RESUMO
The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli. The gene was expressed in E. coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics. The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA.
Assuntos
Resistência a Ampicilina/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Bactérias Gram-Negativas/genética , beta-Lactamases/genética , Southern Blotting , Centrifugação com Gradiente de Concentração , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Genes Bacterianos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Hibridização de Ácido Nucleico , Mapeamento por Restrição , beta-Lactamases/biossínteseRESUMO
Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K. W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol., 1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the wild type strain. Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size. Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis. Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location. This is the first report on plasmid localization of genes in Azospirillum.
Assuntos
Genes Bacterianos , Bactérias Aeróbias Gram-Negativas/genética , Lipopolissacarídeos/biossíntese , Plasmídeos , Rhizobium/genética , Elementos de DNA Transponíveis , Genes , Canamicina Quinase , Mutação , Hibridização de Ácido Nucleico , Fosfotransferases/genética , Mapeamento por RestriçãoRESUMO
The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.
Assuntos
Genes Bacterianos , Genes , Mutação , Polissacarídeos Bacterianos/genética , Rhizobium/genética , Spirillum/genética , Teste de Complementação Genética , Nitrogenase/genéticaRESUMO
Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBr17 by mating with Escherichia coli strains carrying suicide plasmid vectors. The sources of Tn5 were the suicide plasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coli at maximum frequencies of 10 per recipient cell. Transposon Tn5 also conferred streptomycin resistance on Azospirillum colonies as was observed earlier for Rhizobium sp. Eight Tn5-induced Km SmA. brasilense Sp7 mutants with reduced nitrogen-fixing capacity were isolated. The potential use of Tn5-Mob for labeling and mobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in the megaplasmids of A. brasilense Sp7.