The Ti Plasmid‐Encoded VirJ Functions as a Lysyl‐Phosphatidylglycerol Hydrolase in Agrobacterium tumefaciens

Abstract

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.