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The AZM-mediated killing of stationary-phase bacterial cells and reduced expression of QS-regulated virulence factors require interaction between AZM and ribosome ,
The effects of AZM on P. aeruginosa can be counteracted by over expression of ErmBP or a peptidyl-tRNA hydrolase, which blocks the interaction between AZM and ribosome by modifying the 23S rRNA or increases the intracellular aminoacyl-tRNA level, respectively, AZM binds in the nascent peptide exit tunnel (NPET), resulting in ribosome stalling and depletion of the intracellular pools of aminoacyl-tRNAs, 2004. ,
In addition, the DExD/H box helicases have been shown to participate in bacterial responses to various stresses, such as cold shock, pH, osmotic, and oxidative stresses (Owttrim, 2013). And several DEAD family RNA helicases, which belong to a specific subfamily of DExD/H box helicases, have been shown to regulate virulence factors in Escherichia coli, Borrelia burgdorferi, 2004. ,
, , 2011.
The pleiotropic functions of DExD/H box family RNA helicases intrigued us to suspect that they might be involved in the bacterial response to AZM treatment. In this study, we found that deficiency in a DEAH box helicase, PA3297, renders P. aeruginosa more susceptible to the killing and virulence suppression by AZM. Our results suggest that the expression of PA3297 was up regulated in the presence of AZM, which might promote 23S rRNA maturation to counteract the inhibitory effect of AZM on protein elongation, MATERIALS AND METHODS Strains and Plasmids The bacterial strains and plasmids used in this study are listed in Table 1, 1983. ,
Table 2), respectively. Deletion of the PA3297 gene was confirmed by PCR with primers PA14-PA3297-FF and PA14-PA3297-FR (Table 2). For the complementation of PA3297, the PA3297 gene was amplified from the PA14 chromosome by PCR with the primers PA14-PA3297-FF and PA14-PA3297-FR (Table 2). The PCR product was ligated into the EcoRI-SacI sites of pUC18t-mini-Tn7T-Gm, resulting in pTH1502. The plasmid was introduced into the PA3297 mutant by electroporation, along with the helper plasmid pTNS3, The E. coli strains DH5?, S17-1 and P. aeruginosa strains were routinely cultured in Luria-Bertani (LB) broth at 37 ? C. Antibiotics were used at the following concentrations: for E. coli, ampicillin 100 µg/ml, 1998. ,
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