RESUMO
We have constructed a vector to assess the ability of heterologous signal sequences to function in the endophytic bacterium Clavibacter xyli subsp. cynodontis. This secretion reporter contains the phoA gene from Escherichia coli from which the promoter and signal sequences have been deleted. Signal sequences of streptomycete origin were cloned into the vector and the level of secreted alkaline phosphatase determined enzymatically and by Western blotting. We show that a number of the signal sequences of streptomycete origin function well in C. xyli subsp. cynodontis. By inoculating corn plants with C. xyli subsp. cynodontis expressing the sti2-phoA fusion, we demonstrated alkaline phosphatase activity in planta. Our data show that phoA can be used to detect endophytic bacteria in some locations in planta, and is therefore useful in the study of plant-microbe interactions. Furthermore, our data illustrate how phoA fusions can be useful as a reporter for protein secretion activity of C. xyli subsp. cynodontis in planta.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium/metabolismo , Vetores Genéticos , Sinais Direcionadores de Proteínas/genética , Streptomyces/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/imunologia , Fosfatase Alcalina/isolamento & purificação , Proteínas de Bactérias/genética , Sequência de Bases , Corynebacterium/genética , Genes Reporter , Histocitoquímica , Dados de Sequência Molecular , Caules de Planta/microbiologia , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Streptomyces/isolamento & purificação , Zea mays/microbiologiaRESUMO
Clavibacter xyli subsp. cynodontis (Cxc) is a xylem-inhabiting bacterial endophyte of Bermudagrass. This organism is classified with Gram-positive, high G + C content, coryneform-actinomycete bacteria. Southern-blot analysis showed that Cxc contains only one copy of the ribosomal RNA-encoding genes (rRNA). A clone containing the rRNA genes was isolated from a genomic library of Cxc DNA cloned in the lambda EMBL3 vector. The gene cluster was partially sequenced, revealing the gene order 5'-16S-23S-5S-3', similar to that found in other prokaryotes. Low-resolution S1 mapping suggested multiple transcription start points of the rRNA operon.
Assuntos
Actinomycetales/genética , Família Multigênica/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 5S/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Ribossômico/genética , Dados de Sequência Molecular , Óperon , Poaceae/microbiologia , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
A 50-kilobase (kb) cryptic plasmid was found in three geographic isolates of Clavibacter xyli subsp. cynodontis (Cxc). The DNA region essential for replication of the plasmid was cloned in an Escherichia coli vector. The resulting vector, which functions as a shuttle vector between E. coli and Cxc, was characterized further with respect to its stability and copy number.
Assuntos
Vetores Genéticos , Bactérias Gram-Positivas/genética , Plasmídeos , Resistência ao Cloranfenicol/genética , Clonagem Molecular , Escherichia coli , Genes Bacterianos , Plasmídeos/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 5S/genética , Mapeamento por RestriçãoRESUMO
A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryIA(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryIA(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control.