Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation

Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed on their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, it was shown that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR-p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.     

Spontaneous Super-Swarming Derivatives of Azospirillum brasilense Sp245 have Different DNA Profiles and Behavior in the Presence of Various Nitrogen Sources

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.     

Negative effect of alkylresorcinols on motility of rhizobacteria Azospirillum brasilense

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.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Serological relationships of azospirilla revealed by their motility patterns in the presence of antibodies to lipopolysaccharides

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.     

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.     

Arsenic Transformation by Azospirillum Brasilense Sp245 in Association with Wheat (Triticum Aestivum L.) Roots

The transformation of sodium arsenite and sodium arsenate by the rhizospheric nitrogen-fixing bacterium Azospirillum brasilense Sp245 in association with wheat (Triticum aestivum L. ‘Saratovskaya 29’) was studied. The effect produced by the A. brasilense strain on the morphological parameters of wheat in an As-polluted environment was examined. The plants were cultivated in a hydroponic system, with glass beads serving as a support for root growth. The plant-growth medium (an artificial soil solution) was deficient in P and Fe. The total initial As concentrations used were 75, 750, and 7500 μg l⁻¹. The As compounds used contained sodium arsenate and sodium arsenite at an As(V):As(III) ratio of 1:3.6 (in terms of As) in all experiments. Inoculation of A. brasilense Sp245 led to a decrease in the overall root length and to the formation of lateral roots; both effects are possibly related to the bacteria’s ability to synthesize auxins. Inoculation also changed the As(V): As(III) ratio of the plant-growth medium. In all experiments, the concentration of As(V) in the nutrient medium increased relative to the initial one and was approximately 1.5-fold higher than that in the medium of uninoculated plants. This value slightly decreased (1.6 > 1.5 > 1.4) with increasing concentration of As in the medium. Azospirillum-inoculated plants accumulated less As than did the surface-sterilized uninoculated plants. This study shows that A. brasilense Sp245 in association with wheat changes the speciation, bioavailability, and plant uptake of As.     

Detection of putative polysaccharide biosynthesis genes in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains from serogroups I and II

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.     

Obtaining phage mini-antibodies and using them for detection of microbial cells with an electroacoustic sensor

Phage mini-antibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained, and the possibility of using them for detection of microbial cells with a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with a suspension of A. brasilense Sp245 cells with the mini-antibodies differ significantly from the dependences of the resonator with a control cell suspension without mini-anti-bodies. The limit of possible determination of the concentration of microbial cells is found to be 10³ cells/mL upon interaction with mini-antibodies. It has been ascertained that detection of A. brasilense Sp245 cells with the aid of mini-antibodies is possible even in the presence of other cultures, for example, E. coli BL-Ril and A. brasilense Sp7. Therefore, it has been shown for the first time that detection of microbial cells with an electroacoustic sensor is feasible.     

Diverse morphological types of dormant cells and conditions for their formation in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Differences in generation of dormant forms (DF) were revealed between two strains of non-sporeforming gram-negative bacteria Azospirillum brasilense, Sp7 (non-endophytic) and Sp245 (endophytic strain). In post-stationary ageing bacterial cultures grown in a synthetic medium with a fivefold decreased initial nitrogen content, strain Sp7 formed two types of cyst-like resting cells (CRC). Strain Sp245 did not form such types of DF under the same conditions. CRC of the first type were formed in strain Sp245 only under phosphorus deficiency (C > P). The endophytic strain was also shown to form structurally differentiated cells under complete starvation, i.e. at a transfer of early stationary cultures, grown in the media with C > N unbalance, to saline solution (pH 7.2). These DF had a complex structure similar to that of azotobacter cysts. The CRC, which are generated by both azospirilla strains and belong to distinct morphological types, possessed the following major features: absence of division; specific ultrastructural organization; long-term maintenance of viability (for 4 months and more); higher heat resistance (50–60°C, 10 min) as compared with vegetative cells, i.e. the important criteria for dormant prokaryotic forms. However, CRC of non-endophytic strain Sp7 had higher heat resistance (50, 55, 60°C). The viability maintenance and the portion of heat-resistant cells depended on the conditions of maturation and storage of CRC populations. Long-term storage (for 4 months and more) of azospirilla DF populations at ?20°C was optimal for maintenance of their colony-forming ability (57% of the CFU number in stationary cultures), whereas the largest percentage of heat-resistant cells was in CRC suspensions incubated in a spent culture medium (but not in saline solution) at room temperature. The data on the intraspecies diversity of azospirilla DF demonstrate the relation between certain type DF formation to the type of interaction (non-endophytic or endophytic) with the plant partner and provide more insight into the adaptation mechanisms that ensure the survival of gram-negative non-spore-forming bacteria in nature.     

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.     

Tyrosinases of motile Azospirillum strains

A number of motile strains of Azospirillum brasilense, A. lipoferum, and A. irakense, were found to possess tyrosinase activity both on the surface of and inside the cells. A. brasilense Sp245, Sp7, and A. irakense KBC-1 each possessed two forms of tyrosinase of different molecular masses; A. lipoferum 43, A. lipoferum 59b, and A. irakense KA-3 each had a single tyrosinase form of approximately the same molecular mass; and A. brasilense Sp107 possessed a single form of tyrosinase different from all the other forms.     

Structural properties of capsular and O-specific polysaccharides of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 under varying cultivation conditions

Effect of the carbon source in the culture medium and of the growth phase on the composition and structure of the capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) of the bacterium Azospirillum brasilense Sp245 was studied. Growth with fructose resulted in an increased carbohydrate content in the CPSs, while long-term cultivation resulted in an increased content of phosphorus in both CPSs and LPSs. The LPSs produced on the medium with fructose (regardless of the cultivation duration) and the LPSs of the bacteria grown with sodium malate until the stationary phase were characterized by higher levels of unsaturated fatty acids than the LPSs of the bacteria grown with sodium malate to the late exponential phase. The structures of the polysaccharides from the isolated glycopolymers were established using monosaccharide analysis, including determination of the absolute configurations and 1D and 2D NMR spectroscopy. This study is the first to report that the CPS of A. brasilense Sp245 grown with sodium malate to the end of the exponential phase is structurally identical to the O-polysaccharide from the LPS of this bacterium and that the LPS and CPS of A. brasilense Sp245 grown with fructose contain an additional homoglucan of the following structure: [→3)-α-D-Glcp-(1→] _n .     

Changes in cell surface properties and biofilm formation efficiency in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 mutants in the putative genes of lipid metabolism <Emphasis Type="Italic">mmsB1</Emphasis> and <Emphasis Type="Italic">fabG1</Emphasis>

The previously obtained insertion mutants of Azospirillum brasilense Sp245 in the genes mmsB1 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 strains Sp245, 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 preparations, 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.     

Effect of bacterial lipopolysaccharides on morphogenetic activity in wheat somatic calluses

We evaluated the effect of lipopolysaccharides from the plant-growth-promoting associative bacterium Azospirillum brasilense Sp245 and from the enteric bacterium Escherichia coli K12 on the morphogenic potential of in vitro-growing somatic calluses of soft spring wheat (Triticum aestivum L. cv. Saratovskaya 29). A genetic model was used that included two near-isogenic lines of T. aestivum L. cv. Saratovskaya 29 with different embryogenic capacities; one of these lines carries the Rht-B1 dwarfing gene, whereas the other lacks it. When added to the nutrient medium, the lipopolysaccharide of A. brasilense Sp245 promoted the formation of calluses with meristematic centers and stimulated the regeneration ability of the cultured tissues in both lines. By contrast, the lipopolysaccharide of the enteric bacterium E. coli K12 barely affected the morphogenetic activity of callus cells and the yield of morphogenic calluses and regenerated plants. These findings indicate that the lipopolysaccharide of the plant-growth-promoting associative bacterium A. brasilense Sp245 specifically enhances the morphogenetic activity of wheat somatic tissues, which increases the efficacy of culturing of genotypes with a relatively low morphogenic potential. The results of the study may contribute to the improvement of the efficacy of plant cell selection and gene engineering and to a better understanding of the mechanisms responsible for plant recognition of lipopolysaccharides of associative bacteria.     

Phenol Oxidase Activity of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 Mutants with Modified Motility and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 Phase Variants with Different Plasmid Composition

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.     

Complementation Analysis of Associative Bacteria Azospirillum brasilense Sp245 and S27 Defective for Motility and Flagellation

Three mutants of Azospirillum brasilense Sp245 incapable of both formation of the polar flagellum (Fla^–-phenotype) and swarming in semisolid media (Swa^–-phenotype) were characterized. These mutants were shown to have lost the 85-MDa plasmid and to carry the Tn5-Mob transposon and pSUP5011 vector in different regions of their genomes. With the use of A. brasilense Sp245 gene bank, the capacity for both polar flagellum formation and swarming was restored in the above mutants and in the previously generated transposon mutants A. brasilense Sp245 and S27. The transconjugants obtained were only slightly motile in the liquid culture. In the gene bank of Sp245, the recombinant plasmids carrying wild-type fla/swa loci were identified.     

Hemagglutinating activity and motility of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> 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.     

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

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.     

Investigation of specific interactions between microbial cells and polyclonal antibodies using a resonator with lateral electric field

The interaction between polyclonal antibodies and Azospirillum brasilense Sp7 cells was studied using a resonator with lateral electric field. To this end, specific polyclonal rabbit antibodies against the O-antigen epitopes of the strain A. brasilense Sp7 were obtained and the possibility of their application for detection of microbial cells using a piezoelectric resonator with lateral electric field was shown. It was established that frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with the suspension of A. brasilense Sp7 cells and antibodies substantially differed from those of the resonator with the control suspension of cells without antibodies. It was shown that the obtained antibodies interacted with azospirilla cells, and the marker was accumulated all over the cell surface. The limit of possible detection of microbial cells during their interaction with antibodies was found to be 10⁴ cells/mL. Detection of A. brasilense Sp7 cells using antibodies proved to be possible in the presence of foreign bacteria. The presented results demonstrate the possibility of recording the interaction between microbial cells and antibodies and developing a biosensor for quantitative detection of microbial cells.