4). To find the MexT-binding site within the nod boxes, we constructed plasmids containing only one of the boxes, such as the 129–143 nod or 151–165 nod, and carried

out gel-shift assays. As expected, the fragment containing the 129–143 nod box showed a clear interaction with MexT but the fragment with the 151–165 nod box did not (data not shown). These results indicated that the 129–143 bp PI3K phosphorylation nod box is needed for both MexT binding and the transcription of the mexEF-oprN operon. On the other hand, the 151–165 nod box is only required for the transcription of the mexEF-oprN operon. We concluded on the basis of these results that the promoter element is located within the region containing the 129–143 nod box and ATCA(N5)GTCGAT(N4)ACYAT sequence. The factors involved in the upregulation of the mexEF-oprN operon were reported 5-Fluoracil in vivo to be (1) a positive regulator, MexT (Maseda et al., 2000; Köhler et al., 2001), (2) two nod boxes in mexT-mexE intergenic DNA (Köhler et al., 1999), and (3) the sequence ATCA(N5)GTCGTA(N4)ACYAT (Tian et al., 2009). However, how these factors are interrelated in the expression of the mexEF-oprN operon remained to be elucidated. This study addressed this interesting issue by constructing a series of mexT-mexE intergenic DNA deletions connected with the mexE∷lacZ

reporter. The reporter assays revealed that the intergenic DNA between the mexT-proximal 114-bp and mexE-proximal 27-bp regions contained an element essential for the mexEF-oprN expression and that the mexE-proximal Farnesyltransferase 27-bp region contains a second regulatory element. The former element was found to be the MexT-binding site featured by two nod boxes. The binding of MexT to this region of DNA was confirmed by DNA mobility shift assay in the presence of purified MexT. Unexpectedly, site-directed mutagenesis of the nod boxes revealed that only mutation in the

mexT-proximal nod box abolished the interaction. MexT bound to the mexT-proximal nod box but not to the mexT-distal nod box. This result firmly ruled out an earlier proposal that MexT interacts with the ATCA(N5)GTCGTA(N4)ACYAT sequence because this sequence contained mexT-distal nod box (Tian et al., 2009). Thus, it is likely that the mexT-distal nod box DNA contains the promoter element, which is consistent with the general consensus that the region −10 to −50 bp from the transcriptional initiation site contains a promoter. However, the putative promoter site lacked any major promoter-binding sequence known in P. aeruginosa. It is possible, therefore, that this site contains a new promoter-binding sequence or is acted on by an uncharacterized sigma factor. As P. aeruginosa encodes over 20 sigma factors, further analysis is needed (Potvin et al., 2008). The latter element seemed to be the repressor-binding site because deletion of this region caused a fourfold increase in mexE expression.