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E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near‐outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL−1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV) previously optimized as a substrate for OmpT, an outer‐membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA‐LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants. [ABSTRACT FROM AUTHOR]

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1′) and nearest-neighbor positions (P2, P2′) and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a kcat/ Km value of 6.1×106 L mol−1 s−1. Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. [ABSTRACT FROM AUTHOR]

Peptide arrays are used to identify highly active substrates for the OmpT protease, as described by M. Mrksich, B. Liedberg, et al. in their Communication on page 16531 ff. By preparing the peptide arrays on self‐assembled monolayers, mass spectrometry (the SAMDI method) was used to characterize the extent of cleavage of each peptide. A commonly used mutant form of the protease, although regarded as having identical activity, instead had an altered specificity for its substrates. [ABSTRACT FROM AUTHOR]