Glyphosate detection system

Genomic adaptation to glyphosate

As outlined in the project description, our topic is all about 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. 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 for 2 days at 37°C, 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evolution experiments show that glyphosate enters the B. subtilis cell via the amino acid transporters GltT and GltP (unpublished data).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 are regulated by 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 (G2L) (http://appmibio.uni-goettingen.de/index.php?sec=g2l). 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 strongly downregulated the genes 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.

A reporters system for the detection of glyphosate

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). In the absence of glyphosate, the

The promotor of a gene that is downregulated upon glyphosate treatment is active without glyphosate. The xylose repressor xylR is therefore transcribed and the enzyme xylosidase is inactive. When there is glyphosate present, the promoter is downregulated and the xylose repressor xylR is not transcribed. This leads to the expression of the xylosidase which results in a color change.

The promoter of TO DO is upon glyphosate treatment 11-fold downregulated. Downstream of this promoter the xylosidase repressor xylR cloned. This would mean, if there is no glyphosate present, the xylosidase repressor is expressed and the xylosidase is not active. However, if glyphosate is present, the promoter is downregulated and so is the expression of the xylR gene. This leads to an active xylosidase that turns ONPX into a yellow color.

With this detector we would also face the problem of dying cells, since they have to take up glyphosate for detection. Also, the formation of a glyphosate resistant B. subtilis via a resistant EPSP-synthase form would rescue this problem.

To avoid dying cells due to their disability to produce the essential aromatic amino acids, we thought about a resistant form of the EPSP-synthase. We found in Eschenburg et al., 2002 that a mutation at position 96 in the peptidechain from glycine to alanine provides a significant higher resistance against glyphosate. This resistant form should rescue the cells from their glyphosate sensitivity. However, this approach would not solve the problem with the blue cells without glyphosate treatment. Unfortunately, we were currently not able to clone this resistant form into Bacillus!

Fluorescence tagging

Since we had problems to realize the lacZ reporter system, we came up with a new reporting system that does not use molecular switches. For this system, we tagged strains that differ in their glyphosate resistance with fluorophores. We used mTagBFP (blue fluorescent protein), GFP (green fluorescent protein), and mOrange (orange fluorescent protein) as markers, which were obtained from the iGEM DNA distribution kit. The detection system is based on a competition assay. Cells that have high glyphosate resistance TO DO are marked orange, cells with moderate glyphosate resistance are marked blue and cells that cannot survive glyphosate treatment are marked green. Grown in medium without glyphosate, all cells show the same fitness. However, grown in medium with glyphosate, only adapted cells would survive, which could be detected by measuring the fluorescence.

This figure shows a possible application of our glyphosate detector. Fluorescence marked spores, in this case the green color indicates high glyphosate resistance, are propagated on a sample and cultivated. If there is a high glyphosate concentration within the sample, only the green marked cells can survive. If the glyphosate concentration is at a low level, the non-marked cells can survive, but are inhibited in their growth. When there is no glyphosate present, both strains will grow equally.

Such detection system as described in the figure above could also be used by customers, albeit the cells have to be observed with fluorescence detecting device. TO DO. Another option would be that customers could send their samples to a public laboratory company that uses our test kit for determining the glyphosate concentration.

Glyphosate degradation

We conducted a survey and it brought our attention to another important topic within our project. People that are not into science questioned if it would be possible to degrade glyphosate with our detector-bacterium. To address this question, we searched in the literature for such a method and found in Castle et al., 2004 a solution: the glyphosate-acetyl-transferase (GAT) from Bacillus licheniformis. This enzyme N-acetylates glyphosate, which then has no herbicidal effect anymore (Franz et al., 1997). Unfortunately, we were currently not able to clone this resistant form into Bacillus!!!!

References

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