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.

To find promoters that are regulated by glyphosate, we performed RNAseq analyses with the help of Dr. Anja Poehlein from the Göttingen Genomics Laboratory. For this purpose, we cultivated B. subtilis with sublethal amounts of the glyphosate and analyzed the transcriptome. Interestingly, we found that the bacteria that were cultivated with the weedkiller downregulated the genes (> 10-fold!) involved in zinc homeostasis. Thus, we identified novel promoters that respond to the weedkiller and are therefore suitable for the construction of a glyphosate detection system. The principle of the detection system is shown below.

An enzyme-based reporter system for glyphosate detection

The reporter system to detect glyphosate is based on our finding that genes, which are involved in zinc homeostasis in B. subtilis are repressed in the presence of the weedkiller. To allow the detection of glyphosate, we want to fuse a glyphosate-dependent promoter to the xylR gene encoding a transcriptional repressor of the xylA-xylB and xynP-xynB operons (13). We expect that the xylR is transcribed in the absence of the weedkiller because the glyphosate-dependent promoter is active. Furthermore, the production of the XylR causes repression of the xynB gene encoding the xylan β-1,4-xylosidase XynB (14). The method for detecting the β-xylosidase activity is very simple. Therefore, lack of β-xylosidase activity indicates the absence of glyphosate. By contrast, during growth of the bacteria in the presence of glyphosate, the xylR gene is repressed and the β-xylosidase is produced. Therefore, the β-xylosidase activity level indicates the presence of glyphosate. Once characterized, the reporter system can also be introduced in B. subtilis that tolerate high amounts of glyphosate (see above).

Gylphosate detection by monitoring intraspecies competition

We also came up with the idea to develop a reporter system for the detection of glyphosate by monitoring intraspecies competition of fluorescently labelled B. subtilis strains that tolerate different amounts of the weedkiller (15). Since we have mutants of B. subtilis in hands that can grow in the presence of high amounts of glyphosate, such a system is very easy to put into practice. For labelling of our strains, we used the genes encoding the blue fluorescent protein mTagBFP, the green fluorescent protein GFP, and the orange fluorescent protein mOrange, which we obtained from the iGEM DNA distribution kit. The detection system is based on an intraspecies competition assay using blue, green and orange B. subtilis strains that are mixed in an 1:1:1 ratio. The blue, green and orange B. subtilis strains tolerate different amounts of glyphosate. Therefore, in the absence of glyphosate, we expect that all strains survive in a liquid culture. By contrast, during growth in the presence of glyphosate only those cells survive that tolerate high amounts of the weedkiller. Thus, the ratio of the fluorescently labelled strains after cultivation tells us about the amount of glyphosate that was present in the culture.

A possible application of the glyphosate detection system. A mixture of spores of the wild type B. subtilisstrain and of GFP-labelled spores of a strain tolerating high amounts of glyphosate are propagated on a sample and cultivated. In case glyphosate is present, only the labelled cells survive. At low levels of glyphosate, both cell types survive but more of the GFP-labelled cells are present. In the absence of glyphosate, both strains grow equally.

Engineering Bacillus subtilis to increase glyphosate tolerance

Many years ago it has been observed that some bacteria, which were isolated from glyphosate contaminated soil, are capable of degrading the weedkiller (16). It has also been found that glyphosate can be inactivated by covalent modification such as phosphorylation (17,18) and acetylation (19). For instance, the glyphosate N-acetyltransferase GAT from Bacillus licheniformis transfers the acetyl group from acetyl-CoA onto the amino group of the weedkiller (19). The N-acetylated form of glyphosate does not inhibit the EPSP synthase (see above). The GAT was also subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide (19,20,21,22). Therefore, bacteria like B. subtilis expressing the GAT should tolerate high amounts of glyphosate. Furthermore, it has been observed that bacteria can become resistant to glyphosate by the accumulation of mutations in the aroA gene in Salmonella typhimurium, which render the encoded EPSP synthase insensitive to glyphosate (5). It has also been shown that the EPSP synthase from E. coli can become insensitive to glyphosate. Especially the replacement of the glycine at position 96 by alanine in the EPSP synthase confers glyphosate insensitivity to the enzyme (23). Thus, co-expression of the gat and the aroAG96A genes in B. subtilis may help to further increase glyphosate tolerance of the bacteria. In this part of the project, we engineer B. subtilis to improve our reporter systems to allow the detection of high amounts of glyphosate. Currently, we are working on the integration of the gat and the aroAG96A genes into the genome of B. subtilis.

References

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