title: Characterization of the Pseudomonas aeruginosa metalloendopeptidase, Mep72, a member of the Vfr regulon.
abstract: BACKGROUND
  Pseudomonas aeruginosa Vfr (the virulence factor regulator) enhances P. aeruginosa virulence by positively regulating the expression of numerous virulence genes. A previous microarray analysis identified numerous genes positively regulated by Vfr in strain PAK, including the yet uncharacterized PA2782 and PA2783.
  
  
  RESULTS
  In this study, we report the detailed characterization of PA2783 in the P. aeruginosa strain PAO1. RT-PCR analysis confirmed that PA2782-PA2783 constitute an operon. A mutation in vfr significantly reduced the expression of both genes. The predicted protein encoded by PA2783 contains a typical leader peptide at its amino terminus end as well as metalloendopeptidase and carbohydrate binding motifs at its amino terminus and carboxy terminus regions, respectively. An in-frame PA2783::phoA fusion encoded a hybrid protein that was exported to the periplasmic space of Escherichia coli and P. aeruginosa. In PAO1, the proteolytic activity of the PA2783-encoded protein was masked by other P. aeruginosa extracellular proteases but an E. coli strain carrying a PA2783 recombinant plasmid produced considerable proteolytic activity. The outer membrane fraction of an E. coli strain in which PA2783 was overexpressed contained specific endopeptidase activity. In the presence of cAMP, purified recombinant Vfr (rVfr) bound to a 98-bp fragment within the PA2782-PA2783 upstream region that carries a putative Vfr consensus sequence. Through a series of electrophoretic mobility shift assays, we localized rVfr binding to a 33-bp fragment that contains part of the Vfr consensus sequence and a 5-bp imperfect (3/5) inverted repeat at its 3' and 5' ends (TGGCG-N22-CGCTG). Deletion of either repeat eliminated Vfr binding.
  
  
  CONCLUSIONS
  PA2782 and PA2783 constitute an operon whose transcription is positively regulated by Vfr. The expression of PA2783 throughout the growth cycle of P. aeruginosa follows a unique pattern. PA2783 codes for a secreted metalloendopeptidase, which we named Mep72. Mep72, which has metalloendopeptidase and carbohydrate-binding domains, produced proteolytic and endopeptidase activities in E. coli. Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region. However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.
authors.affiliation: Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX, USA. abdul.hamood@ttuhsc.edu.
authors.forename: Aysegul
authors.initials: A
authors.lastname: Balyimez
authors.forename: Jane A
authors.initials: JA
authors.lastname: Colmer-Hamood
authors.forename: Michael
authors.initials: M
authors.lastname: San Francisco
authors.forename: Abdul N
authors.initials: AN
authors.lastname: Hamood
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021382:Protein Sorting Signals; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018085:Regulon; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D033903:Periplasmic Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C091090:Vfr protein, Pseudomonas aeruginosa; D008666:Metalloendopeptidases