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

Abstract

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 genetic 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 β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor AmpR. Loss of the putative ampD orthologue, 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 the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 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 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 β-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit β-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.

Diversification of LytM Protein Functions in Polar Elongation and Cell Division of Agrobacterium tumefaciens

“…Together, these strong converging phenotypes suggest that the EnvC ortholog may activate AmiC in A. tumefaciens , and, moreover, that A. tumefaciens can complete division without an amidase. AmpD (Atu2113) is the only other canonical amidase in the A. tumefaciens genome and has been previously shown to function in the PG recycling pathway ( Figueroa-Cuilan et al, 2020 ). Moreover, Δ ampD had no morphological defects and the Δ ampD Δ amiC double mutant morphologically phenocopied Δ amiC ( Supplementary Figures 6A–C ).…”

“…pathway (Figueroa-Cuilan et al, 2020). Moreover, ampD had no morphological defects and the ampD amiC double mutant morphologically phenocopied amiC (Supplementary Figures 6A-C).…”