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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.