2%, 0.02%, 0.002%, and 0.0002%) to an OD600 of about 0.5, harvested the cells, and analyzed the protein profile of the lysate using SDS-PAGE. We examined the gels for a unique band that exists in the lysate from induced but not uninduced cultures. We obtained optimum induction using LB broth containing 0.002% arabinose (data not shown). LMG194/pAB4 was grown in RM minimal medium supplemented with glucose overnight and subcultured into fresh RM minimal medium. At an OD600 of 0.5, 0.002% arabinose was added to induce expression of PA2783 and incubation continued for 5 h. Initial examination of total proteins

from the whole cell lysate confirmed the overproduction of the protein. As shown in Figure 6B, P505-15 compared with proteins from the uninduced culture, a unique band that corresponds to the predicted 70.5-kDa recombinant PA2783 protein (rPA2783) was detected in the induced culture. We extracted the band and determined the amino acid sequence of an internal peptide. The sequence matched (100%) that of the predicted protein (data Quisinostat not shown). Using the cold osmotic shock procedure [36, 42], we fractionated the cells into supernatant, periplasmic, cytoplasmic, and outer membrane fractions and separated the proteins by SDS-PAGE. Recombinant PA2783 was localized to the membrane fraction (data not shown). As overproduction of foreign

proteins in E. coli often results in their seclusion in inclusion bodies, which localize with the membrane fraction, we attempted to solubilize rPA2783. Despite trying numerous protocols, we failed to obtain a soluble protein with proteolytic activity. As an alternative, Depsipeptide in vivo we purified the outer membrane fraction of LMG/pAB4 and examined it for enzymatic activity [41, 42]. We detected the 70.5-kDa

rPA2783 within the outer membrane preparation of the arabinose-induced cells only (Figure 6C). This was confirmed by amino acid sequence analysis of an internal peptide obtained from the eluted protein (data not shown). Similarly, we detected the endopeptidase activity within the outer membrane of the arabinose-induced cultures only (Figure 6D). These results suggest that P. aeruginosa PA2783 encodes a membrane-bound 65-kDa protein with endopeptidase activity. We propose the name Mep72 for this protein that belongs to the metalloendopeptidase family M72.001, and mep72 for the gene encoding it. Vfr regulates mep72 expression by specifically binding to its upstream region Vfr is a DNA binding protein that regulates the expression of several genes including lasR, toxR, pvdS, and ptxR by binding to the promoter region of these genes [15, 16, 18, 43]. Thus, Vfr may regulate mep72 expression directly by binding to the upstream region of the PA2782-mep72 operon. Analysis of the upstream region revealed the presence of a potential Vfr-binding sequence located from −58 to −38 bp 5′ of the PA2782 GTG codon and between the −10 and −35 sequences (Figure 7A) [18].