Difference between revisions of "Team:Goettingen/Results"

Identification of glyphosate uptake systems

Genomic adaptation of Bacillus subtilis to glyphosate

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 strain 168, we performed growth experiments in CS-Glc minimal medium that was supplemented with increasing amounts of glyphosate. CS-Glc medium contains glucose and succinate as carbon sources and ammonium as the nitrogen source (see Notebook). As shown in Figure 1B, at a glyphosate concentration of about 1 mM the growth rate 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), this study revealed 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 1. (A) Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase in B. subtilis. (B) Inhibition of B. subtilis by GS.

Therefore, we plated a B. subtilis wild type strain on agar plates supplemented with different amounts of glyphosate (0 mM - 60 mM). We observed that the growth of the bacteria was strongly inhibited on agar plates containing 5 mM glyphosate and there was no growth at concentrations higher than 10 mM. After further incubation of the agar plates, we observed that a high number of mutants appeared on the plates. Whole genome sequencing analyses uncovered that the mutants had inactivated the gltT gene encoding a high-affinity transporter GltT, which is involved in amino acid uptake (1). By analyzing additional mutants that tolerate high amounts of glyphosate, we found a variety of different mutations (transitions, transversions, deletions, insertions and duplications) in the gltT gene. However, the mutations have one thing in common: they all led to the inactivation of the gltT gene! Further adaptation of the a mutant lacking the gltT allowed us to isolate variants of B. subtilis that even tolerate higher amounts of glyphosate. Again, we analyzed the genomes of the evolved bacteria to identify the mutations causing the phenotypes. These analyses revealed that the isolated mutants had inactivated the gltP gene encoding a low-affinity amino acid transporter GltP (2). Our evolution experiments show that glyphosate enters the B. subtilis cell via the amino acid transporters GltT and GltP (unpublished data). To conclude, genomic adaptation to weedkiller led to the identification of the first glyphosate uptake systems!

References

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