These results strongly suggest that the unique pattern of mep72 expression is due to the effect of Vfr-independent translational/post-translational regulation. This pattern of expression is not a feature of the Vfr regulon. Many genes of the Vfr regulon including
lasB, lasA, lasR are part of the quorum sensing system and as such, expression is induced at later rather than earlier stages of growth [16, 54]. The significance of this pattern of expression is not known at this time. However, during our analysis of the P. Entospletinib in vivo aeruginosa global regulator PtxR (using ptxR-lacZ transcriptional fusions), we previously reported a pattern of expression that mimics that of PA2782-mep72. The expression of one of the ptxR-promoter nested deletions reached a peak at early stage of growth, sharply declined after that, and continued a low level of expression toward the end of growth cycle . Similar
to mep72, Vfr binds selleck chemicals llc to the ptxR upstream and directly regulates ptxR expression . Through the examination of the promoter regions of genes regulated by Vfr including lasR, toxA, pvdS, prpL, and algD, Kanack et al. developed a 21-bp Vfr binding consensus sequence that consist of two halves and contain several conserved nucleotides within each half . Experimental evidence revealed that changing one or more of these conserved nucleotides within the lasR or fleQ promoters affected the expression of these genes and their P5091 in vitro regulation by Vfr [16, 18, 44]. Our current analysis confirmed that Vfr specifically binds to the PA2782-mep72 promoter (Figure 7C). As with other Vfr-regulated genes, Vfr binding to the PA2782-mep72 promoter is cAMP dependent (Figure 7C). However, in contrast to all previously identified Vfr binding sites, the potential Vfr binding region Nutlin-3 supplier within PA2782-mep72 does not contain the intact Vfr consensus sequence (Figure 7D and E). Rather, we localized Vfr binding within the PA2782-mep72 promoter to a 33-bp sequence (probe VI), which contains only 6 bp from the left half of the Vfr consensus sequence (Figure 7E). Careful examination of the sequence revealed the presence of a 5-bp imperfect inverted repeat, with two bp
mismatch (underscored), at either end of the 33-bp sequence: TGGCG-N22-CGCTG (Figure 7E). Compromising either of the repeats eliminated Vfr binding (Figure 7D and E). Thus, this sequence may constitute an alternative Vfr binding site. The TGGCG-N22-CGCTG sequence overlaps the −35 region (Figure 7E). Additionally, the 33-bp sequence contains two direct repeats (TG/TG and CA/CA) (Figure 7E). Furthermore, the 33-bp sequence contains another imperfect (7/9) inverted repeat consisting of 9 bp, TGGCGCAAA-N9-TTGCCGCCA. Probe VII, which lost the ability to bind Vfr, lacks only one bp (A) from the right side of this repeat (Figure 7E). Further analysis including DNA foot printing experiments will be done to determine the exact sequence to which Vfr binds.