Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism

Abstract

Glyphosate (GS) specifically inhibits the 5‐enolpyruvyl‐shikimate‐3‐phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate‐3‐phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP‐derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho‐Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina.

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

“…A plausible answer to this question could be that more than one target exists in B. subtilis. To test this hypothesis, it is necessary to study the influence of GS on the growth of an aroE mutant in minimal medium (Wicke et al, 2019;Schwedt et al, 2023). Unfortunately, our attempts to cultivate the aroE mutant in minimal medium failed.…”

“…accumulate mutations in the EPSP synthase gene (Schwedt et al, 2023). In all isolated GS-resistant mutants, the intracellular PEP concentrations were increased due to the inactivation of the ppsR gene, encoding a regulator of the PEP synthetase PpsA (Schwedt et al, 2023).…”

“…accumulate mutations in the EPSP synthase gene (Schwedt et al, 2023). In all isolated GS-resistant mutants, the intracellular PEP concentrations were increased due to the inactivation of the ppsR gene, encoding a regulator of the PEP synthetase PpsA (Schwedt et al, 2023). Very likely, the increased cellular PEP concentration hinders GS from inhibiting the EPSP synthase in the evolved B. anthina mutants.…”

“…Next, we analysed the growth of the isolates on C-Glc plates, which is a prerequisite for investigating GS resistance. Previously, it has been shown that the growth medium must be free of amino acids like glutamate and aspartate because they interfere with the uptake of GS via glutamate transporters in B. subtilis (Wicke et al, 2019;Schwedt et al, 2023).…”

“…For instance, the modification of the GS target through the acquisition of mutations in the EPSP synthase gene is a widespread resistance mechanism (Barry et al, 1992;Chekan et al, 2016;Comai et al, 1983;Della-Cioppa et al, 1987). Moreover, organisms can develop GS resistance due to mutations resulting in overproduction of the EPSP synthase or PEP, the cosubstrate of the enzyme (Gaines et al, 2010;Dillon et al, 2017;Wicke et al, 2019;Schwedt et al, 2023). The ability of bacteria to degrade GS also confers resistance to the herbicide (Hove-Jensen et al, 2014;Hertel et al, 2021).…”