Induction of AmpC-mediated-βlactam resistance requires a single lytic transglycosylase in Agrobacterium tumefaciens

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here we use a reverse genetics approach to identify genes involved in ampicillin resistance with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased {beta}-lactamase production, a response dependent on the broad-spectrum {beta}-lactamase AmpC and its transcription factor AmpR. Loss of the putative ampD orthologue, atu2113, led to constitutive production of AmpC-dependent {beta}-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent {beta}-lactamase activity and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101{Delta} mltB3 transforms plants with comparable efficiency to wildtype but can be cleared with sub-lethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium. IMPORTANCEAgrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in {beta}-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit {beta}-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.