RESUMO
Haemophilus parasuis is a member of the family Pasteurellaceae and an important respiratory-tract pathogen of swine, which is the etiological agent of Glasser's disease. Because no genetic manipulation system is available for H. parasuis so far, in vivo studies about the role of its genes involved in virulence are unfeasible. Here we demonstrate that H. parasuis has a cyclic AMP (cAMP)-dependent natural transformation system that enables the uptake of DNA in which the ACCGAACTC sequence signal must be present. After improving DNA transformation parameters, such as cAMP and DNA concentration and exposition time of the exogenous DNA, a knockout mutant of H. parasuis defective in the thy gene, encoding the thymidylate synthase enzyme, has been constructed. Data presented in this work open the possibility for the functional analysis of genes involved in the infectious process of this animal pathogen.
Assuntos
Haemophilus parasuis/genética , Transformação Bacteriana , Animais , Sequência de Bases , AMP Cíclico/genética , AMP Cíclico/fisiologia , DNA Bacteriano/análise , Haemophilus parasuis/patogenicidade , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação , Proteínas Ribossômicas/genética , Suínos , Timidilato Sintase/genética , Virulência/genéticaRESUMO
It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium. In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S. typhimurium fur mutant than in the wild-type strain. According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an alpha-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S. typhimurium fur cells in comparison with wild-type cells. Introduction of an additional mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S. typhimurium fur mutant. Likewise, expression of pepE and the E. coli lac operon was the same in the S. typhimurium fur cpdA double mutant and the wild-type strain. Moreover, these results also demonstrate that the S. typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon. This positive control must be mediated by binding of the S. typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay.
