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
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.
RESUMO
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
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
The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.
Assuntos
Bacillus subtilis/genética , Desidrogenases de Carboidrato/genética , Genes , Glucose Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Regulação da Expressão Gênica , Glucose 1-Desidrogenase , Glucose Desidrogenase/análise , Glucose Desidrogenase/isolamento & purificação , Esporos Bacterianos/fisiologiaRESUMO
We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis.
Assuntos
Bacillus subtilis/genética , Guanina/metabolismo , Mutação , NADH NADPH Oxirredutases/genética , Purinas/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/crescimento & desenvolvimento , GMP Redutase , Genótipo , Especificidade da EspécieRESUMO
The sporulation of Bacillus subtilis can be induced in the presence of amino acids and glucose by partially depriving the cells of guanine nucleotides. This can be achieved, e.g., by the addition of decoyinine, a specific inhibitor of GMP synthetase. To determine the effect of this and other inhibitors on cell wall synthesis, we measured in their presence the incorporation of acetylglucosamine into acid-precipitable material. The rate of wall synthesis decreased by 50% within 5 min after decoyinine addition; this decrease was prevented by the presence of guanosine. A comparison with the effects of other inhibitors of cell wall synthesis indicated that decoyinine inhibited the final portion of the cell wall biosynthetic pathway, i.e., after the steps inhibited by bacitracin or vancomycin. Decoyinine addition also prevented cellular autolysis and cell wall turnover. It is not known whether these two effects of decoyinine on cell wall synthesis are causally related.
Assuntos
Bacillus subtilis/metabolismo , Carbono-Nitrogênio Ligases , Peptidoglicano/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Bacillus subtilis/efeitos dos fármacos , Parede Celular/metabolismo , Nucleotídeos de Guanina/metabolismo , Ligases/antagonistas & inibidoresRESUMO
Glucose dehydrogenase of Bacillus subtilis is a developmental enzyme that is not found in growing (vegetative) cells but is synthesized after the differentiation process that leads to the production of endospores has started. We have isolated the gene coding for this enzyme from a lambda Charon 4A phage library of B. subtilis DNA. It is transcribed and translated in vegetative cells of the nondifferentiating organism Escherichia coli into enzymatically active glucose dehydrogenase that has the same physicochemical properties as the enzyme produced in B. subtilis during sporulation. Subcloning of the lambda DNA insert into pBR322 plasmid derivatives showed that the glucose dehydrogenase gene was transcribed in E. coli from a promoter within the B. subtilis genome.
