RESUMO
The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.
Assuntos
Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pasteurella multocida/efeitos dos fármacos , Pasteurella multocida/patogenicidade , Transcrição Gênica/efeitos dos fármacos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Análise por Conglomerados , Farmacorresistência Bacteriana Múltipla/genética , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , VirulênciaRESUMO
One of the most efficient systems for the high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulated transcription unit supplying the specific T7 RNA polymerase. Various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase are in use. Most of them show high levels of expression in non-induced cells, low factor of induction or impaired growth of host cells. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is suppressed by the use of the genes for the Lac repressor and T7 lysozyme, integrated on the expression vector. T7 lysozyme expression is probably down-regulated in the induced expression system by antisense RNA. This overcomes the inhibitory effect of T7 lysozyme on T7 RNA polymerase as shown by SDS PAGE and flow cytometry analysis of expressed GFP. The main features of the expression vector compared with other systems are low background, high factor of induction and unaffected growth of non-induced cells.
Assuntos
Bacteriófago T7/genética , Regulação da Expressão Gênica , Muramidase/genética , Clonagem Molecular/métodos , DNA Recombinante/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichia coli is composed of 13 different subunits. The corresponding genes are organized in the nuo-operon (from NADH:ubiquinone oxidoreductase) at min 51 of the E. coli chromosome. To study the structure and function of this complex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promotor region with the phage-T7 RNA polymerase promotor and by expressing the genes with the T7 RNA polymerase coded on an inducible plasmid. It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membranes that complex I is overproduced 4-fold after induction. Complex I was isolated by chromatographic steps performed in the presence of dodecyl maltoside. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability over a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.