RESUMO
The inducibility of two promoter systems, one heterologous and one homologous, has been assessed in the Gram-negative bacterium Myxococcus xanthus. The heterologous system involved the hybrid tac promoter and the presence of lacIq, the lac repressor from Escherichia coli. This system is inducible in its natural host with isopropyl-beta-D-thiogalactopyranoside (IPTG). The homologous promoter system involves the light-inducible carQRS promoter, which is normally involved in the expression of the regulators of the light-inducible light-protective carotenoid synthesis regulon in M. xanthus. In each case, promoter activity and strength was assayed using the E. coli gene lacZ. In our constructs, which were present in a single copy in the M. xanthus chromosome, the carQRS promoter yielded at least a 47-fold increase in beta-galactosidase production upon light induction, whilst IPTG increased by 8-fold the amount of enzyme produced under the control of the ptac-lacIq system. Regulation by the latter was significantly higher than that obtained with the unmodified lacZ promoter.
Assuntos
Galactosidases/biossíntese , Isopropiltiogalactosídeo/farmacologia , Myxococcales/enzimologia , Estimulação Luminosa , Tioglicosídeos/farmacologia , beta-Galactosidase/biossíntese , Indução Enzimática/efeitos dos fármacos , Genética Microbiana , Técnicas In Vitro , Myxococcales/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
The secretion of numerous proteins during vegetative growth of Myxococcus xanthus, and the multicellular development cycle induced upon starvation of these bacteria, are partially interrelated in so far as mutants impaired in extracellular protein production are unable to undergo development. We have cloned and sequenced a gene in which a Tn5 insertion leads to a decrease in the production of most, if not all, extracellular proteins, and prevents development and sporulation. The deduced protein is homologous to the putative ubiquinone-binding subunit of bacterial and mitochondrial NADH:ubiquinone oxidoreductases (complex I). This is the first example of the presence of this complex in a bacterium from subclass delta of the proteobacteria. This gene is expressed during growth and during early development. As its disruption by Tn5 does not impair growth of the mutant strain, we assume the presence of a second alternative NADH oxidoreductase, and suggest that the phenotypic alterations caused by the mutation are due to a decrease in the proton-motive force.
Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Bacteriófago lambda/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , Sondas de DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Mitocôndrias/genética , Dados de Sequência Molecular , Mutagênese Insercional , Myxococcus xanthus/crescimento & desenvolvimento , Hibridização de Ácido Nucleico , Plasmídeos , Reação em Cadeia da Polimerase , Recombinação Genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Transformação Genética , beta-Galactosidase/metabolismoRESUMO
A recombinant Myxococcus xanthus strain was constructed that constitutively produces two proteins from Escherichia coli, the cytoplasmic beta-galactosidase and the periplasmic pH 2.5 acid phosphatase (AppA protein). We have previously shown that during vegetative growth, AppA protein is partly accumulated in the periplasm of M. xanthus and partly released into the medium. We demonstrate here that during starvation-induced development, release of periplasmic AppA protein to the medium did not occur over a period of 20 h. This was coincident with, but not caused by, the arrest of the synthesis of the foreign proteins. We have shown that this lack of secretion could be attributed to starvation per se and did not depend on the ability of the cells to undergo development. Our findings suggest that protein secretion which occurs during the first hours of starvation-induced development might therefore take place via a different route from that which occurs in vegetative cells.
Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Proteínas de Bactérias/genética , Permeabilidade da Membrana Celular , Meios de Cultura , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Óperon Lac , Myxococcus xanthus/genética , Myxococcus xanthus/crescimento & desenvolvimento , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.