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 behaves 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 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 2A and 2B, 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 glyphosate (GS). (B) Growth of the B. subtilis wild type strain SP1 with increasing amounts of GS. The figure inlays show the relationship between the growth rate (μ) and the 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 (GS)-resistant mutants. (A) Emergence of 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.

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 (GS)-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 GS.

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 illustrated 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 7B). 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 (GS). (A) Growth of the B. subtilis wild type (WT) strain 168 in CS-Glc minimal medium supplemented with increasing amounts of GS. (B) Growth of the ΔgltT mutant strain BP233 in CS-Glc minimal medium supplemented with increasing amounts of GS. The figure inlay shows the relationship between the growth rate (µ) and the GS concentration. (C) Growth of the ΔgltP mutant strain BP234 in CS-Glc minimal medium supplemented with increasing amounts of GS. (D) Growth of the ΔgltT ΔgltP mutant strain BP235 in CS-Glc minimal medium supplemented with increasing amounts of GS. The figure inlay shows the relationship between the growth rate (µ) and the 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 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). (D) Killing two birds with one stone: the ΔgltT mutation confers resistance to glyphosate and glufosinate!

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, respectively; 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 illustrated 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

To test what effect glyphosate on the transcriptome of Bacillus subtilis has, we performed RNA-Seq with the help of the Göttingen Genomics Laboratory. This has been done for Escherichia coli in the past, but it has not yet been studied in Bacillus subtilis (58). For this purpose, we cultivated SP1 in CS-Glc minimal medium containing 0 mM glyphosate and 0.75 mM glyphosate.This concentration was chosen because it was previously shown that at a glyphosate concentration of about 1 mM the growth rate of SP1 is reduced by 50%. By choosing a lower concentration, we made sure that the bacteria were under stress, but still had sufficient growth rates.

Figure 10. Differential gene expression of SP1 in presence of glyphosate. A. Diagram shownig the fold change of every RNA in presence of glyphosate. The strongest regulated operon has been marked with yellow circles. B. Table containing genes underlying strong regulation when SP1 grows in the presence of glyphosate. Shown are the 4 genes belonging to the stongest downregulated operon (marked in A) and the most 4 upregulated genes.

Performing RNA-Seq allowed us to make predictions about the changes in gene expression of SP1 when in contact with glyphosate. As illustrated in Figure 10 A, only a few genes were subject to strong regulation. While a 5 fold increase in gene expression was the highest increase, we had an up to 11 fold decrease in gene expression. Nearly all upregulated genes were involved in sporulation, while the operon that showed the biggest fold change in expression is related to zinc deprivation responses (yciA-yciB-yczL-yciC) (59). Unexpectedly, we could not detect any major differential gene expression of genes encoding proteins involved in the shikimate and specific aromatic amino acid pathways.

The promoters involved in the regulation of the yciA-yciB-yczL-yciC operon were identified as PyciAB and PyciC (60)

3. A reporter-based glyphosate detection system

3.1 Utilising PyciAB and PyciC to build a reporter-based glyphosate detection system

Previously, it has been shown that the operon containing the genes yciA-yciB-yczL-yciC was the most differentially expressed operon when Bacillus subtilis was exposed to glyphosate. The promotors involved in the regulation of these genes were identified as PyciAB and PyciC. Based on this new data, we constructed a reporter based glyphosate detection system. For this purpose, we fused the identified promotors to the transcriptional repressor of the xylA-xylB and xynP-xynB operons xylR (Figure 11A). The Bacillus subtilis strain lacking this repressor was transformed with the plasmid pAC7 containing these contructs. Now, when the new strains come into contact with glyphosate, the expression of xylR will be downregulated, which will lead to the expression of xlyA and ultimately to the utilization of xlyose. This can be measured by performing a xylose assay using the colourimetric substrate 4-Nitrophenyl-β-D-xylopyranoside (PNPX). The measured β-xylosidase activity can be directly linked to the expression of xylR which depends on the activity of the effect of glyphosate of the promotor (Figure 11 B). The higher the β-xylosidase activity, the higher the amount of glyphosate in the medium.

Figure 11. A. Sequence of the promotores PyciAB and PyciC including the ribosome-binding site (RBS) were fused to the xylR gene by PCR. B. working scheme of the detection system with and without glyphosate

4. Competition assay for glyphosate detection

4.1. Competition assay with fluorophores

Unfortunately, current analytical methods, which are based on enzyme-linked immunosorbent assays (ELISAs) and mass spectrometry (MS) are very expansive, slow, need sophisticated equipment and well-trained technical staff. Therefore, the development of a rapid and straightforward method to detect glyphosate is very attractive because the intensive use of the herbicide may lead to its accumulation in the soil and food. The basic idea of the competition assay for the detection of glyphosate is depicted in figure X.

Figure X. The basic principle of the competition assay for glyphosate detection. In the absence of glyphosate (no selective pressure), both strains grow with the same rate. When glyphosate is present, the herbicide-resistant strain (expressing an orange fluorophore gene) will outcompete the wild type strain. Thus, the velocity at which the wild type strain is eliminated from the co-culture is indicative for the glyphosate present in the culture or test sample.

Two B. subtilis strains that tolerate different amounts of glyphosate are labelled with different fluorophore genes. The fluorescence labelling of the bacteria allows to count the amount of each strain when co-cultivated in the absence and in the presence of selective pressure that is exerted by glyphosate using fluorescence microscopy. In the absence of selective pressure, we expect that both strains will survive the co-cultivation. By contrast, in the presence of glyphosate, the strain that tolerates glyphosate will outcompete the glyphosate-sensitive B. subtiles strain. Thus, the selective pressure (≈ [glyphosate]) correlates with the amount of glyphosate-resistant cells and glyphosate-sensitive cells.

Figure X. Integration of the fluorophore genes behind an σA–dependent artificial promoter (Plij2) into the amyE-locus in the Bacillus genome.

Characterization of mOrange

The fluorophore genes derived from the DNA distribution kit from iGEM. Previous work using BBa_E2050 has only focused on its function in yeast. The aim of the experiments was to transform mERP into differentBacillus strains. Because the plasmid pSB1C3 does not contain an origin of replication for Bacillus, we cloned the fluorophore into the plasmid pAC7 and transformed it into competent DH5α. The fluorophore was additionally coupled to a self-made promoter (Plij2, Figure X), which is characterized by a perfect consensus sequence and perfect RBS for Bacillus.

Figure X: The wildtype strain 168 from Bacillus subtilis does not show orange color, while the tagged strains are orange colored.

The E. coli strain has an increased transformation efficiency, where plasmid insertion is enhanced due to mutations in recA1 and endA1. pAC7 is a vector for the construction of translational lacZ fusions that can be integrated at the amyE-locus. Transformation with pAC7 allows direct integration of the fluorophore into the genome of Bacillus. In a further step, mERP was transformed into WT Bacillus subtilis strain 168, BP233 (trpC2 ΔgltT::spc) and BP235 (trpC2 ΔgltT::spc ΔgltP::cat), which are characterized by a resistance towards the herbicide glyphosate.

Figure X: On the left side are cells from strain iGEM24 (mOrange-tagged WT) in bright field microscopy. Mag:40x. Exp: 0.85 s. Filter: none. On the right side are cells from strain iGEM24 in fluorescence microscopy. Mag: 40x. Exp: 44.33 s. Filter: DAPI.

Activity of the fluorophore in the WT 168 was further analysed with fluorescence microscopy. Therefore, strains were incubated overnight at 37°C with agitation in darkness. 10 mL of the culture was transferred on microscope slides with solid 1% agarose in H2O to lock the cells in place for long exposure times.

To create a powerful glyphosate detection system, which is based on intraspecies competition, we need to have strains with similar fitness . Strains having a different fitness will not allow to generate a stable co-culture because the strain with the higher fitness will alway outcompete the other strain, even in the absence of a selective pressure. To characterize our strains and to determine their fitness we performed different growth experiments. First, we integrated fluorophore genes into the amyE gene of the B. subtilis strains 168, (wild type), ΔgltT::spc (BP233) and ΔgltT::spc ΔgltP::cat (BP235) (Figure X). The wild type strain 168 is very sensitive to glyphosate. By contrast, the ΔgltT single mutant and ΔgltT ΔgltP double mutant lacking the glyphosate transport systems tolerate high amounts of the herbicide (see above). We used the gfp, bfp and morange genes encoding the fluorophores GFP, BFP and mOrange, respectively, for strain labeling. To drive the expression of the fluorophore genes, we attached an artificial promoter to the genes during PCR (see Fig. X). The sigma factor A-dependent artificial promoter is constitutively active and contains a ribosome binding site for B. subtilis. Next, we cultivated the constructed strains in a multi-well plate reader, to determine their fitness based on the growth rates that are achieved in the medium.

Figure X. Growth of strains: 168: wildtype. iGEM24: mOrange-tagged wildtype (trpC2 amyE::(mOrange-lacZ aphA3)). iGEM28: mOrange-tagged ΔgltT mutant (trpC2 ΔgltT::spc amyE::(mOrange-lacZ aphA3)). iGEM36: mOrange-tagged ΔgltT ΔgltP mutant (trpC2 ΔgltT::spc ΔgltP::cat amyE::(mOrange-lacZ aphA3)). All strains are tagged with mOrange and show the same growth behavior. On the abscissa is the time in [h] given and on the ordinate the optical density at 600 nm (OD600).

As shown in Fig. X, all strains have similar growth rates in CS-Glc minimal medium. Thus, the expression of the mOrange gene and the absence of the glyphosate transporters GltT and GltP does not affect the fitness of the strains!

To detect glyphosate by intraspecies competition, we mixed the wild type strain synthesizing the GFP protein with the ΔgltT ΔgltP double mutant synthesizing the mOrange fluorescence protein in a 1:1 ratio. As shown in Fig. X, the amount of wild type cells decreased in the co-culture with increasing glyphosate concentrations.

By contrast, the amount of cells of the strain carrying the mOrange gene increased with increasing glyphosate concentrations since the ΔgltT ΔgltP mutant tolerates the herbicide glyphosate. Thus, the intraspecies competition assay, which is based on fluorescently labelled B. subtilis strains seems to be suitable to detect glyphosate!

Figure X: Green cells represent the wildtype strain (trpC2 amyE::(morange-lacZ aphA3)) and orange cells the double transporter mutant (trpC2 ΔgltT::spc ΔgltP::cat amyE::(morange-lacZ aphA3)). The ratio indicates the relative amount of orange cells in comparison to green cells.
A: Merged channels orange and green from culture with 0 mM glyphosate after 4 h. There are nearly equal amounts of green and orange cells.
B: Merged channels orange and green from culture with 2 mM glyphosate after 4 h. The number of orange cells is 4.21-fold higher.
C: Merged channels orange and green from culture with 3.5 mM glyphosate after 4 h. Nearly no green cells are visible.
D: Calculated ratio between the number of orange and green cells with increasing glyphosate concentrations.

The number of green cells in comparison to the orange cells decreases with increasing glyphosate concentrations.

To perform a competition assay in a greater scale, we used a platereader for our growth experiments. This device enabled us to measure different fluorophores and the optical density simultaneously. Strains were cultivated in CS-glucose (CS-Glu) medium to an OD600 of 1–1.5. A 96–well plate was prepared containing 100 µL of CS-Glu medium with the desired concentrations of glyphosate. 10 µL of the cell mixture (1:1 ratio) were transferred into the wells to obtain an OD600 of 0.1 in the wells. We used the SynergyMx by BioTek for our measurements. GFP was excited at 488 nm and emission was measured at 509 nm. for mOrange, the excitation wavelength was 529 nm and emission was detected at 562 nm.

Figure X:The fluorescence is normed to the optical density, which equals the cell number. A: With increasing glyphosate concentrations (0–2 mM glyphosate) decreases the GFP fluorescence. B: With increasing glyphosate concentrations (0–2 mM glyphosate) is the fluorescence of mOrange also increased.

This competition assay enables us to calculate the concentration of glyphosate in samples from people that are curious, how much glyphosate is in their soil.

4.2. Competition with β–galactosidase expressing strains

Another approach using competition assays is to use enzymatic indication systems. Therefore, we used the strain BP235 (ΔgltT ΔgltP) and the strain BP193 (palf4::lacZ) (61). As previously described, BP235 is characterized by high-level glyphosate resistance, while BP193 resembles the wildtype despite the lacZ gene. The promoter palf4 is an artificial one and harbors a perfect consensus sequence for Bacillus subtilis and a ribosome binding site. This leads to constitutive expression of the lacZ gene and to blue cells if the medium is supplemented with X-Gal. Cultures of both strains in CS-Glu medium without glyphosate were mixed to obtain a 1:1 ratio of both strains and incubated with increasing glyphosate concentrations (0–5 mM) for 24 h (Figure X).

Figure X: Cell mixture (1:1) of strains BP193 and BP235 were incubated with increasing glyphosate concentrations (0–5 mM). Strain BP193 harbors the lacZ gene behind an artificial promoter, but does not tolerate glyphosate in contrast to strain BP235. The intensity of the blue color decreases with increasing glyphosate concentration. Strains BP193 and BP235 were also incubated without glyphosate as a control.

The intensity of the blue color drecreases with increasing glyphosate concentrations (Figure X). The change and intensity of the blue color correlates with the glyphosate concentration and can therefore be used as detection and quantification system. Furthermore, a β–galactosidase assay can quantify the activity of the promoter palf4 and thus, the concentration of glyphosate.

The promoter-reporter gene fusion of palf4 and lacZ allows to determine the activity of the promoter. Since palf4 is constitutively active this is directly linked to the survival of the cells. Transcription results in synthesis of ß-galactosidase which in turn hydrolyses o-nitrophenyl-ß-D-galactopyranoside (ONPG), resulting in the formation of o-nitrophenol. Formed o-nitrophenol absorbs light with a wavelength of 420 nm, and can therefore be used as an indicator for the amount of cells and subsequently for the concentration of glyphosate. We determined ß-galactosidase activity for the mixture of BP193 and BP235 in presence of increasing amounts of glyphosate. In addition, we used BP193 and BP235 as controls. Cells were grown in a 1:1 ratio. ß-galactosidase activity was high in the BP193 control and at low glyphosate concentration. Increasing glyphosate concentration is directly linked to cell death of BP193 and a corresponding decrease in ß-galactosidase activity (Figure Y).

Figure X: Cell mixture (1:1) of strains BP193 and BP235 were incubated with increasing glyphosate concentrations (0–5 mM). Strain BP193 harbors the lacZ gene behind an artificial promoter, but does not tolerate glyphosate in contrast to strain BP235. ß-galactosidase activity can be measured at a wavelength of 420 nm. Activity decreases with increasing glyphosate concentration. BP235 does not contain the lacZ gene, hence no activity can be observed.

In an additional step, we verified that the change in coloration occurred due to the death of the sensitive strain on the one hand and the increased growth of the resistant BP235 strain on the other hand. Hence, we adopted the experimental set up and analysed growth of the two strains in the platereader. To analyse growth of BP235, we used the strain constitutively expressing GFP, which was constructed similarly to the previously used strains. For the analysis we again used 100 µL CS-Glu medium supplemented with the appropriate glyphosate concentration. The cells were added in a 1:1 ratio. GFP was excited at 488 nm and emission was measured at 509 nm.

Figure X: Cell mixture (1:1) of strains BP193 and BP235 were incubated with increasing glyphosate concentrations (0–5 mM). Strain BP193 harbors the lacZ gene behind an artificial promoter, but does not tolerate glyphosate in contrast to strain BP235.

5. Engineering bacteria to disarm glyphosate

5.1. The glyphosate N-acetyltransferase 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).

Figure X. A. Sequence alignment showing the similarities of the wild type (wt) gat alleles from Bacillus licheniformis and Bacillus subtilis as well as the gat alleles R7 and R11 from B. licheniformis that were obtained by 11 rounds of gene shuffling. The red arrows indicate the 31 amino acid exchanges in the glyphosate N-acetyltransferase (Gat) variant R11. The optimized Gat variants shown an up to a 4,500-fold increase in catalytic efficiency (kcat/Km) relative to the native enzyme. The GenBank accession numbers for the sequences: B. licheniformis ST401 GAT, AX543338; R7 GAT, AY597417; R11 GAT, AY597418; B. subtilis YitI, CAA70664. B. Structure of the Gat R7 variant (PDBid: 2JDD) in ternary complex with acetyl-CoA and the competitive inhibitor 3-phosphoglycerate (3PG). The four active site residues (Arg-21, Arg-73, Arg-111, and His-138), which are labelled in black color, contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. C. Top view on the active site of Gat R7. D. Construction of a plasmid for the expression of the gat R11 variant in B. subtilis. Expression is driven by the constitutively active σA-dependent Palf4 promoter The plasmid pAC7::gat R11 integrates into the amyE locus of the B. subtilis chromosome in single copy. lacZ, beta-galactosidase; amyE-5' and amyE-3', fragments of the B. subtilis amyE for homologous recombination; amp, ampicillin resistance gene for the selection in E. coli; kan, kanamycin resistance gene for the selection in B. subtilis; ori pBR322, origin of replication for E. coli.

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 R11 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.

Cultivation of the different strains in liquid media allows more detailed characterisation. We analysed growth of the bacteria in CS-Glu medium supplemented with increasing amounts of glyphosate, using a platereader.
In general, the analysis supports previous results. The strain containing aroA* confers only a slight increase in resistance in comparison to the WT strain and hence a significantly lower resistance in comparison the ∆gltT strain (Figure X A, B and D ). However, for the strain combining aroA* and ∆gltT a 9-fold higher glyphosate concentration is needed to reduce the growth rate by 50% (Figure X F. Figure Y ).
Further characterisation of the Gat enzyme supports previous results and underline significant increased resistance in both strains conferring gat or a combination of gat and ∆gltT (Figure X C and E, Figure Y).

Figure X. A. Growth of the B. subtilis wild type strain in presence of 0 - 3.5 mM Glyphosate. B. Growth of the ∆gltT B. subtilis strain with increasing glyphosate concentration (0-30 mM). C. Growth of the gat B. subtilis strain with increasing glyphosate concentration (0-30 mM. D.Growth of the aroA* B. subtilis strain with increasing glyphosate concentration (0-30 mM E. Growth of the aroA* B. subtilis strain with increasing glyphosate concentration (0-30 mM). Growth of the B. subtilis strain containing both ∆gltT and gat with increasing glyphosate concentration (0-30 mM). F. Growth of the B. subtilis strain containing both ∆gltT and aoA* with increasing glyphosate concentration (0-30 mM). Bacteria were cultivated in liquid Cs-Glu medium.

Figure Y. The relationship between the growth rate (µ) and the glyphosate (GS) concentration for the B. subtilis wild type (WT) strain SP1, the ΔgltT mutant strain BP233, the gat B. subtilis strain, the aroA* B. subtilis strain, the B. subtilis strain containing both ∆gltT and aroA* and the the B. subtilis strain containing both ∆gltT and gat in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS).

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. Unfortunately, also the second ITC measurement using freshly purified AroA and AroE enzymes from E. coli and B. subtilis, respectively, did not reveal whether glyphosate may interact with the EPSP synthases. Nevertheless, we are grateful to the iGEM Team Marburg for performing the ITC measurements.

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.

References

  1. Fischer et al. (1986) J. Bacteriol. 168: 1147-1154
  2. Zeigler et al. (2008) J. Bacteriol. 190: 6983-6995.
  3. Barbe et al. (2009) Microbiology 155: 1758-1775
  4. Kearse et al. (2012) Bioinformatics 28: 1647-1649.
  5. Omasits et al. (2014) Bioinformatics. 30: 884-886.
  6. Michna et al. (2014) Nucleic Acids Res. 42: D692–D698.
  7. Zaprasis et al. (2015) Appl. Environ. Microbiol. 81: 250-259.
  8. Tolner et al. (1995) J. Bacteriol. 177: 2863-2869.
  9. Amrhein et al. (1980) Naturwissenschaften 67: 356-357.
  10. Comai et al. (1983) Science 221: 370-371.
  11. Stalker et al. (1985) J. Biol. Chem. 260: 4724-4728.
  12. Gundlach et al. (2017) Sci. Signal. 10: eaal3011.
  13. Priestmann et al., (2005) FEBS Lett. 579: 728-732.
  14. Cao et al. (2012) PLoS One. 7: e38718.
  15. Light et al. (2016) Biochemistry 55: 1239-1245.
  16. Liu & Cao, (2018) Biotechnol Lett. 40: 855-864.
  17. Rogers et al. (1983) Appl. Envrion. Microbiol. 46: 37-43.
  18. Gaines et al. (2011) J Agric Food Chem. 59: 5886-5889.
  19. Juglam et al. (2014) Plant Physiol. 166: 1200-1207.
  20. Sammons & Gaines (2014) Pest. Manag. Sci. 70: 1367-1377.
  21. Dillon et al. (2017) Plant Physiol. 173: 1226-1234.
  22. Sost & Amrhein (1990) Arch. Biochem. Biophys. 282: 433-436.
  23. Padgette et al. (1991) J. Biol. Chem. 266: 22364-22369.
  24. He et al. (2001) Biochim. Biophys. Acta 1568: 1-6.
  25. He et al. (2003) Biosci. Biotechnol. Biochem. 67: 1405-1409.
  26. Bearson et al. (2002) Plant Physiol. 129: 1265-1275.
  27. Eschenburg et al. (2002) Planta 216: 129-135.
  28. Sun et al. (2005) Appl. Envrion. Microbiol. 71: 4771-4776.
  29. Zhou et al. (2006) Plant Physiol. 140: 184-195.
  30. Healy-Fried et al. (2007) J. Biol. Chem. 282: 32949-32955.
  31. Vande Berg et al. (2008) Pest. Manag. Sci. 64: 340-345.
  32. Funke et al. (2009) J. Biol. Chem. 284: 9854-9860.
  33. Tian et al. (2010) Appl. Environ. Microbiol. 76: 6001-6005.
  34. Pollegioni et al. (2011) FEBS J. 278: 2753-2766.
  35. Chekan et al. (2016) MedChemComm. 7: 28-36.
  36. Castle et al. (2004) Science 304: 1151-1154.
  37. Siehl et al. (2005) Pest. Manag. Sci. 61: 235-240.
  38. Siehl et al. (2007) J. Biol. Chem. 282: 11446-11455.
  39. Norris et al. (2009) Appl. Environ. Microbiol. 75: 6062-6075.
  40. Rao et al. (1983) Antimicrob. Agents Chemother. 24: 689-695.
  41. Penaloza-Vazquez et al. (1995) Appl. Environ. Microbiol. 61: 538-543.
  42. Liu et al. (2015) Transgenic Res. 24: 753-763.
  43. Staub et al. (2012) J. Ind. Microbiol. Biotechnol. 39: 641-647.
  44. Tao et al. (2017) Pestic. Biochem. Physiol. 140: 65-68.
  45. Fitzgibbon & Braymer, (1988) Appl. Environ. Microbiol. 54: 1886-1888.
  46. Fitzgibbon & Braymer, (1990) Appl. Environ. Microbiol. 56: 3382-3388.
  47. Fartyal et al. (2018) Front. Plant. Sci. 13: 144.
  48. Pipke et al. (1980) Appl. Environ. Microbiol. 53: 974-978.
  49. Shinabarger et al. (1986) J. Bacteriol. 168: 702-707.
  50. Liu et al. (1991) Appl. Environ. Microbiol. 57: 1799-1804.
  51. Dick & Quinn, (1995) Appl. Microbiol. Biotechnol. 43: 545-550.
  52. Singh & Walker, (2006) FEMS Microbiol. Rev. 30: 428-471.
  53. Castro et al. (2007) J. Environ. Sci. Health B. 42: 883-886.
  54. Hove-Jensen et al. (2014) Microbiol. Mol. Biol. Rev. 78: 176-197.
  55. Kryuchkova et al. (2014) Microbiol. Res. 169: 99-105.
  56. Sviridov et al., (2011) Biochemistry (Mosc). 76: 720-725.
  57. Sviridov et al. (2015) Prikl. Biokhim. Mikrobiol. 51: 183-190.
  58. Lu et al. (2013) Mol Biosyst. 9: 522–30.
  59. Gaballa & Helmann (1998) J Bacteriol. 180: 5815–5821.
  60. Gaballa et al. (2002) J Bacteriol. 184: 6508–6514.
  61. Gundlach et al. (2017) Science Signaling 10: eaal3011