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 (WT) 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 (WT) 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.

Our 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; see below). To conclude, B. subtilis rapidly adapts to toxic glyphosate concentrations at the genome level!

Figure 4. Characterization of glyphosate-resistant B. subtilis mutants. The wild type (WT) strain (A) and its derivatives iGEM1 (B), iGEM7 (C), and iGEM13 (D) were cultivated for 10 h at 37°C in CS-Glc minimal medium supplemented with increasing amounts of glyphosate.

1.3. Identification of mutations by genome and Sanger sequencing

To identify the underlying genomic alterations causing glyphosate resistance the strains iGEM1, iGEM7 and iGEM13, we performed whole-genome sequencing with the help of the Göttingen Genomics Laboratory (Figure 5A). By analysing the genome sequences we found that the bacteria had deleted a 1240 bp-long fragment containing a part of the 5’ ends of the yhfE and gltT genes and the entire yhfF gene (Figure 5B).

Figure 5. The mutations of the glyphosate-resistant mutants and the link between glutamate and glyphosate transport. (A) The illumina sequencing reads were mapped onto the reference the B. subtilis 168 reference genome NC_000964 from GenBank (3) using the Geneious software package (Biomatters Ltd.) (4). (B) The mutations in the glyphosate (GS)-resistant B. subtilis mutants identified by genome sequencing. (C) Effect of the identified mutations on the glutamate transporters GltT and GltP in the strains iGEM1, 7 and 13. Membrane topologies of the transporters were predicted using the web-based protein topology tool Protter (5). (D) Comparison of glutamate and glyphosate.

The 1240 bp-long deletion is also present in the strains iGEM7 and iGEM13 because they were derived from the strain iGEM1 (Figure 5B). In addition to the deletion affecting the yhfE, yhfF and gltT genes, the strains iGEM7 and iGEM13 had replaced a single nucleotide and deleted 11 nucleotides, respectively, in the gltP gene. The yhfE gene encodes a putative aminopeptidase and the function of the yhfF gene is unknown (SubtiWiki; 6). The gltT and gltP genes code for the high-affinity sodium-coupled glutamate/aspartate symporter GltT and for the low-affinity proton/glutamate symporter GltP, respectively (7,8). The fact that all strains had acquired mutations in genes encoding transporters that are involved in glutamate and aspartate uptake suggests that the proteins also transport glyphosate (Figure 5B). The 1240 bp- and 11 bp-long deletions in the gltT and gltP genes truncate the encoded proteins after 282 and 214 amino acids, respectively (Figure 5C). The G232A mutation in the gltP gene results in a replacement of a glutamate residue by a lysine residue at position 78, which is adjacent to the second transmembrane helix of the GltP transporter (Figure 5C). While the deletions certainly destroy the amino acid transporters, the E78K replacement probably reduces the transport of glyphosate via GltP. This idea is in line with the observation that the strain iGEM7 carrying the G232A mutation in the gltP gene did not grow as good as the strain iGEM13 with the deletion in gltP on plates with high amounts of glyphosate (Figure 5C). A structural comparison revealed that glutamate and glyphosate resemble each other because the molecules contain negatively charged groups encompassing positively charged groups (Figure 5D). Thus, both, GltT and GltP could be involved in glyphosate uptake. To conclude, B. subtilis rapidly adapts to toxic glyphosate concentrations by inactivating the gltT and gltP genes!

1.4. Glyphosate-resistant mutants preferentially inactivate the gltT gene

Previously, it has been shown that bacteria like Salmonella typhimurium can develop glyphosate resistance due to the acquisition of mutations in the promoter region and in the coding region of the aroA gene, elevating the EPSP synthase levels and rendering the encoded enzyme insensitive to glyphosate, respectively (9-11). Our genome sequencing analyses of the isolated iGEM1, iGEM7 and iGEM13 strains did not identify mutations that alter the synthesis or the coding sequence of the EPSP synthase. To assess whether B. subtilis can develop glyphosate resistance by the acquisition of mutations that alter the target of the herbicide, we isolated 12 additional mutants from CS-Glc plates containing 10 mM glyphosate. DNA sequencing analysis revealed that each strain had mutations within the gltT coding sequence that would truncate or lengthen GltT transporter (Figure 6). Thus, the inactivation of the gltT gene is the predominant mutational event allowing B. subtilis to grow in the presence of the weedkiller glyphosate.

Figure 6. The mutations in the gltT gene and the consequences for the encoded transporter GltT in the B. subtilis mutants that were isolated from CS-Glc medium agar plates containing 10 mM glyphosate.

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

To create a proper detection system using the Gram-positive

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