EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

We found that cells from Mycobacterium smegmatis , a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l- tryptophan, l -phenylalanine, and l -tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs.

Phylogenetic Occurrence of the Phenylpropanoid Pathway and Lignin Biosynthesis in Plants

The phenylpropanoid pathway serves as a rich source of metabolites in plants and provides precursors for lignin biosynthesis. Lignin first appeared in tracheophytes and has been hypothesized to have played pivotal roles in land plant colonization. In this review, we summarize recent progress in defining the lignin biosynthetic pathway in lycophytes, monilophytes, gymnosperms, and angiosperms. In particular, we review the key structural genes involved in p-hydroxyphenyl-, guaiacyl-, and syringyl-lignin biosynthesis across plant taxa and consider and integrate new insights on major transcription factors, such as NACs and MYBs. We also review insight regarding a new transcriptional regulator, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, canonically identified as a key enzyme in the shikimate pathway. We use several case studies, including EPSP synthase, to illustrate the evolution processes of gene duplication and neo-functionalization in lignin biosynthesis. This review provides new insights into the genetic engineering of the lignin biosynthetic pathway to overcome biomass recalcitrance in bioenergy crops.

Detection of Genetically Modified Protein in Soy-containing Foods

The use of genetically modified organisms (GMO) in food is a highly public and controversial issue. We have used an enzyme-linked immunosorbent assay (ELISA) to detect the genetically modified protein 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase within soy-containing food products from the grocery store. We found that EPSP synthase is detectable in 3 out of 5 food products tested. Of specific interest, we found contamination levels of EPSP synthase (0.36%) in Heinz' Pablum Soya Cereal, which is currently deemed to be free of genetic modifications by the company as well as by Greenpeace Canada. These results demonstrate that genetically modified organisms are present in foods commonly available for human consumption and that the widespread use of this technology may make it difficult to ensure that any given product is free of all traces of genetically modified protein.

Tillage, Glyphosate and Beneficial Arbuscular Mycorrhizal Fungi: Optimising Crop Management for Plant–Fungal Symbiosis

Zero till cropping systems typically apply broad-spectrum herbicides such as glyphosate as an alternative weed control strategy to the physical inversion of the soil provided by cultivation. Glyphosate targets 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in plants. There is growing evidence that this may have a detrimental impact on non-target organisms such as those present in the soil microbiome. Species of commercial importance, such as arbuscular mycorrhizal (AM) fungi that form a symbiotic relationship with plant roots are an important example. This study investigates the impact of soil cultivation and glyphosate application associated with conventional tillage (CT) and zero tillage (ZT) respectively on AM fungi populations under field and glasshouse conditions. Topsoil (<10 cm) was extracted from CT and ZT fields cropped with winter wheat, plus non-cropped control plots within the same field boundary, throughout the cropping year. Glyphosate was applied in glasshouse experiments at rates between 0 and 350 g L−1. Ergosterol, an indicator of fungal biomass, was measured using high performance liquid chromatography before and after glyphosate application. Fungal root arbuscules, an indicator of AM fungi–root symbiosis, were quantified from the roots of wheat plants. Under glasshouse conditions root arbuscules were consistently higher in wheat grown in ZT field extracted soils (P = 0.01) compared to CT. Glyphosate application however inhibited fungal biomass in both the ZT (P < 0.00001) and CT (P < 0.001) treatments. In the absence of glyphosate, the number of stained root arbuscules increased significantly. Ergosterol levels, used as a proxy for fungal biomass, remained lower in the soil post glyphosate application. The results suggest that CT has a greater negative impact on AM fungal growth than ZT and glyphosate, but that glyphosate is also detrimental to AM fungal growth and hinders subsequent population recovery.

Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase

ABSTRACTGlyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (EPSPS) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in autotrophs. Our results reveal that bacteria easily become resistant to glyphosate through changes in the EPSPS active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes.

Evolution of target and non-target based multiple herbicide resistance in a single Palmer amaranth (Amaranthus palmeri) population from Kansas

The evolution of resistance to multiple herbicides in Palmer amaranth is a major challenge for its management. In this study, a Palmer amaranth population from Hutchinson, Kansas (HMR), was characterized for resistance to inhibitors of photosystem II (PSII) (e.g., atrazine), acetolactate synthase (ALS) (e.g., chlorsulfuron), and EPSP synthase (EPSPS) (e.g., glyphosate), and this resistance was investigated. About 100 HMR plants were treated with field-recommended doses (1×) of atrazine, chlorsulfuron, and glyphosate, separately along with Hutchinson multiple-herbicide (atrazine, chlorsulfuron, and glyphosate)–susceptible (HMS) Palmer amaranth as control. The mechanism of resistance to these herbicides was investigated by sequencing or amplifying the psbA, ALS, and EPSPS genes, the molecular targets of atrazine, chlorsulfuron, and glyphosate, respectively. Fifty-two percent of plants survived a 1× (2,240 g ai ha−1) atrazine application with no known psbA gene mutation, indicating the predominance of a non–target site resistance mechanism to this herbicide. Forty-two percent of plants survived a 1× (18 g ai ha−1) dose of chlorsulfuron with proline197serine, proline197threonine, proline197alanine, and proline197asparagine, or tryptophan574leucine mutations in the ALS gene. About 40% of the plants survived a 1× (840 g ae ha−1) dose of glyphosate with no known mutations in the EPSPS gene. Quantitative PCR results revealed increased EPSPS copy number (50 to 140) as the mechanism of glyphosate resistance in the survivors. The most important finding of this study was the evolution of resistance to at least two sites of action (SOAs) (~50% of plants) and to all three herbicides due to target site as well as non–target site mechanisms. The high incidence of individual plants with resistance to multiple SOAs poses a challenge for effective management of this weed.

Homogeneity among glyphosate-resistant Amaranthus palmeri in geographically distant locations

Since the initial report of glyphosate-resistant (GR) Amaranthus palmeri (S) Wats. in 2006, resistant populations have been reported in 28 states. The mechanism of resistance is amplification of a 399-kb extrachromosomal circular DNA, called the EPSPS replicon, and is unique to glyphosate-resistant plants. The replicon contains a single copy of the 10-kb 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which causes the concomitant increased expression of EPSP synthase, the target enzyme of glyphosate. It is not known whether the resistance by this amplification mechanism evolved once and then spread across the country or evolved independently in several locations. To compare genomic representation and variation across the EPSPS replicon, whole genome shotgun sequencing (WGS) and mapping of sequences from both GR and susceptible (GS) biotypes to the replicon consensus sequence was performed. Sampling of GR biotypes from AZ, KS, GA, MD and DE and GS biotypes from AZ, KS and GA revealed complete contiguity and deep representation with sequences from GR plants, but lack of homogeneity and contiguity with breaks in coverage were observed with sequences from GS biotypes. The high sequence conservation among GR biotypes with very few polymorphisms which were widely distributed across the USA further supports the hypothesis that glyphosate resistance most likely originated from a single population. We show that the replicon from different populations was unique to GR plants and had similar levels of amplification.

Evaluating aroA gene essentiality and EPSP synthase vulnerability in Mycobacterium smegmatis under different nutritional conditions

The epidemiological importance of bacteria from the genus Mycobacterium is indisputable and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important metabolites in bacteria, represents a target for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene knockout, gene knockdown and kinetic assays to evaluate aroA gene essentiality and the vulnerability of its protein product, EPSPS synthase from Mycobacterium smegmatis (MsEPSPS), under different nutritional conditions. We demonstrate by an allelic exchange-based gene knockout approach the essentiality of MsEPSPS under rich and poor nutritional conditions. By performing gene complementation experiments with wild-type (WT) and point mutant versions of aroA gene, together with kinetic assays using WT and mutant recombinant proteins, we show that aroA gene essentiality depends on MsEPSPS activity. To evaluate MsEPSPS vulnerability, we performed gene knockdown experiments using the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system. The experiments were performed in both rich and defined (poor) media, using three different repression forces for aroA gene. We only observed growth impairment when bacteria were grown in defined medium without supplementation of aromatic amino acids, thereby indicating that MsEPSPS vulnerability depends on the environment conditions.ImportanceWe evaluated both gene essentiality and target vulnerability of the enzyme that catalyzes the sixth step of the shikimate pathway, the aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium smegmatis (MsEPSPS). Combining gene knockout experiments and kinetic assays, we established a causal link between aroA gene essentiality and the biological function of EPSPS protein, which we advocate is an indispensable step for target validation. Moreover, we characterized MsEPSPS vulnerability under different nutritional conditions and found it is a vulnerable target only when M. smegmatis is grown under poor nutritional conditions without supplementation with aromatic amino acids. Based on our findings, we suggest that gene essentiality information should be obtained from gene knockout experiments and not knockdown approaches, as even low levels of a protein after gene silencing can lead to a different growth phenotype when compared to that under its complete absence, as was the case with aroA and MsEPSPS in our study.

Investigation of the target-site resistance of EPSP synthase mutants P106T and T102I/P106S against glyphosate

Single or double EPSP synthase mutations lead glyphosate to undergo conformational changes that limit its inhibitory action.