The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates

• Published in: Environmental microbiology 2023-12, Vol.25 (12), p.3604-3622
• Main Authors: Schwedt, Inge, Schöne, Kerstin, Eckert, Maike, Pizzinato, Manon, Winkler, Laura, Knotkova, Barbora, Richts, Björn, Hau, Jann‐Louis, Steuber, Julia, Mireles, Raul, Noda‐Garcia, Lianet, Fritz, Günter, Mittelstädt, Carolin, Hertel, Robert, Commichau, Fabian M.
• Format: Article
• Language: English
• Subjects: 3-Phosphoshikimate 1-Carboxyvinyltransferase - genetics; 3-Phosphoshikimate 1-Carboxyvinyltransferase - metabolism; Amino acids; Aromatic compounds; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Biosynthesis; Cell death; Cell viability; E coli; Escherichia coli; Escherichia coli - metabolism; Folic acid; Folic Acid - metabolism; Glycine - metabolism; Glyphosate; Growth conditions; Herbicides; Intermediates; Menaquinones; Metabolites; Microbiological strains; Phenylalanine; Phosphates; Quinones; Shikimic Acid - metabolism; Tyrosine; Uptake
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/1462-2920.16518

Glyphosate (GS) inhibits the 5‐enolpyruvyl‐shikimate‐3‐phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS‐insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient‐poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS‐resistant EPSP synthase under non‐selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis. Laboratory, environmental and undomesticated Bacillus subtilis isolates exclusively adapt to the herbicide glyphosate (GS) by the mutational inactivation of glutamate transporters. Even though Escherichia coli rapidly acquires GS resistance by target modification, the continuous evolution of GS‐resistant EPSP synthase under non‐selective growth conditions indicates that GS resistance causes substantial fitness costs. Thus, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability.