Difference between revisions of "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

Previously, it has been reported that the growth rate of a B. subtilis culture containing 2.5 mM glyphosate strongly increases after 16 h of incubation (1). It has been suggested that this phenomenon might be either due to a physiological adaptation of the bacteria to glyphosate or due to the emergence of glyphosate-resistant mutants. To assess whether B. subtilis is able to adapt to toxic levels of glyphosate at the genome level, we propagated the wild type strain 168 on CS-Glc agar plates supplemented with increasing amounts of the herbicide (0, 10, 20 30 and 40 mM). After incubation for 24 h at 37°C, bacterial growth was only visible on the plates lacking glyphosate. When the plates were further incubated for 48 h, several glyphosate-resistant mutants appeared on the plates supplemented with 10 mM glyphosate (Figure 3A). No mutants appeared on the plates with glyphosate concentrations higher than 10 mM. Next, we repeated the plating experiment with a mutant (designated as iGEM1) that was isolated from the 10 mM glyphosate plate. After 48 h of incubation, the glyphosate-resistant strain iGEM1 formed mutants on plates containing 30 mM and 40 mM glyphosate (Fig. 2A). Again we isolated mutants from the 30 mM and the 40 mM glyphosate plates that were designated as iGEM 7 and iGEM13, respectively (Figure 3B). Our attempts to further increase the glyphosate resistance of the strains iGEM7 and iGEM13 failed . To ensure that the acquired mutations in the strains iGEM1, iGEM7 and iGEM13 are stable, the mutants were passaged three times on SP rich medium plates. The glyphosate resistance of the mutants was confirmed by streaking the bacteria on CS-Glc medium supplemented with increasing amounts of glyphosate. As in Figure 3C, all strains that were adapted to glyphosate grew on plates with 10 mM glyphosate. The strains iGEM7 and iGEM13 also formed single colonies on 20 mM glyphosate plates. Moreover, albeit weak, the strain iGEM13 showed even growth with 40 mM glyphosate.

Figure 3. Isolation and characterization of glyphosate-resistant mutants. (A) Emergence of glyphosate (GS)-resistant B. subtilis mutants on CS-Glc minimal medium agar plates that were incubated for 48 h at 37°C. WT, B. subtilis wild type strain 168; iGEM, GS-resistant B. subtilis strain that was isolated from plates containing 10 mM GS. (B) Genealogy of the GS-resistant B. subtilis mutants. (C) Evaluation of growth of the GS-resistant mutants on CS-Glc minimal medium plates supplemented with increasing amounts of GS. The plates were incubated for 48 h at 37°C.

The growth experiments using CS-Glc liquid medium with increasing amounts of glyphosate also confirmed that the strains iGEM7 and iGEM13 tolerate higher amounts of the herbicide than their progenitor strain iGEM1 (Figure 4). To conclude, B. subtilis rapidly adapts to toxic glyphosate concentrations at the genome level!

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.