Induction of AmpC-mediated β-lactam resistance requires a single lytic transglycosylase inAgrobacterium tumefaciens

Abstract

ABSTRACTThe remarkable ability ofAgrobacterium tumefaciensto transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge ofA. 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 genetic approach to identify genes involved in ampicillin resistance with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treatingA. tumefaciensC58 with ampicillin led to increased β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor AmpR. Loss of the putativeampDorthologue,atu2113, led to constitutive production of AmpC-dependent β-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent β-lactamase activity and its loss elicited ampicillin and carbenicillin sensitivity in theA. tumefaciensC58 and GV3101 strains. Furthermore, GV3101ΔmltB3transforms 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 ofA. tumefaciensconstitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium.IMPORTANCEAgrobacterium tumefaciens, a significant biotechnological tool for transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination ofA. tumefaciensfrom transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in β-lactam resistance inA. tumefaciens. Knowledge about proteins that promote or inhibit β-lactam resistance will enable the development of strains to improve the efficiency ofAgrobacterium-mediated plant genetic transformations. Effective removal ofAgrobacteriumfrom transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.