Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella.

Microbial proteases play diverse and important roles in bacterial virulence but their detection and characterisation is often hampered by their limited abundance or lack of expression in the absence of suitable environmental signals. We describe here a sensitive proteomic approach to detect proteases that are under the control of a virulence regulator and to characterise their recognition motifs. Using MG++-depleted growth media or a mutant strain of Salmonella in which the PhoP-PhoQ virulence regulatory system is constitutively active, truncated forms of DnaK, elongation factor G, elongation factor Tu and ribosomal protein S1 proteins were detected. Two other global regulatory mutants and cells exposed to acid or to oxidative stress failed to produce the truncated proteins, indicating specific control of the protease activity by the PhoP-PhoQ system. Our results suggest that at least two proteases are induced. To define the proteolytic cleavage sites of one of the proteases, peptides from each of the truncated proteins were identified by tryptic mass fingerprinting/nanoelectrospray mass spectrometry and mapped onto the sequence of the intact protein. Alignment of the regions around the cut site indicates that the protease recognises a dibasic amino acid motif characteristic of the omptin protease family. The induction of such proteases in bacteria depleted of Mg++ ions may contribute to the PhoPQ-mediated resistance of Salmonella to cationic antimicrobial peptides. Additionally, our results suggest it would be prudent to keep the concentration of this ion above micromolar levels during bacterial sample preparation for proteomic analyses.