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. 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!

Identification of promoters that respond to glyphosate

Since we planned to create a system for the detection of glyphosate, we took a closer look at the pathway for biosynthesis of the essential aromatic amino acids tryptophan, tyrosine, and phenylalanine. This pathway involves the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates the precursor for the de novo synthesis of the three amino acids (3). Glyphosate specifically inhibits the EPSP synthase in plants, fungi, bacteria and archaea (4,5,6,7). The EPSP synthase converts phospoenolpyruvate (PEP) and shikimic acid-3-phosphate (S3P) into EPSP. Therefore, inhibition of the EPSP synthase by glyphosate results in the depletion of the cellular levels of aromatic amino acids unless they are provided by the environment (4,8,9,10). In B. subtilis the EPSP synthase is encoded by the aroE gene for which we could show that it is essential for growth of the bacteria (unpublished data)! We came up with the hypothesis that treatment of B. subtilis with glyphosate inhibits the EPSP synthase AroE, which probably lead to upregulation of the genes involved biosynthesis of the aromatic amino acids. We were particularly interested in the trpE and trpP genes, encoding the subunit I of the anthranilate synthase TrpE (11) and the tryptophane transporter TrpP (12), respectively. In case the of the trpE and trpP genes are upregulated in the presence of glyphosate, the promoters driving the expression of these genes are suitable to generate reporter systems to detect the weedkiller. To assess the expression of the trpE and trpP genes, the promoters were fused to the lacZ gene encoding the β-galactosidase from Escherichia coli. The constructs were integrated into the genome of B. subtilis and the resulting strains were used for enzyme activity assays. Surprisingly, the activities of the two selected promoters did not change when the bacteria were cultivated with glyphosate. Thus, the promoters do not respond to the weedkiller in B. subtilis.

References

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