RESUMO
A Xoo recA insertion inactivation mutant was constructed. The mutant, lacking RecA, showed increased sensitivity towards mutagen killing. This phenotype could be complemented by a cloned, functional recA. Unlike other bacteria, both the recA mutant and the parental strain had similar level of resistance to H2O2 killing and peroxide-induced mutagenesis.
Assuntos
Mutagênese Insercional , Recombinases Rec A/genética , Xanthomonas/genética , Southern Blotting , Western Blotting , Peróxido de Hidrogênio/farmacologia , Reação em Cadeia da Polimerase , Fatores de Tempo , Xanthomonas/efeitos dos fármacos , Xanthomonas/fisiologiaRESUMO
Nucleotide sequence of Xanthomonas oryzae pv. oryzae (Xoo) DNA from pSM-A1 was determined and sequence analysis revealed an ORF with high homology to RecA proteins. Expression analysis using an anti-RecA antibody demonstrates that MMS treatment induces recA in Xanthomonas strains but not in an Escherichia coli harbouring cloned Xoo recA. This indicates the existence of a recA regulatory mechanism in Xanthomonas that is not function in E. coli. In Xoo, recA was highly induced by treatments with chemical mutagens, UV and peroxides, while superoxides, a thiol agent, a heavy metal and heat shock were not inducers. The increased amount of RecA induced by H2O2 or MMS treatments were due to increased transcription of recA. recA showed no growth phase or starvation regulation. The pattern of recA regulation in Xoo could play important roles in stress survival in the environment and during plant-microbe interactions.
Assuntos
Regulação Bacteriana da Expressão Gênica , Recombinases Rec A/genética , Xanthomonas/genética , Proteínas de Bactérias/genética , Northern Blotting , Dano ao DNA , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Mutagênicos/farmacologia , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Recombinases Rec A/análise , Análise de Sequência de DNA , Raios Ultravioleta , Xanthomonas/efeitos dos fármacos , Xanthomonas/crescimento & desenvolvimentoRESUMO
We have modified the positive-selection cloning vector pUN121 to expand the numbers of unique cloning sites by insertion of a multiple cloning site into the lambda cI gene without disrupting its repressor function, resulting in plasmid pSKM10. Plasmid pSKM10 has seven restriction enzyme sites suitable for general cloning purposes. A mobilizable version (pSKM11) of pSKM10 was constructed by insertion of the IncP mob sequence which permitted mobilization of the plasmid into a wide variety of Gram- bacteria.
Assuntos
Bacteriófago lambda/genética , Clonagem Molecular/métodos , Vetores Genéticos , Mutagênese Insercional/métodos , Plasmídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis , DNA Viral , Genes Virais , Dados de Sequência Molecular , Proteínas Repressoras/genéticaRESUMO
The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows lambda red- gam- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.
Assuntos
Recombinases Rec A/genética , Xanthomonas/genética , Bacteriófago lambda/fisiologia , Southern Blotting , Western Blotting , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Teste de Complementação Genética , Metanossulfonato de Metila/farmacologia , Mitomicina/farmacologia , Recombinases Rec A/isolamento & purificação , Recombinases Rec A/metabolismo , Especificidade da Espécie , Ensaio de Placa ViralRESUMO
We have built a series of useful gene cassette plasmids to facilitate the construction of generalized and specialized cloning vectors. The gene cassettes consist of two promoter-less genes, cat and gus, a selectable marker gene (tet) and an IncP mob sequence. All of these genes in the cassette vectors are flanked by many unique restriction sites to facilitate their use in the construction of cloning vectors.
