Estudo do sistema BlaR/Blal e de dois operons codificando sistemas de efluxo RND em Caulobacter crescentus. / Study of the BlaR/BlaI system and two operons encoding RND efflux systems from Caulobacter crescentus.

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

27/04/2012

RESUMO

The aim of this work is to characterize three clusters of genes from the alpha proteobacterium Caulobacter crescentus involved in metal and antibiotics response. We analyzed the cluster comprising genes CC1637-CC1640, which contains a BlaR/BlaI signal transduction system, and we performed a comparative analysis with two RND efflux systems consisting on genes CC2720-CC2725 and CC2388-CC2390, which are probably involved in cadmium and zinc response. Mutant strains for one or two of these genes were obtained, and the study of promoter activity was performed by b-galactosidase activity assays using lacZ as reporter gene. RT-PCR and b-galactosidase activity assays revealed that the CC1638 gene probably does not possess an exclusive promoter, and that the genes CC1637-CC1640 may constitute an operon. The promoter of CC1640 does not respond to H2O2, Cd2+ and Zn2+, but the DCC1640 strain presented low viability in the presence of Cd2+. The DCC1637 and DCC1638 strains showed sensitivity to t-butyl-hydroperoxide. The DCC1640 strain showed sensitivity to the antibiotics CTX and PPT, and b-galactosidase activity assays performed both on plates and liquid medium showed induction of the expression by the presence of antibiotics CTX and CFE. We observed auto-regulation of the operon by the protein encoded by the CC1640 gene (BlaI), which was confirmed by EMSA assays. The presence of BlaR inhibits the binding of the BlaI protein to the promoter, suggesting that both BlaI/BlaR regulate the CC1640 gene promoter. In silico analyses for the TTACGNNCGTAA consensus, located on CC1640 promoter, identified this sequence in promoter regions of other genes. Relative expression analyses indicate that the genes CC1568, CC1230 and CC2661 are regulated by the BlaI protein, suggesting that BlaI regulates the expression of other genes, possibly involved in b-lactamic antibiotics response. b-galactosidase activity assays of the intergenic region CC2720-CC2721 showed that it does not possess promoter activity, and a RT-PCR analysis confirmed that the genes CC2720-CC2721 are co-transcribed and belong to the CC2720-CC2725 operon. The expression of the operon showed significant induction in the presence of Cd2+, moderate induction in the presence of Zn2+ and Co2+, and a slight induction in the presence of Ni2+. The expression of the CC2388-CC2390 operon is highly induced in the presence of nickel and cobalt, not induced by cadmium and moderately induced by zinc. The DCC2724 strain is not sensitive to zinc, cobalt or nickel. The DCC2390 strain is sensitive to cobalt, slightly sensitive to nickel and not sensitive to zinc, and both strains are sensitive to cadmium. A double mutant was constructed, as well as a complemented strain, and results suggest that these are two efflux systems with distinct metal responses.

ASSUNTO(S)

ativação enzimática bacterial genetics cádmio cadmium enzyme activation gene regulation genética bacteriana regulação gênica zinco zinc

ACESSO AO ARTIGO

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