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Revealing novel protein interaction partners of glyphosate in Escherichia coli.

Despite all debates about its safe use, glyphosate remains the most widely applied active ingredient in herbicide products, with renewed approval in the European Union until 2033. Non-target organisms are commonly exposed to glyphosate as a matter of its mode of application, with its broader environmental and biological impacts remaining under investigation. Glyphosate displays structural similarity to phosphoenolpyruvate (PEP), thereby competitively inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), crucial for the synthesis of aromatic amino acids in plants, fungi, bacteria, and archaea. Most microbes, including the gut bacterium Escherichia coli (E. coli), possess a glyphosate-sensitive class I EPSPS, making them vulnerable to glyphosate's effects. Yet, little is known about glyphosate's interactions with other bacterial proteins or its broader modes of action at the proteome level. Here, we employed a quantitative proteomics and thermal proteome profiling (TPP) approach to identify novel protein binding partners of glyphosate in the E. coli proteome. Glyphosate exposure significantly altered amino acid synthesizing pathways. The abundance of shikimate pathway proteins was increased, suggesting a compensatory mechanism. Extracellular riboflavin concentrations were elevated upon glyphosate exposure, while intracellular levels remained stable. Beyond the target enzyme EPSPS, thermal proteome profiling indicated an effect of glyphosate on the thermal stability of certain proteins, including AroH and ProA, indicating interactions. Similar to the competitive binding between PEP and glyphosate at EPSPS, one reason for the interaction of AroH and ProA with the herbicide could be a high structural similarity between their substrates and glyphosate. Overall, glyphosate induced metabolic disturbances in E. coli, extending beyond its primary target, thereby providing new insights into glyphosate's broader impact on microbial systems.

Glyphosate

Simultaneous detection of glyphosate and glufosinate target-site resistance in Eleusine indica via multiplex TaqMan qPCR.

BACKGROUND: Continuous use of glyphosate followed by glufosinate-ammonium has selected for multiple resistance to both herbicides in Eleusine indica worldwide. Managing such resistant weeds requires fast, accurate molecular detection assay. To address this critical need, we developed a robust multiplex TaqMan quantitative (q)PCR assay that simultaneously detects five well-characterized target-site resistance markers in E. indica: EPSPS copy number variation; T102I in EPSPS; P106A and P106S in EPSPS; and S59G in GS1-1. RESULTS: The multiplex qPCR assay showed analytical specificity when tested on genomic DNA from nine reference accessions: three susceptible, three glyphosate-resistant (with EPSPS CNV) and three multiple-resistant. Subsequent analysis of 56 field-collected samples demonstrated 98.2% concordance (55 of 56) with Sanger sequencing across all five resistance-associated markers: EPSPS CNV, T102I, P106A, P106S and GS1-1 S59G, confirming the reliability and practical value of the multiplex qPCR assay. Only samples 7-8 showed discordance at EPSPS position 102, where Sanger chromatograms showed overlapping peaks at this position, which is likely to be a result of heterozygous mutation distribution among amplified EPSPS gene copies. This case further underscores the advantages of the multiplex qPCR assay over Sanger sequencing in detection sensitivity and accuracy. Moreover, a strong correlation (R2 = 0.8935) in gene copy number estimation between the two methods across all samples further supports the reliability of the qPCR assay. CONCLUSIONS: In summary, this study delivers a simple, robust and high-throughput diagnostic tool for the rapid, simultaneous identification of dual herbicide target-site resistance in goosegrass, offering superior sensitivity, quantitative resolution and throughput compared with Sanger sequencing. © 2026 Society of Chemical Industry.

Herbicides

Kinetics of nitrosation of the herbicide glyphosate.

The herbicide glyphosate was nitrosated by third-order kinetics to N-nitrosoglyphosate. The nitrosation at 25 degrees C was maximum at the reaction pH of 2.5 and had a pH-dependent rate constant of 2.43 M-2 sec-1. An activation energy of 9.5 kcal mole-1 also suggested that glyphosate is nitrosated very readily. Thiocyanate increased the rate 4.6 fold. The possibility of using these results to predict the formation of N-nitrosoglyphosate under normal agricultural practice is discussed.

Chemical Phenomena

Ectopic recombination: a novel mechanism of EPSPS gene amplification in glyphosate-resistant Chloris truncata.

Amplification of 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene confers resistance to the herbicide glyphosate in the tetraploid Chloris truncata in Australia. To study the mechanism of amplification, the genomic organization of the EPSPS gene was investigated using fluorescence in situ hybridization (FISH) in one susceptible (Ct-S) and two resistant (Ct-R1 and Ct-R2) biotypes of C. truncata. FISH analysis revealed faint signals of the EPSPS gene on the telomeric regions of a single pair of homologous chromosomes in Ct-S plants. However, much brighter hybridization signals of the EPSPS gene were detected on three pairs of homologous chromosomes in Ct-R1 and on four pairs in the Ct-R2 plants. Thus, there was gene amplification on the native EPSPS locus as well as spread of EPSPS loci to additional chromosomes. All loci were detected in terminal regions which are hotspots of recombination. This local as well as ectopic EPSPS amplification to specific regions of chromosomes is a novel mechanism resistance to herbicides. We hypothesize that, during the bouquet stage of meiosis, telomeres come together forming a bouquet and this may provide an opportunity for ectopic recombination, supported by FISH analyses in interphase nuclei. Overall, the gene amplification appears to have occurred in two steps. First, there was tandem EPSPS amplification at the native locus, possibly via unequal recombination. Second, the amplified locus underwent ectopic recombination and spread to two additional chromosomes in Ct-R1 and three additional chromosomes in Ct-R2 plants.

Glyphosate

An acute dose of glyphosate alters novel object exploration and hippocampal cFos expression in a sex-dependent manner in wildtype mice.

Glyphosate (GLY) is the active ingredient in most herbicides, including off-the-shelf weed killers such as Roundup®. GLY crosses the blood-brain barrier, increases oxidative stress and genotoxicity, and impacts reproduction, but the extent of its effects remains unclear. Previous research reports conflicting evidence on sex-specific susceptibility to GLY's effects, and very few investigate the effects of a single, acute dose on learning, memory, and neuronal activation. In vitro studies have found GLY interferes with gene expression and is uniquely capable of inducing DNA double strand breaks (DSBs) compared to other herbicides. DSBs can induce expression of immediate early genes (IEGs), which are important for synaptic plasticity, learning, and memory. However, a clear connection between GLY, IEGs, and learning and memory has yet to be made. To explore this, we tested male and female wildtype mice in novel object recognition after they received an acute, oral dose of 0, 250, or 500 mg/kg of GLY and assessed hippocampal DSB and IEG levels. We hypothesized that a single dose of GLY would impair memory by disrupting IEG expression and would affect males more than females. We did not find robust evidence that GLY impaired memory, though females that received 500 mg/kg did not explore the novel object more than the familiar. Hippocampal DSBs were decreased following 500 mg/kg in both sexes, yet hippocampal IEG immunoreactivity was decreased in GLY-exposed males only, revealing a complex sex-dependent relationship. These data add to the literature that GLY is potentially detrimental, highlighting the need for further investigations.

Animals

Retraction of the landmark glyphosate safety publication by Williams, Kroes and Munro (2000) should be reversed.

The decision by the co-Editor-in-Chief of Regulatory Toxicology and Pharmacology, Prof. Martin van den Berg, to retract the 2000 review article by Williams, Kroes, and Munro has elicited widespread criticism within the scientific community. Issued in late 2025, the retraction decision cites procedural concerns including potential ghostwriting, undisclosed conflicts of interest, and omission of certain unpublished studies, invoking Committee on Publication Ethics guidelines despite lacking evidence of fraud or scientific flaws. This editorial argues that the retraction decision involves editorial overreach and misapplication of the guidelines. The alleged omissions stemmed from proprietary data access limitations that were disclosed in the original paper. Subsequent reviews by several independent expert panels and regulatory authorities with access to all glyphosate data, including the studies cited by the retracting editor, reached similar conclusions. Claims of ghostwriting were previously investigated and found lacking, including a declaration by EFSA as to the clarity of the conflict disclosures. The retraction's timing, reliance on litigation documents, and apparent biases that were not disclosed in the retraction notice raise questions of ideological interference. Absent substantive rebuttals based on scientific merit rather than speculative claims of inappropriate authorship and data access, this retraction decision sets a dangerous precedent for retroactive censorship, potentially chilling beneficial industry-academic collaborations and eroding trust in the integrity of scientific publishing. With the strongest conviction, we assert that retracting a paper without scientific flaws isn't protection-it is censorship. We therefore call for the immediate reversal of this flawed and unjustified retraction to preserve trust in peer-reviewed literature.

COPE

Influence of pesticides on the growth of cyanobacteria.

Two unicellular and two filamentous cyanobacteria (blue-green algae) were exposed under conditions optimal for photoautotrophic growth to eleven pesticides. Low concentrations (0.01 to 5 ppm) of diuron, atrazine, and paraquat inhibited growth. With MCPA, MCPP, 2,4-D, milstem and ethrel, marked inhibitory effects were achieved only at concentrations above 100 ppm. Growth was inhibited by glyphosate, DDT, and thiram at intermediate concentrations. In some cases, the effective concentration of the pesticide varied considerably with the organism tested.

Cyanobacteria

Complete genome sequences of three effective nitrogen-fixing strains of Bradyrhizobium ottawaense from Canada.

We report complete genome sequences of three nitrogen-fixing Bradyrhizobium ottawaense strains isolated from soybeans in Canada. Each ~9.0 Mb genome (chromosome and plasmid) harbors predicted genes for nodulation, nitrogen fixation, N2O mitigation, phosphate solubilization, iron acquisition, phytohormone production, and stress tolerance, highlighting their potential for sustainable agriculture.

Bradyrhizobium ottawaense