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Induction of AmpC-Mediated β-Lactam 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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9238390/ https://www.ncbi.nlm.nih.gov/pubmed/35638841 http://dx.doi.org/10.1128/aem.00333-22 |
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author | Figueroa-Cuilan, Wanda M. Howell, Matthew Richards, Christopher Randich, Amelia Yadav, Akhilesh K. Cava, Felipe Brown, Pamela J. B. |
author_facet | Figueroa-Cuilan, Wanda M. Howell, Matthew Richards, Christopher Randich, Amelia Yadav, Akhilesh K. Cava, Felipe Brown, Pamela J. B. |
author_sort | Figueroa-Cuilan, Wanda M. |
collection | PubMed |
description | 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 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 efficiency comparable to that of the wild type but can be cleared with sublethal 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. IMPORTANCE Agrobacterium 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 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. |
format | Online Article Text |
id | pubmed-9238390 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-92383902022-06-29 Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens Figueroa-Cuilan, Wanda M. Howell, Matthew Richards, Christopher Randich, Amelia Yadav, Akhilesh K. Cava, Felipe Brown, Pamela J. B. Appl Environ Microbiol Genetics and Molecular Biology 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 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 efficiency comparable to that of the wild type but can be cleared with sublethal 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. IMPORTANCE Agrobacterium 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 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. American Society for Microbiology 2022-05-31 /pmc/articles/PMC9238390/ /pubmed/35638841 http://dx.doi.org/10.1128/aem.00333-22 Text en Copyright © 2022 Figueroa-Cuilan et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Genetics and Molecular Biology Figueroa-Cuilan, Wanda M. Howell, Matthew Richards, Christopher Randich, Amelia Yadav, Akhilesh K. Cava, Felipe Brown, Pamela J. B. Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens |
title | Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens |
title_full | Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens |
title_fullStr | Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens |
title_full_unstemmed | Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens |
title_short | Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens |
title_sort | induction of ampc-mediated β-lactam resistance requires a single lytic transglycosylase in agrobacterium tumefaciens |
topic | Genetics and Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9238390/ https://www.ncbi.nlm.nih.gov/pubmed/35638841 http://dx.doi.org/10.1128/aem.00333-22 |
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