Identification of MltG as a Prc Protease Substrate Whose Dysregulation Contributes to the Conditional Growth Defect of Prc-Deficient Escherichia coli

Abstract

Microbial proteases play pivotal roles in many aspects of bacterial physiological processes. Because a protease exerts its biological function by proteolytically regulating its substrates, the identification and characterization of the physiological substrates of a protease advance our understanding of the biological roles of the protease. Prc (also named Tsp) is an <i>Escherichia coli</i> periplasmic protease thought to be indispensable for <i>E. coli</i> to survive under low osmolality at 42°C. The accumulation of the Prc substrate MepS due to Prc deficiency contributes to the conditional growth defect. Because preventing MepS accumulation only partially restored the growth of Prc-deficient <i>E. coli</i>, we hypothesized that other unidentified Prc substrates intracellularly accumulate due to Prc deficiency and contribute to the conditional growth defect. To identify previously undiscovered substrates, 85 <i>E. coli</i> proteins able to physically interact with Prc were identified using <i>E. coli</i> proteome arrays. Ten proteins were shown to be cleavable by Prc <i>in vitro</i>. Among these candidates, MltG was able to interact with Prc in <i>E. coli</i>. Prc regulated the intracellular level of MltG, indicating that MltG is a physiological substrate of Prc. Prc deficiency induced the accumulation of MltG in the bacteria. Blocking MltG accumulation by deleting <i>mltG</i> partially restored the growth of Prc-deficient <i>E. coli</i>. In addition, Prc-deficient <i>E. coli</i> with blocked MltG and MepS expression exhibited higher growth levels than those with only the MltG or MepS expression blocked under low osmolality at 42°C, suggesting that these accumulated substrates additively contributed to the conditional growth defect. MltG is a lytic transglycosylase involved in the biogenesis of peptidoglycan (PG). In addition to MltG, the previously identified physiological Prc substrates MepS and PBP3 are involved in PG biogenesis, suggesting a potential role of Prc in regulating PG biogenesis.