSII, were previously identified. These lipopolysaccharides share the same repeating O-polysaccharide unit but have different antigenic structures and different charges of their O-polysaccharides and/or core oligosaccharides. Several dozens of predicted genes involved in the biosynthesis of polysaccharides have been localized in the AZOBR_p6 plasmid of strain Sp245 (GenBank accession no. HE577333). In the present work, it was demonstrated that an artificial transposon Omegon-Km had inserted into the central region of the AZOBR_p60120 gene in the A. brasilense Sp245 LPSI− Cal− KM252 mutant. In A. brasilense strain Sp245, this plasmid gene encodes a putative glycosyltransferase containing conserved domains characteristic of the enzymes participating in the synthesis of O-polysaccharides and capsular polysaccharides (accession no. YP004987664). In mutant KM252, a respective predicted protein is expected to be completely inactivated. As a result of the analysis of the EcoRI fragment of the AZOBR_p6 plasmid, encompassing the AZOBR_p60120 gene and a number of other loci, novel data on the structure of AZOBR_p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.

Bacteria Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327–HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1:: Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase–response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.

Motility of the serologically different Azospirillum brasilense strains Sp245 (serogroup I) and Sp7 (serogroup II) was studied in the presence of antibodies to their lipopolysaccharides (LPS). A procedure was proposed in order to determine the motility patterns indicating the specificity of the interaction between the anti-LPS antibodies and bacteria. Analysis of the effect of such antibodies on motility of 25 strains (A. brasilense, A. lipoferum, A. irakense, and Azospirillum sp.) revealed bacteria exhibiting antigenic cross reactions with A. brasilense Sp7 or Sp245. The effect of anti-LPS antibodies on motility of azospirilla was in agreement with the results of immune agglutination analysis of bacterial cells and of immunodiffusion analysis of the LPS preparations. According to our results, strains Azospirillum sp. SR81 and A. brasilense SR14 should be included into serogroups I and II, respectively.

Based on an example of Azospirillum brasilense Sp245, it was shown that, in bacteria with mixed flagellation, insertional mutagenesis of one of the copies of the flhB gene, which encodes a component of the flagellar protein export apparatus, may be concurrent with defects in the formation of both a constitutive polar flagellum and inducible lateral flagella. Despite the presence of a second copy of flhB in the plasmid-located gene cluster, which seems necessary for the formation of lateral flagella, the flhB1::Omegon-Km Sp245 mutant completely lost the ability to produce them. The described effect of the inactivation of flhB1 might be explained by the use of FlhBl for the assembly of both types of flagella. Since the open reading frame AZOBR_150176, which is transcribed in the same direction and codes for a hypothetical multisensor hybrid histidine kinase/response regulator, adjoins to the 3’-end of flhB1, the participation of the latter protein in the induction of the lateral flagellar synthesis in response to the increase in the density of the environment was not excluded.

In liquid media, rhizobacteria Azospirillum brasilense are motile by means of a polar flagellum and in semisolid media, also by means of additionally synthesized lateral flagella. In the presence of 4-n-hexylre-sorcinol (0.04–0.10 mM), the swimming rate of A. brasilense Sp245 decreased significantly. At the concentrations over 0.10 mM, motility was completely suppressed. The presence of 4-n-hexylresorcinol in liquid medium also had a negative effect on motility of the cells of Sp245 and other A. brasilense strains (Sp7, Cd, and SR75). The concentrations of 5-methylresorcinol which negatively affected the rate of bacterial spreading in semisolid media were an order of magnitude higher than those of 4-n-hexylresorcinol. Alkylresorcinols, which belong to alkylhydroxybenzene autoregulators, probably affect the cellular mechanisms (systems) responsible for flagella rotation in azospirilla.

Earlier such Azospirillum brasilense Sp245 mutants as flagellation-defective SK051, SK248 with immobilized flagella, and BK570 swimming and swarming faster than Sp245 were obtained. In SK051 and SK248 the self-killer vector pJFF350 integrated into the 18.3-kb XhoI fragment ofplasmid 85MDa (p85) while in BK570, it integrated into the 9.1-kb XhoI-fragment of p85. In the present work, analysis of the nucleotide sequence of fusion products of p85 and pJFF350 was performed. In p85, in addition to three IS elements (two of which caused cointegrate formation) and phage integrase gene, 22 open reading frames with coding sequence properties were identified. Possible participation of predicted translation products of several p85 genes in bacterial motility detection is discussed. Since differences in the primary structure of p85::pJFF350 cointegrates from SK051 and SK248 cells are localized within pJFF350 DNA, different effects of DNA-folding changes on expression of corresponding p85 genes are suggested.

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.

In an 85-MDa plasmid (p85) of the plant-associated bacteria Azospirillum brasilense Sp245 the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I of cytochrome c oxidase (ccoN); presumable NO sensor carrying two hemerythrin domains (orf181); and an enzyme, required for synthesis of presumable NO antagonist, homocysteine (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164 encoding conserved secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA of A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A region homologous to p85 nirK—orf208-orf181 genes was detected in an 115-MDa plasmid of A. brasilense Sp7 type strain.

Lipopolysaccharides LpsI and LpsII containing the same O-specific polysaccharide (OPS), yet different in antigenic structure and charge, have been revealed in the rhizobacterium Azospirillum brasilense Sp245. In the present work, four putative glycosyltransferase genes were identified in a 14-kbp fragment of a 120-MDa plasmid of Sp245, p120-lpsKM348X. Insertional mutagenesis of one of them, encoding the predicted ADP-heptose:LPS-heptosyltransferase, resulted in LpsI loss. By means of DNA hybridizations and PCR with primers specific towards several sites of p120-lpsKM348X, it was demonstrated that homologous segments of 120-MDa plasmids of A. brasilense strains Sp245 and Sp107, which are characterized by identical structures of the OPS repeating units, are practically identically organized. In an 85-MDa plasmid of Sp245, a locus was identified with high homology to the plasmid genes of glycosyltransferases and conserved membrane-bound proteins from a wide range of soil bacteria.

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.

Summary The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa + phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri + phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri + mutant BK759.G gave rise to stable Swa ++ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa ++ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates.

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.

Summary The plant-beneficial bacterium Azospirillum brasilense can swim in liquids and swarm or migrate with the formation of microcolonies in soft media. To get closer to understanding the influence of natural environments on A. brasilense motility, we studied the individual and social movement of the bacterium in the presence of various plant lectins. The lectins with specificity for N -acetyl- β - d -glucosamine oligomers (wheat germ, Solanum tuberosum and Ulex europeus agglutinins) decreased A. brasilense swimming speed and induced the formation of branched–granular colonies instead of the swarming rings. These effects seemed to be a consequence of specific interactions between the agglutinins and the lectin-binding polymers present in the A. brasilense cell envelope. Concanavalin A (with an affinity for terminal α - d -mannosyl and α - d -glucosyl residues) and Phaseolus vulgaris phytohemagglutinin P (with unknown specificity) almost did not affect the motility of A. 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.

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.

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.

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of theAzospirillum brasilenseSp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilenseSp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.

Azospirilla are well-known plant-beneficial α-proteobacteria. However, they have been isolated not only from a wide range of plant, soil, and water locations, but also from human tissues. Thus, it is not surprising that these highly adaptable bacteria have flexible genomes, composed of several large replicons. In this chapter, experimental data on plasmid plasticity in the type strain A. brasilense Sp7 and in the facultative endophyte A. brasilense Sp245 are discussed. It is shown that certain changes in the primary structure of an 85-MDa plasmid of strain Sp245 were accompanied by alterations in flagellar formation and in bacterial social motility on soft media; e.g., shifts between flagellar-dependent swarming or flagellar-independent microcolonial spreading were noticed. A spontaneous reorganization of 85- and 120-MDa plasmids gave rise to an A. brasilense Sp245 derivative with a novel cell-surface architecture and with altered social motility and biofilm formation. Spontaneous changes in the plasmid profile of A. brasilense Sp7 derivatives encompassing 90- and 115-MDa replicons were concurrent with variations in the colony morphology, cell antigenic structure, swarming, and biofilm formation on abiotic surfaces and plant roots. Such genetic and physiological plasticity of azospirilla is expected to be of significance for their survival in natural environments.

In this introductory chapter, we briefly characterize major groups of plant-associated bacteria and discuss what is currently known about genome organization and plasticity in such versatile and resourceful microbes as plant pathogenic and plant-growth-promoting bacteria.

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri+phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa+Gri–, or Swa–Gri–, or Swa–Gri+phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.

Azospirillum brasilense swims in liquid environments and swarms in semisolid media. Five variants of A. brasilense Sp245, Sp245.P1–Sp245.P5, which swarmed faster than Sp245 in a semisolid malate–salt medium, have been isolated. In Sp245.P1–Sp245.P4, a new megaplasmid was revealed instead of an indigenous 85-MDa plasmid (p85). By polymerase chain reactions (PCR) with primers to the segments of p85 important for proper bacterial motility/flagellation and for dissimilatory nitrite and NO reduction, that DNA of p85 was found retained by all the variants. In ERIC- and RAPD-PCR, microdiversity between the total DNAs of Sp245 and its variants was detected. Interstrain differences in growth characteristics in liquid peptone–succinate–salt medium with KNO3 or KNO2 and in KNO2 production/consumption were revealed. Although all the variants swam and swarmed faster than Sp245 in the medium supplemented with NH4Cl or KNO3, not all of them could do so in MPSS with KNO2.

The rhizobacteria Azospirillum brasilense Sp245 produce antigenically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeated chains of O-specific oligosaccharides (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 A. brasilense mutant Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid: [Formula: see text].

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.