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Identification of MltG as a Prc Protease Substrate Whose Dysregulation Contributes to the Conditional Growth Defect
Po-Chuen Hsu1, Chien-Sheng Chen2, Shuying Wang3,4,5,6
1Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
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.
Insights
The bacterial protease Prc (also known as Tsp) in Escherichia coli has a new substrate, MltG. Accumulation of MltG and MepS in Prc-deficient bacteria contributes to conditional growth defects.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Microbial proteases regulate essential bacterial physiological processes.
- The Escherichia coli protease Prc (Tsp) is crucial for survival under specific stress conditions (low osmolality at 42°C).
- Prc deficiency leads to the accumulation of its substrate MepS, causing a conditional growth defect.
Purpose of the Study:
- To identify novel physiological substrates of the E. coli protease Prc.
- To investigate the contribution of these substrates to the conditional growth defect observed in Prc-deficient bacteria.
- To elucidate the role of Prc in regulating peptidoglycan (PG) biogenesis.
Main Methods:
- Proteome arrays were used to identify E. coli proteins that physically interact with Prc.
- In vitro assays were performed to determine protease cleavage activity.
- Gene deletion and growth assays were conducted to assess the physiological impact of substrate accumulation.
Main Results:
- Eighty-five potential Prc-interacting proteins were identified, with ten showing in vitro cleavage.
- MltG was confirmed as a physiological Prc substrate, accumulating in Prc-deficient E. coli.
- Blocking MltG and MepS accumulation partially restored bacterial growth, indicating an additive effect on the conditional growth defect.
Conclusions:
- MltG is a newly identified physiological substrate of the E. coli protease Prc.
- The accumulation of Prc substrates, including MltG and MepS, contributes additively to the conditional growth defect.
- Prc may play a significant role in regulating peptidoglycan biogenesis through its substrates like MltG, MepS, and PBP3.
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