<b>Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases</b>

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in <i>Vibrio cholerae</i>. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. <i>dacA1</i> is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in <i>dacA1</i>. A subsequent suppressor screen to restore viability of <i>∆dacA1</i> in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.肽聚糖（Peptidoglycan, PG）是细菌细胞壁的主要组成成分，既可维持细胞形态，又能保护细胞抵御内部渗透压与外界环境胁迫。肽聚糖的合成对细菌的生长与存活至关重要，而肽聚糖囊的延展需要一系列肽聚糖修饰过程。内肽酶（Endopeptidases, EPs）可切割相邻肽聚糖链之间的交联键，从而允许新合成的肽聚糖嵌入其中。内肽酶整体上对细菌生长不可或缺，因此必须受到严格调控，以避免肽聚糖囊降解与细胞死亡。然而，目前学界对内肽酶的调控机制尚知之甚少。本研究利用转座子测序（TnSeq）技术，在霍乱弧菌（Vibrio cholerae）中挖掘新型内肽酶调控因子。该筛选实验发现，羧肽酶DacA1（青霉素结合蛋白5，PBP5）可缓解内肽酶的毒性。dacA1基因对细菌在LB培养基中的存活至关重要，而内肽酶过表达可抑制这种必需性，这表明内肽酶毒性既可因dacA1缺陷而加剧，也可被dacA1缺陷所缓解。后续为恢复ΔdacA1突变株在LB培养基中的存活能力而开展的抑制筛选实验，鉴定出了肽聚糖合成通路中的功能减弱型突变体，以及可促进内肽酶激活的突变。综上，本研究的数据表明，DacA1在维持肽聚糖稳态中的作用比此前预想的更为复杂。