[Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense 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 (86­99%) 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.

[Insertional mutation in the AZOBR_p60120 gene is accompanied by defects in the synthesis of lipopolysaccharide and calcofluor-binding polysaccharides in the bacterium Azospirillum brasilense Sp245].

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.

[Genome Rearrangements in Azospirillum brasilense Sp7 with the Involvement of the Plasmid pRhico and the Prophage phiAb-Cd].

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.

[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 in their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, we showed 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.

[Insertional mutagenesis of a chromosomal copy of flhB gene is concurrent with defects in the formation of polar and lateral flagella in bacterium Azospirillum brasilense Sp245].

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 FlhB1 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.

[Alterations in the primary structure of an 85-MDa plasmid affecting flagellation and motility of bacterium Azospirillum brasilense Sp245].

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.

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

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].

[Localization of denitrification genes in plasmid DNA of bacteria Azospirillum brasilense].

In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, 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 ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (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 conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA 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 site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.

[Changes in Cell Surface Properties and Biofilm Formation Efficiency in Azospirillum brasilense Sp245 Mutants in the Putative Genes of Lipid Metabolism mmsB1 and fabG1].

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.

[Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

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.

[Search for conjugative plasmids in Azospirillum brasilense and Azospirillum lipoferum associated with plants]. / Poisk kon''iugativnykh plazmid u assotsiirovannykh s rasteniiami bakterii Azospirillum brasilense i Azospirillum lipoferum.

The conjugative plasmids in Azospirillum brasilense strains S17. Sp107, Sp245, SpBr14, JM6B2, JM82Al, UQ1794, UQI796 and in Azospirillum lipoferum strain RG20 were prove to exist for the first time in connection with their potency to mobilize a non-conjugative IncQ-plasmid pVZ361 from IncQ-group (ori RSF1010, KmR, SuR. 11.4 kb) for conjugated transfer to aplasmid strains Agrobacterium tumefaciens and Pseudomonas putida at high frequencies.

[Plasmid P85 from Azospirillum brasilense SP245: study of the circle of possible hosts and incompatibility with plasmids from Azospirillum brasilense SP7]. / Plazmida P85 Azospirillum brasilense SP245: izuchenie kruga vozmozhnykh khoziaev i nesovmestimosti s plazmidami Azospirillum brasilense SP7.

The possibility of the stable inheritance of the plasmid p85 mobilized derivatives from Azospirillum brasilense Sp245 in the cells of the bacterial genera Rizobiaceae (Agrobacterium tumfaciens) and Pseudomonadaceae (Pseudomonas putida) has been shown. The plasmid p85 participates in coding for the physiologically active products (the plant hormones). It is not inherited by the Escherichia coli strains. For the first time the incompatibility of azospirillium plasmids has been demonstrated on the example of the plasmid p85 from Azospirillum brasilense Sp245 and the plasmid p115 from Azospirillum brasilense Sp7.

[Transposon Tn5 and its derivatives used in genetic analysis of bacteria]. / Transpozon Tn5 i ego proizvodnye, ispol'zuemye v geneticheskom analize bakterii.

The review deals with the derivatives of transposon Tn5 carrying new genes of antibiotic resistance. The derivatives were constructed for mobilization of genetically labeled replicons, for direct selection of mutants having lost the marked plasmids, for obtaining the genes with the strong constitutive or regulated expression, for isolation of conditional mutations, for faster physical mapping of megaplasmids.

[Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation]. / Transpozonovyi tnPhoA mutagenez kak sposob polucheniia mutantov Azospirillum brasilense po podvizhnosti i zhgutikovaniiu.

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.

[Transposon mutagenesis, elimination and mobilization of plasmids in nitrogen-fixating bacterium Azospirillum brasilense Sp245]. / Transpozonovyi mutagenez, éliminatsiia i mobilizatsiia plazmid azotfiksiruiushchei bakterii Azospirillum brasilense Sp245.

The expressed difference in the plasmid profile of A. brasilense Sp245 is registered as a result of Tn5-Mob-mutability. Integration of the vector pSUP5011 into one of the A. brasilense Sp245 plasmid and using of the Tn5-Mob transposon to mobilize the 85Md cryptic plasmid are reported. The properties of A. brasilense Sp245 with the mutant plasmids composition (surface structure, acetylene and nitrate reduction, ability to a number of carbohydrates utilization, formation of melanin, antibiotics resistance specter) have been analyzed. The transposon Tn5-Mob insertion into the 85Md plasmid resulted in isolation of a mutant excreting a melanin-like pigment into the medium. The results suppose 85Md plasmid participation in melaninogenesis.

[Effect of plasmid composition on the chemotaxis reaction of Azospirillum brasilense Sp245 associated with gramineae]. / Vliianie plazmidnogo sostava na reaktsii khemotaksisa assotsiirovannykh so zlakami bakterii Azospirillum brasilense Sp245.

Correlation between the loss of the 120 and/or 130 kb plasmids by the spontaneous Azospirillum brasilense mutant and appearance of the Che(-)-phenotype towards 9-mono- and disaccharides has been established. The plasmid rearrangement is concomitant with the appearance of a new 44 Kda protein among the membrane proteins of azospirilli. Chemotaxis to alanine, glutamic acid, and to organic acids is not impaired.

Novel classes of Azospirillum brasilense mutants with defects in the assembly and functioning of polar and lateral flagella.

Azospirillum brasilense Sp245 has a mixed type of flagellation: a single polar flagellum (Fla) is synthesized constitutively, and abundant lateral flagella (Laf) are produced only in solid and semisolid media. In the present study, Omegon-Km Fla-Laf-, Fla-Laf+, Fla-/Mot-Laf-. Fla-/Mot-Laf+, Mot-Laf-, and Che- mutants of Sp245 were constructed in vivo. In some of the mutants, a number of cells possessed from 1 to 5 subpolar long flagella (Sfl). These Sfl provided the host cells with unusual motility patterns. Mutants producing shortened Fla and Laf were also detected. In the mutants still producing Laf, their expression retained the wild-type inducibility. However, all the mutants described were unable to form swarm rings in semisolid media. The results suggest the existence of common control or structural elements in assembly and rotation apparatus of both Fla and Laf systems. Single Omegon-Km insertions were localized in 85 MD (in two mutants) and in 120 MD (in one mutant) indigenous plasmids, as well as to least in two different regions of chromosomal DNA (in other mutants).

[Azospirillum brasilense SP245 mutants in production of anthranilic and indolyl-3-acetic acids]. / Mutanty azospirillum brasilense SP245 po produktsii antranilovoi i indolil-3-ukususnoi kislot.

The mutants of Azospirillum brasilense Sp245 altered in the production of anthranilic (Ant) and indolyl-3-acetic (IAA) acids were selected after the chemical or transposon facilitated mutagenesis and divided into the following three classes: Ant+IAA+, Ant+IAA- and Ant-IAA-. A hypothesis on the existence of a pattern for tryptophan conversion to anthranilate that is different from the classic pattern, and on the connection of the indolyl-3-acetic synthesis with this process is suggested.

[Characteristics of genes identified in the 120 MDa plasmid DNA in a mutant of Azospirillum brasilense Sp245 bacteria, defective in polar flagellation and swarming]. / Kharakteristika genov, vyiavlennykh v DNK 120-MDa plazmidy u mutanta bakterii Azospirillum brasilense Sp245, defektnogo po produktsii poliarnogo zhgutika i roeniiu.

The sequencing data were analyzed for two regions of the 120-MDa plasmid (p120) of Azospirillum brasilense Sp245. The 2420-bp region I, which flanks the omegon insertion in the SK048 mutant defective in production of the polar flagellum (Fla-) and swarming (Swa-), was shown to contain a cluster of two open reading frames (orf) that possess properties of coding sequences (CDSs). The NtrA (sigma 54) boxes were found in their upstream regions. The products of orf1 and orf2 are 16.5 and 15.5 kDa in molecular weight and consist of 151 and 152 amino-acid residues, respectively. The PRF1 polypeptide was found to contain a region homologous to the cysteine- and glycine-rich zinc-binding domain of the DnaJ heat-shock protein. ORF2 showed a homology to Haemophilus ducreyi pilin, fragments of Streptomyces and Mycobacterium integral membrane proteins, and eukaryotic transcriptional regulators. The omegon proved to be inserted into orfX1/X2 which possibly has a deletion and shows a GC content untypical for A. brasilense genes. The deduced ORFX2 polypeptide is homologous to fragments of arsenite-translocating ATPase and signal-transducing histidine kinase of archaebacteria. Possible causes of the Fla-Swa- phenotype of the A. brasilense SK048 mutant are considered. One coding orf was identified in the 1194-bp region II located approximately 4 kb away from the omegon insertion. The N-terminal region of the deduced product of this partly sequenced orf was shown to contain a signal sequence typical for secreted proteins.

[Effect of integrating the pJFF350 vector into the 85-MDa plasmid of Azospirillum brasilense Sp245 on bacterial flagellation and mobility]. / Vliianie integratsii vektora pJFF350 v 85-MDa plamidu Azospirillum brasilense Sp245 na zhgutikovanie i podvizhnost' bakterii.

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.