RESUMO
In this study, the putative target genes of the Arc two-component system of the rumen bacterium Mannheimia succiniciproducens were determined by analyzing the transcriptome of the ArcA overexpression strain and by the in silico scanning of the entire genome sequence with the position weight matrix of the ArcA binding sequence developed for Escherichia coli. The majority of 79 repressed genes were involved in energy metabolism and carbohydrate transport and metabolism, while the majority of 82 induced genes were involved in hypothetical or unknown functions. Our results suggest that the Arc system in M. succiniciproducens has a specific function that differs from that in E. coli.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mannheimia/genética , Regulon , Proteínas Repressoras/genética , Transativadores/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biologia Computacional/métodos , Escherichia coli/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Proteínas Repressoras/metabolismo , Transativadores/metabolismoRESUMO
The ArcB/A two-component signal transduction system of Escherichia coli modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the putative arcA and arcB genes of Mannheimia succiniciproducens MBEL55E, a capnophilic (CO2-loving) rumen bacterium, encode functional proteins that specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro, and the arcA and arcB genes of M. succiniciproducens confer toluidine blue resistance to E. coli arcA and arcB mutants. However, neither the quinone analogs (ubiquinone 0 and menadione) nor the cytosolic effectors (d-lactate, acetate, and pyruvate) affect the net phosphorylation of M. succiniciproducens ArcB. Our results indicate that different types of signaling molecules and distinct modes of kinase regulation are used by the ArcB proteins of E. coli and M. succiniciproducens.
Assuntos
Mannheimia/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Teste de Complementação Genética , Mannheimia/genética , Dados de Sequência Molecular , Fosforilação , Homologia de Sequência de Aminoácidos , Cloreto de Tolônio/metabolismoRESUMO
Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.