Alvarado Obando Manuela, Rey-Varela Diego, Cava Felipe, Dörr Tobias
Department of Microbiology, Cornell University, Ithaca, New York, United States of America.
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.
PLoS Genet. 2024 Apr 10;20(4):e1011234. doi: 10.1371/journal.pgen.1011234. eCollection 2024 Apr.
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 Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 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 dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 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（PG）是细菌细胞壁的主要成分；它维持细胞形状，同时保护细胞免受内部渗透压和外部环境挑战。PG合成对于细菌生长和存活至关重要，并且需要一系列PG修饰来使细胞壁扩张。例如，内肽酶（EPs）切割相邻PG链之间的交联以允许掺入新合成的PG。EPs对细菌生长是必不可少的，并且很可能必须受到严格调控以防止细胞壁降解和细胞死亡。然而，EP调控机制却知之甚少。在这里，我们使用转座子测序（TnSeq）来揭示霍乱弧菌中的新型EP调节因子。该筛选表明羧肽酶DacA1（PBP5）减轻了EP毒性。dacA1对于在LB培养基上的生存能力至关重要，并且这种必要性被EP过表达所抑制，这表明EP毒性既减轻了dacA1缺陷，又被dacA1缺陷所减轻。随后的一个抑制子筛选，以恢复ΔdacA1在LB培养基中的生存能力，鉴定出PG合成途径中的次等位基因突变体以及促进EP激活的突变。因此，我们的数据揭示了DacA1在维持PG稳态方面的作用比以前认为的更为复杂。
