Team:Goettingen/Results

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

Next, we tested whether the clean deletion of either the gltT gene or the gltT gltP genes is indeed sufficient to confer high-level resistance of B. subtilis to glyphosate. For this purpose, we constructed the mutant strains BP233 (gltT), BP234 (gltP) and BP235 (gltT gltP). To assess the glyphosate resistance of the gltT, gltP and gltT gltP mutants, we cultivated the bacteria in CS-Glc minimal medium supplemented with increasing amounts of glyphosate. As shown in Figure 7A and 7C, growth of the wild type and the gltP mutant strains, respectively, was inhibited by 5 mM glyphosate. This observation indicates that GltP is a low-affinity glyphosate transporter and that the cell still contains a transport system for the herbicide. The transporter with a high-affinity for glyphosate seems to be in fact GltT because the deletion of the gltT gene conferred high-level resistance to the herbicide (Figure 7C). Moreover, the deletion of the gltP gene in the background of a gltT mutant strain further enhanced glyphosate tolerance and the growth rates of the strains BP233 (gltT) and BP235 (gltT gltP) were reduced by 50% at herbicide concentrations of 6.1 mM and 8.0 mM, respectively (Figure 7B and 7D). Thus, in comparison to the wild type strain, 6- to 8-fold higher glyphosate concentrations are needed to reduce the growth rate of the strains BP233 (gltT) and BP235 (gltT gltP) by 50% (Figure 8).

Figure 7. Inactivation of the glutamate transporters confers high-level resistance to glyphosate. (A) Growth of the B. subtilis wild type (WT) strain 168 in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS). (B) Growth of the ΔgltT mutant strain BP233 in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS). The figure inlay shows the relationship between the growth rate (µ) and the glyphosate (GS) concentration. (C) Growth of the ΔgltP mutant strain BP234 in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS). (D) Growth of the ΔgltT ΔgltP mutant strain BP235 in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS). The figure inlay shows the relationship between the growth rate (µ) and the glyphosate (GS) concentration.

Figure 8. The relationship between the growth rate (µ) and the glyphosate (GS) concentration for the B. subtilis wild type (WT) strain 168, the ΔgltT mutant strain BP233 and the the ΔgltT ΔgltP mutant strain BP235 in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS).

We also tested whether the glyphosate that is present in commercially available Roundup® is toxic for B. subtilis. Roundup® was ordered from the german company Westfalia. 1 l of Roundup® Alphee contains 9.4 g of the isopropylamine salt of glyphosate (which corresponds to 7.2 g/l pure glyphosate), 5 g surfactant (undefined) and water. To test the effect of Roundup® Alphee on growth of the bacteria, we propagated the B. subtilis strains 168 (wild type ), BP233 (ΔgltT), BP234 (ΔgltP) and BP235 (ΔgltT ΔgltP) on CS-Glc minimal medium agar plates that were supplemented with glyphosate (control) and with equimolar amounts of glyphosate present in Roundup (test conditions). As shown in Figure 9, Roundup® Alphee killed the B. subtilis wild type strain and the strain lacking the low-affinity glutamate transporter GltP already at a concentration of 2 mM. By contrast, all strains lacking the high-affinity transporter GltT grew in the presence of 2 - 6 mM glyphosate that is present in Roundup.

Figure 9. A. Growth of the strains 168 (wild type), BP233 (ΔgltT), BP234 (ΔgltP) and BP235 (ΔgltT ΔgltP) on CS-Glc minimal medium in the presence of different amounts of glyphosate. B. Roundup® Alphee purchased from Westfalia. C. Growth of the strains 168, BP233, BP234 and BP235 on CS-Glc minimal medium in the presence of different amounts of Roundup® Alphee.

1.6. Complementation of the gltT mutation

We also performed a complementation experiment to provide further evidence that GltT is the major glyphosate transporter in B. subtilis. For this purpose, we fused the artificial Palf4 promoter (12) together with the gapA ribosome-binding site to the gltT gene and integrated the construct into the amyE locus of the gltT mutant (Figure 8A). Next, we propagated the strains BP233 (gltT) and BP237 (gltT Palf4-gltT) together with the wild type strain 168 on agar plates without and with glyphosate (Figure 8B). As expected, all strains grew in the absence of glyphosate. By contrast, only the gltT mutant strain BP233 grew with glyphosate. Thus, like the wild type also the complementation strain BP237 take up glyphosate via GltT. To conclude, under the tested growth conditions the high-affinity glutamate transporter GltT is the major entryway of glyphosate into B. subtilis!.

Figure 8. Complementation of the ΔgltT mutant strain. A. The artificial Palf4 promoter (12) and the ribosome-binding site (RBS) of the B. subtilis gapA gene were fused by PCR to the gltT gene. B. Growth of the B. subtilis wild type 168 (WT), the ΔgltT mutant BP233 and the ΔgltT Palf4-gltT complementation strain BP237 on CS-Glc minimal medium agar plates without glyphosate (- GS) and with 10 mM glyphosate (+ GS). The plates were incubated for 24 h at 37°C.

1.7. GltT is also involved in the uptake of the herbicide glufosinate.

Our structural comparison of glutamate and glyphosate revealed that the two molecules resemble each other because they contain negatively charged groups encompassing positively charged groups (see Figure 5D). However, the structural similarity of glutamate and glyphosate is not very high. During a scientific discussion with our collaborator Dr. Till Ischebeck from the Department of Plant Biochemistry at the University of Göttingen, we came up with the idea that GltT may also transport the herbicide glufosinate, which is structurally more similar to glutamate than glyphosate and inhibits the glutamine synthetase (Figure 9A) (Fraser and Ridley, 1984). Glufosinate (phosphinothricin) is a naturally occurring broad-spectrum systemic herbicide produced by several species of Streptomyces soil bacteria. Plants may also metabolize bialaphos (L-Alanyl-L-alanyl-phosphinothricin), another naturally occurring herbicide, directly into glufosinate (Fraser and Ridley, 1984). The compound irreversibly inhibits glutamine synthetase, an enzyme necessary for the de novo synthesis of glutamine from ammonia and glutamate, giving it antibacterial, antifungal and herbicidal properties. Application of glufosinate to plants leads to reduced glutamine and elevated ammonia levels in tissues, halting photosynthesis, resulting in plant death. In the 1960s and early 1970s, scientists at University of Tübingen and at the Meiji Seika Kaisha Company independently discovered that species of Streptomyces bacteria produce a tripeptide they called bialaphos that inhibits bacteria; it consists of two alanine residues and a unique amino acid that is an analog of glutamate that they named "phosphinothricin". The inactivation of the gltT gene indeed allowed B. subtilis to tolerate glufosinate (Figures 9B and 9C)! Moreover, the observation that the expression of two copies of the gltT gene in strain BP236 (amyE::Palf4-gltT) increased the sensitivity of the bacteria toward glufosinate further supports the idea that GltT is involved in glufosinate uptake by B. subtilis (Figures 9B and 9C). In contrast to glyphosate, however, the simultaneous deletion of the gltT and gltP genes did not increase glufosinate tolerance of B. subtilis (Figures 9B and 9C). To conclude, under the tested growth conditions the high-affinity glutamate transporter GltT is the major entryway of glyphosate into B. subtilis and GltT is also involved in Glufosinate uptake!

Figure 9. Glufosinate is also taken up by B. subtilis via GltT. (A) Comparison of glutamate and glufosinate. (B) Disk diffusion assay to test whether glufosinate is transported via GltT and/or GltP. The strains 168 (wild type), BP233 (ΔgltT), BP234 (ΔgltP, BP235 (ΔgltT ΔgltP), BP237 (ΔgltT amyE::Palf4-gltT) and BP236 (amyE::Palf4-gltT) were cultivated in LB medium, washed once in 1 X C-salts and propagated (100 µl cell suspension, OD600 ≈ 2) on CS-Glc minimal medium agar plates. A filter disk containing 5 µl of a 0.5 M glufosinate solution was placed in the center of the plates, which were incubated for 2 days at 37°C. (C) Evaluation of the disk diffusion assay shown in (B).

1.8. A novel nechanism conferring resistance to glyphosate.

In the past years, the underlying molecular mechanisms conferring resistance to glyphosate have been intensively studied in bacteria and plants. In many cases glyphosate resistance is directly linked to the target of the herbicide. For instance, bacteria like S. aureus are a priori resistant to glyphosate because this organism synthesizes an insensitive EPSP synthase (13-16). Moreover, increased cellular levels of the EPSP synthase, which can be achieved either by overexpression of the coding gene or by gene amplification, can confer resistance to glyphosate (Figure 9) (9, 17-21). Due to the increased cellular levels of the EPSP synthase, the glyphosate is probably titrated away and sufficient amounts of the precursor for aromatic amino acid biosynthesis can be produced. As mentioned in the design section, several glyphosate-insensitive EPSP synthase mutant variants have been isolated and engineered (11, 22-35). Often, a single amino acid exchange is sufficient to render the EPSP synthase insensitive to glyphosate. However, although glyphosate resistance is frequently linked to the target of the herbicide, resistance against the herbicide may occur by other means. Several studies have demonstrated that glyphosate can be detoxified by covalent modification. For instance, the Gat glyphosate N-acetyltransferase from Bacillus licheniformis, which was subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide, converts glyphosate to N-acetylglyphosate, which is not herbicidal and is not an effective inhibitor of EPSP synthases (Figure 9) (36-39). Moreover, the hygromycin phosphotransferases Hph and GlpA from E. coli and Pseudomonas pseudomallei, respectively, phosphorylate glyphosate and thus confer tolerance to the herbicide (Fig. 8) (40,41). Interestingly, the gat gene has also been used as a selection marker for genetic engineering of bacteria (39). The enzymes that covalently modify glyphosate have been successfully introduced into crops to increase herbicide resistance (42). Recently, it has been also demonstrated that enhanced export of glyphosate can reduce toxicity of the herbicide. For instance, the overexpression of the uncharacterized membrane proteins MFS40 and YhhS from Aspergillus oryzae and E. coli, respectively, with similarity to the major facilitator secondary transporter superfamily, enhance glyphosate tolerance of E. coli (Figure 9) (43,44). It will be interesting to test whether these transporters are suitable for engineering crops to enhance glyphosate tolerance. Proteins of unknown function can also increase glyphosate tolerance. For instance, the overexpression of the igrA gene product from the Pseudomonas sp. strain PG2982 increases glyphosate tolerance in transgenic rice (45-47). Finally, many bacteria can survive in the presence of glyphosate because they are able to degrade the herbicide (Figure 9) (48-57). To conclude, several mechanisms of glyphosate resistance have been described over the past years and novel mechanisms allowing survival with the herbicide are certainly identified in the future. We have observed that the deletion of the gltT gene in B. subtilis confers high-level glyphosate resistance to the bacteria. The B. subtilis gltT mutant strain could be a suitable host to identify glyphosate uptake systems from plants by expressing cDNA libraries and by screening for transformants that are unable to grow with glyphosate.

Figure 9. Mechanisms conferring resistance to glyphosate. GltT and GltP, B. subtilis glutamate transporters; YhhS and MFS40, major facilitator secondary transporters from E. coli and A. oryzae; GAT, B. licheniformis glyphosate N-acetyltransferase; GlpA, P. pseudomallei hygromycin phosphotransferase; PEP, phosphoenolpyruvate; 3PS, 3-phosphoshikimate; EPSP, enolpyruvylshikimate-3-phosphate; EPSPS, enolpyruvylshikimate-3-phosphate synthase. Membrane topologies of the transporters were predicted using the web-based protein topology tool Protter (5).

2. Identification of glyphosate-regulated promoters

2.1. Effect of glyphosate on the Bacillus subtilis transcriptome

3. A reporter-based glyphosate detection system

3.1. XXX

4. Competition assay for glyphosate detection

4.1. XXX

5. Engineering bacteria to disarm glyphosate

5.1. Overexpression of the glyphosate N-acetyltransferase in B. subtilis confers resistance to weedkiller

Many years ago it has been found that glyphosate can be inactivated by acetylation yielding N-acetylglyphosate (see Figure 9) (36). Because the acetylated form of glyphosate has low affinity for the active site of EPSP synthase, it is nonherbicidal. The glyphosate N-acetyltransferase GAT from the Gram-positive soil bacterium Bacillus licheniformis transfers the acetyl group from acetyl-CoA onto the amino group of the weedkiller (36). Interestingly, B. subtilis possesses a putative glyphosate N-acetyltransferase, which is designated as YitI and shares 59% overall amino acid identity with GAT of B. licheniformis. However, the native GAT enzymes are very poor catalysts for N-acetylation of glyphosate (36). Moreover, despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (Siehl et al., 2007 JBC). As an alternative strategy for glyphosate resistance involving enzymatic conversion of glyphosate to N-acetylglyphosate the GAT was also subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide (36). When introduced into plants, optimized gat genes confer robust tolerance to glyphosate (36). Therefore, we expect that bacteria like B. subtilis expressing the optimized GAT should tolerate high amounts of glyphosate even though it is taken up from the environment via the high-affinity glyphosate transporter GltT (see above). To evaluate the potential of the GAT to allow B. subtilis in the presence of glyphosate concentrations that are toxic for the wild type bacteria, we wanted to express the gat gene in B. subtilis (Figure X). For this purpose, we ordered the gat gene as a g-Block from Integrated DNA Technologies, which was free of charge because the company supports the iGEM competition. Next, we amplified the gat gene by PCR using the g-Block as template DNA. An artificial promoter and a ribosome binding site was attached during the PCR (Figure X). The promoter-gene fusion was cloned and introduced in single copy into the genomes of the B. subtilis wild type strain and the gltT mutant strain to further increase its glyphosate tolerance (Figure X). To assess the potential of the GAT to disarm glyphosate in the different B. subtilis strains, we propagated the bacteria on CS-Glc minimal medium agar plates that were supplemented with different amounts of glyphosate. As shown in Figure X, the strains synthesizing the GAT tolerated significantly more glyphosate than the wild type strain. Moreover, the inactivation of the gltT glyphosate transporter gene and the overexpression of the gat gene confers high-level glyphosate tolerance to the bacteria. Thus, when introduced into B. subtilis, the optimized gat gene confers indeed robust tolerance to glyphosate. To conclude, the engineered bacteria possess two interesting properties: (i) B. subtilis promotes plant growth by protecting plants against pathogens and (ii), the bacteria take up glyphosate from the environment and inactivate the weedkiller!

Figure X. A. Growth of the wild type and ΔgltT mutant and of the isogenic strains overexpressing the gat and aroA* genes on CS-Glc minimal medium in the presence of different amounts of glyphosate. B. Growth of the strains described in A on CS-Glc minimal medium in the presence of different amounts of Roundup® Alphee.

6. Characterization of EPSP synthases

6.1. Essentiality of the B. subtilis aroE EPSP synthase gene

To our surprise, we did not find glyphosate-resistant mutants of B. subtilis that had accumulated mutations in the aroE gene rendering the encoded EPSP synthase insensitive to the herbicide. Since AroE from B. subtilis belongs to the class II EPSP synthases, which are less sensitive to glyphosate, the potential of enzyme to further evolve herbicidal insensitivity might be limited (Figure X) (Barry et al., 1992; Schönbrunn et al., 2001; Light et al., 2016).

Figure X. The EPSP synthase fall into three different groups. While the E. coli EPSP synthase glyphosate sensitive (group I), the B. subtilis enzyme belongs to the group II of glyphosate-insensitive EPSP synthases. Not much is known about the glyphosate-dependent inhibition of the EPSP synthases that belong to group III. The phylogenetic three has been constructed using the Geneious software package (Biomatters Ltd.). Sequences of the EPSP synthases were derived from the UniProt database.

Moreover, experimental evidence suggests that the B. subtilis aroE gene is essential for growth even under conditions that do not require the enzymatic function of the EPSP synthase (Koo et al., 2017). As essential bacterial genes appear to be more conserved than nonessential genes, the aroE gene probably does not permit the accumulation of mutations, which can be beneficial (Jordan et al., 2002). To confirm that the aroE gene is indeed essential, we transformed the B. subtilis with a PCR product consisting of DNA fragments flanking the target gene and the intervening spc spectinomycin gene (aroE::spc deletion cassette) and propagated the bacteria on SP rich medium plates (see Notebook). Several tiny colonies appeared on the transformation plates after 24 h of incubation. However, the potential transformants were not viable and supplementation of the agar plates with casamino acids also did not improve growth of the bacteria. Thus, under the tested conditions aroE seems to be essential in B. subtilis. Next, we tested whether the chromosomal copy of the aroE gene becomes dispensable for the bacteria carrying an EPSP synthase gene on a plasmid. For this purpose, we transformed the B. subtilis wild type strain 168 with the plasmids pIGEM2 and pBP143 containing the aroE and aroA EPSP synthase genes from B. subtilis and E. coli, respectively. The wild type carrying the empty plasmid pBQ200 served as a control. Next, we transformed the three strains with the aroE::spc deletion cassette and with chromosomal DNA of strain BP233 (gltT::spc), of which the latter served as the positive control. While we did not get transformants without DNA, many transformants appeared with chromosomal DNA of strain BP233 (Figure X). Moreover, the chromosomal copy of the aroE gene was only dispensable in strains carrying an extra EPSP synthase gene on a plasmid. To conclude, the aroE EPSP synthase gene is essential in B. subtilis. Moreover, the bacteria seem to require the enzymatic function of the EPSP synthase because also the enzyme from E. coli permits growth of the aroE mutant (Figure X).

Figure X. The EPSP synthase is essential in B. subtilis. Transformation experiment to evaluate the essentiality of the EPSP synthase in B. subtilis. The bacteria were propagated on SP plates supplemented with spectinomycin and incubated for 24 h at 37°C.

6.2. Purification and characterization of EPSP synthases

This year, our team came toghether with the iGEM team of Marburg, to investigate the interaction of the EPSP synthases AroE and AroA from B. subtilis and E. coli, respectively, with glyphosate. Glyphosate targets AroE and AroA in B. subtilis and E. coli, respectively (see Background information). Since the members of the Bange lab, which is hosting the iGEM team of Marburg, are experts in the field of Isothermal titration calorimetry (ITC), we did not hesitate to contact them for support. Our team cloned the aroE and aroA genes B. subtilis and E. coli, respectively, and the resulting constructs are suitable for overexpression of the Strep-tagged AroE and AroA enzymes in the E. coli strain BL21. The N-terminally Strep-tagged proteins can be purified by Streptactin:Strep-tag affinity purification system from the IBA, Göttingen. The purified proteins were send to the iGEM team in Marburg (Figure X). The ITC measurments were performed in Marburg. Unfortunately, not interaction between glyphosate and the EPSP synthases could be detected. The experiments will be repeated using freshly purified proteins.

Figure X. (A) Evaluation of the purification of the N-terminally Strep-tagged EPSP synthases from B. subtilis and E. coli Strep-AroE and Strep-AroA, respectively, by 12% SDS PAGE. The proteins were stained with Coomassie Brilliant Blue. M, unstained protein molecular weight marker Thermo Scientific; CE, crude extract; FT, flow through, W, washing steps; E, elution steps. (B) ITC measurements with Strep-AroE and Strep-AroA and glyphosate. (C) Control experiments with water and the calcium chelator EDTA.

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