Elucidating the Substrate Preference of the Bi-Functional Repressor-Protease LexA

Abstract

The rise of drug resistance in bacteria is a growing problem, and poses a formidable medical and scientific challenge. Mutations - the driving force behind evolution and drug-resistance - have long been thought to arise spontaneously in bacteria, leading to the pessimistic view that the rise of drug resistance is inevitable. However, the acquisition of mutations is in fact a regulated process, in part tied to the bacterial stress response or SOS pathway.The key regulatory protein governing the SOS pathway is the bi-functional repressor protease, LexA. In its basal state, LexA represses genes involved in the SOS response (SOS genes). When stimulated following genotoxic stress, LexA auto-proteolyzes via a serine protease mechanism at an internal cleavage region. Self-cleavage leads to de-repression of SOS genes, activating pro-mutagenic pathways, such as genetic recombination and error-prone DNA replication. This increased mutation rate, in turn, can promote the development of drug resistance under stress.Here we employ structural modeling, saturation mutagenesis, and kinetics to systematically elucidate substrate preference of the LexA enzyme in Pseudomonas aeruginosa. Our studies elucidate the key determinants for substrate recognition and demonstrate that overall, LexA is a relatively tolerant protease. Furthermore, this study reveals positions in the LexA protein that are amenable for labeling with fluorescent probes, which can be used for conformational studies and inhibitor screens. Understanding the enzymes that regulate mutation can lead to insights about the evolution of bacteria and potentially open up new avenues for combating the rise of drug resistance