LD-transpeptidation is crucial for fitness and polar growth in Agrobacterium tumefaciens.

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. Author summary: Peptidoglycan (PG) is essential for bacterial cell wall integrity. While most bacteria use penicillin binding proteins (PBPs) for crosslinking, some also employ LD-transpeptidases (LDTs); however, the essentiality and functions of LDTs remain poorly understood. Interestingly, Hyphomicrobiales bacteria have PG rich in LD-crosslinks, suggesting that LDTs play a vital role in maintaining cell wall integrity in these organisms. Our study of Agrobacterium tumefaciens revealed that LD-transpeptidation, mediated by at least one of 14 putative LDTs, is essential for maintaining both viability and morphogenesis. A mutant lacking 7 Hyphomicrobiales-specific LDTs suffered fitness and shape loss due to reduced LD-crosslinking and PG tethering to the outer membrane. Our results provide new insights into LD-transpeptidase specialized functions and conditional essentiality. The conservation of these enzymes across diverse polar-growing species suggests broader implications for understanding microbial cell wall growth and adaptation to environmental stresses. [ABSTRACT FROM AUTHOR]