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 Escherichia coli periplasmic protease thought to be indispensable for E. coli 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 E. coli, 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 E. coli proteins able to physically interact with Prc were identified using E. coli proteome arrays. Ten proteins were shown to be cleavable by Prc in vitro. Among these candidates, MltG was able to interact with Prc in E. coli. 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 mltG partially restored the growth of Prc-deficient E. coli. In addition, Prc-deficient E. coli 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.
Highlights
Microbial intracellular proteases govern vital physiological processes through diverse actions, such as protein quality control and the promotion of turnover, maturation and modification, which are crucial for proper function or localization of bacterial proteins (Weichart et al, 2003; Clausen et al, 2011; Dalbey et al, 2012)
To confirm that low osmolality contributed to the growth defect of Prc-deficient E. coli at 42◦C, we measured the growth of the wild-type E. coli K12 strain BW25113 (WT-BW25113) and its prc mutant in salt-free 1/2 Luria-Bertani broth (LB) medium with the osmolality adjusted to the level of the regular LB medium by using different solutes
The strains were cultured in normal LB medium and salt-free 1/2 LB medium in which the osmolality was adjusted to approximately 390 milliosmoles per liter per kilogram (mOsm/kg) using NaCl (172.5 mM), KCl (172.5 mM), MgCl2 (114.1 mM), LiCl (172.7 mM), sorbitol (300 mM), and glucose (344 mM)
Summary
Microbial intracellular proteases govern vital physiological processes through diverse actions, such as protein quality control and the promotion of turnover, maturation and modification, which are crucial for proper function or localization of bacterial proteins (Weichart et al, 2003; Clausen et al, 2011; Dalbey et al, 2012). The PDZ domain is essential for substrate recognition by recognizing and binding to the nonpolar or hydrophobic C-terminal residues of substrate proteins (Beebe et al, 2000). Penicillin-binding protein 3 (PBP 3) and MepS ( named Spr) have been identified as physiological substrates of Prc in E. coli (Hara et al, 1989, 1991; Nagasawa et al, 1989; Singh et al, 2015; Chueh et al, 2019). The fates of these natural substrates are different; MepS is fully degraded after being proteolyzed by Prc, while only 11 residues in the C-terminal of PBP3 are cleaved (Hara et al, 1989; Nagasawa et al, 1989)
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