RESUMO
Pseudomonas aeruginosa shows multidrug resistance, which is mainly attributable to its expression of xenobiotic efflux pumps. However, it is unclear how silent pumps are expressed in clinical isolates. Here, we sequenced the complete genome of P. aeruginosa strain 8380, which was isolated from a human gut.
RESUMO
The NfxC-type mutant of Pseudomonas aeruginosa produces the MexEF-OprN efflux pump and down-regulates expression of the quorum-sensing-dependent efflux pump MexAB-OprM and production of virulence factors in the presence of an active transcriptional regulator, MexT. Consequently, these cells are resistant to chloramphenicol and hypersusceptible to ß-lactam antibiotics. An upper negative regulator, MexS, has been assumed to inactivate MexT in wild-type strains, hence shutting down production of the MexEF-OprN pump. This observation was, however, reported in only one clinical strain and not confirmed in well-characterized laboratory strains. Moreover, it is not known whether MexS is involved in the quorum-sensing-dependent regulation of virulence factor production. To assess these issues, a plasmid carrying wild-type mexS was introduced into three NfxC-type mutants from laboratory strains, which carry an impaired mexS and unimpaired mexT. Unexpectedly, all the transformants produced an increased amount of MexEF-OprN proteins. Three clinical NfxC strains were similarly transformed and although MexEF-OprN was undetectable in two of these strains, one produced an increased amount of these proteins, similar to the laboratory strains. These results were interpreted to mean that P. aeruginosa takes two separate routes in MexT-mediated regulation of mexEF-oprN expression: the MexS-bypassed pathway and MexS-mediated pathway. On the other hand, the transformants of both the laboratory and clinically derived NfxC-type cells produced increased amounts of MexAB-OprM and virulence factors, suggesting that production of these proteins occurs via the MexS-mediated pathway.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/genética , Análise de Sequência de DNA , Transformação Bacteriana , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Expression of the MexEF-OprN efflux pump in Pseudomonas aeruginosa seems to be upregulated by MexT. The region upstream of the mexEF-oprN operon contains mexT arranged in tandem with mexEF-oprN and separated by 230 bp of mexT-mexE intergenic DNA. Therefore, it is likely that this intergenic DNA contains the promoter-operator element of mexEF-oprN. To characterize how the expression of the mexEF-oprN operon was controlled, we analyzed the mexT-mexE intergenic DNA by constructing a series of intergenic DNA deletions connected with the mexEâ·lacZ reporter and made two important discoveries. The first was that the central region of the DNA contained two nod boxes. The mexT-proximal nod box was identified as the MexT-binding site by gel-shift assays using purified MexT. The mexT-distal nod box was required for the transcription of the mexEF-oprN operon, but not for the binding of MexT, suggesting that this region accommodates the binding of the RNA polymerase. The second observation is that there is a 13 bp inverted repeat sequence separated by 10 bp immediately upstream of the mexE gene. Deletion of this region caused a sudden rise in MexEF-OprN production, suggesting that this region accommodates the binding of a putative repressor protein.