Specificity Determinants for Autoproteolysis of LexA, a Key Regulator of Bacterial SOS Mutagenesis

Abstract

Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA’s self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme’s protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA’s internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1–P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1′ show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2′) that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.

Systematically Altering Bacterial SOS Activity under Stress Reveals Therapeutic Strategies for Potentiating Antibiotics

“…To access one extreme, the constitutively active SOS pathway, we first constructed a Δ lexA strain in the SulA-null background (defined as the Delta strain). This Delta strain was subsequently used as the parent for genetic recombineering at the lexA locus to yield four previously described LexA cleavage rate variants: a “hyperactive” E86P mutant, the reconstituted wild-type (WT) enzyme, a “hypoactive” G80A mutant, and finally the SOS-inactive S119A mutant, which is completely incompetent in self-cleavage ( 41 , 42 ). The relative in vitro self-cleavage activities of these LexA variants were confirmed in biochemical assays, with G80A ~5-fold reduced and E86P ~10-fold enhanced relative to WT LexA and with S119A unable to self-cleave under both base- and RecA-mediated conditions (see Fig.…”

A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage

“…For experiments with purified LexA, a previously described expression vector was used as a basis for mutagenesis, encoding LexA with an N-terminal His-tag with or without the S119A mutation. 32 Linker variations were introduced by overlap extension PCR. Proteins were expressed in E. coli BL21 pLysS, followed by one-step purification using the His-tag as previously described.…”

Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments

“…The SOS response is more complex in P. aeruginosa than in E. coli due to the participation of two other chromosomal regulators (PrtR and PA0906), which are present in most P. aeruginosa isolates and other species of the genus, in addition to LexA (197)(198)(199)(200)(201)(202). In P. aeruginosa, DNA damage causes autocleavage of LexA by RecA and the elimination of repression of the LexA regulon, as in E. coli (198,201,203). The induction of PrtR-managed genes adversely affects survival during genotoxic stress, decreases antimicrobial resistance, and reduces resistance to oxidant agents (204).…”