The Ti Plasmid‐Encoded <scp>VirJ</scp> Functions as a Lysyl‐Phosphatidylglycerol Hydrolase in <scp> <i>Agrobacterium tumefaciens</i> </scp>

Agrobacterium tumefaciens delivers oncogenic transfer DNA (T-DNA) into plants via a type IV secretion system (T4SS). This process requires virulence factors from the tumor-inducing (Ti) plasmid and chromosomal genes such as acvB. We previously identified AcvB as a lysyl-phosphatidylglycerol (L-PG) hydrolase. Loss of AcvB in the nopaline-type strain C58 increases membrane L-PG levels, thereby compromising T-DNA transfer. Interestingly, octopine-type strains harbor an additional truncated acvB homologue, virJ, on the Ti plasmid. The established L-PG hydrolase function of AcvB and its similarity to VirJ motivated us to investigate the enzymatic function of VirJ. Purified recombinant VirJ hydrolyzed L-PG to phosphatidylglycerol and lysine. Transient virJ expression in an acvB deletion strain reduced elevated L-PG levels, rescuing impaired growth under acidic conditions and defective T-DNA transfer. These results provide the first direct evidence that VirJ functions as an L-PG hydrolase and demonstrate that its enzymatic activity in maintaining L-PG homeostasis is crucial for T-DNA transfer. Importantly, elevated L-PG levels did not disrupt T4SS assembly but likely compromised its functionality once formed. In contrast, L-PG excess did not affect the conjugative RP4 T4SS or the type VI secretion system. Together, these findings highlight a secretion system-specific reliance on membrane lipid composition.