Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, SciLifeLab, Umeå University, Umeå, Sweden.
Division of Biological Sciences, University of Missouri-Columbia, Columbia, Missouri, United States of America.
PLoS Genet. 2024 Oct 21;20(10):e1011449. doi: 10.1371/journal.pgen.1011449. eCollection 2024 Oct.
Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.
肽聚糖（PG）是一种网格状结构，是细菌细胞壁的主要成分，对于维持细胞完整性和形状至关重要。虽然大多数细菌依赖青霉素结合蛋白（PBPs）进行交联，但有些物种也使用 LD-转肽酶（LDTs）。与 PBPs 不同，LDTs 的必要性和生物学功能在很大程度上仍不清楚。α变形菌的 Hyphomicrobiales 目以极性生长而闻名，其 PG 中 LD 交联异常丰富，这表明 LDTs 可能在这些细菌的 PG 合成中发挥更重要的作用。在这里，我们研究了植物病原体根癌农杆菌中的 LDTs，发现至少有 14 种假定的 LDTs 之一的 LD 转肽化对于其生存是必不可少的。值得注意的是，一种缺乏广泛存在于 Hyphomicrobiales 中的一组独特的 7 种 LDTs 的突变体表现出 LD 交联减少和 PG 与外膜 β-桶蛋白的连接减少。因此，该突变体遭受严重的适应性丧失和细胞形状变圆，突出了这些 Hyphomicrobiales 特异性 LDTs 在维持细胞壁完整性和促进伸长方面的关键作用。Tn 测序筛选进一步揭示了根癌农杆菌 LDTs 的非冗余功能。具体来说，Hyphomicrobiales 特异性 LDTs 与分裂和细胞周期蛋白表现出合成遗传相互作用，而另一个组的单个 LDT 也是如此。此外，我们的发现表明，除了一种以外，缺乏所有 LDTs 的菌株表现出独特的表型特征和遗传相互作用。总的来说，我们的工作强调了 LD 交联在根癌农杆菌细胞壁完整性和生长中的关键作用，并提供了对这些交联活性功能专业化的深入了解。
