Team:Goettingen/Results

1. Identification of glyphosate uptake systems

1.1. Interaction between glyphosate and Bacillus subtilis

To create a proper detection system using the Gram-positive model bacterium Bacillus subtilis, we first had to evaluate how this organism grows in the presence of glyphosate. Previously, it was shown that glyphosate negatively affects growth of B. subtilis due to the inhibition of the EPSP synthase AroE (Figure 1A) (1). Moreover, it has been demonstrated that 1.8 mM of glyphosate was required to inhibit the growth rate by 50%. To re-evaluate the effect of glyphosate on growth of our B. subtilis laboratory strains 168 and SP1, we performed growth experiments in CS-Glc minimal medium that was supplemented with increasing amounts of glyphosate (Figure 1B). CS-Glc medium contains glucose and succinate as carbon sources and ammonium as the nitrogen source (see Notebook). While the B. subtilis strain 168 is auxotrophic for tryptophan due to a mutation in the trpC gene, the strain SP1 can grow in the absence of exogenous tryptophan (2).

Figure 1. (A) Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which converts phosphoenolpyruvate (PEP) and 3-P-shikimate into EPSP and P in B. subtilis. EPSP is a precursor for the aromatic amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp). (B) How does glyphosate affect growth of our B. subtilis wild type strains?

As shown in Figures 2B and 2C, at a glyphosate concentration of about 1 mM the growth rate of both strains was reduced by 50% and the bacteria were not able to grow at glyphosate concentrations higher than 3 mM. In contrast to a previous study (1), we have observed that 44% fewer glyphosate is required to reduce the growth rate of B. subtilis by 50%. This discrepancy might be due to differences in the genetic makeup of the B. subtilis strains, in the medium composition, in the purity of glyphosate or due to the different cultivation conditions. However, glyphosate negatively affects growth of B. subtilis in CS-Glc minimal medium.

Figure 2. (A) Growth of the B. subtilis wild type strain 168 with increasing amounts of GS. (B) Growth of the B. subtilis wild type strain SP1 with increasing amounts of GS. The figure inlay shows the relationship between the growth rate (μ) and the glyphosate (GS) concentration. The bacteria were cultivated at 37°C in CS-Glc minimal medium.

1.2. Genomic adaptation of Bacillus subtilis to glyphosate

To create a proper detection system using the Gram-positive

1.3. Identification of mutations by genome and Sanger sequencing

To create a proper detection system using the Gram-positive

1.4. Characterization of gltT, gltP and gltT gltP mutant strains

To create a proper detection system using the Gram-positive

References

  1. Fischer et al. (1986) J. Bacteriol. 168: 1147-1154
  2. Zeigler et al. (2008) J. Bacteriol. 190: 6983-6995.
  3. Zaprasis et al. (2015) Appl. Environ. Microbiol. 81: 250-259.