Comparative action of glyphosate as a trigger of energy drain in eubacteria

Abstract

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycitie], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzynies, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of commonpathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry of energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coi, after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also produced profound effects upon glyphosate sensitivity.Many antimetabolites that act against biosynthetic pathways mimic end products, causing inappropriate regulatory effects upon enzymes having early-pathway positions. In contrast, N-(phosphonomethyl)glycine (PMG), an herbicide known as glyphosate, is an exceedingly effective inhibitor of a mid-pathway enzyme of aromatic amino acid biosynthesis, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase. EPSP synthase has been shown to be a sensitive target of PMG action in procaryotes, lower eucaryotes, and higher plants (see reference 34 for reference citations). Although PMG is now under intensive study in higher plants by virtue of its potent herbicidal properties, antimicrobial studies are few. The presence of PMG during growth c...

Microbial Origin of Plant-Type 2-Keto-3-Deoxy- d - arabino -Heptulosonate 7-Phosphate Synthases, Exemplified by the Chorismate- and Tryptophan-Regulated Enzyme from Xanthomonas campestris

“…Significant derepression of AroA II was not observed when a culture in the mid-exponential stage of growth in rich medium was centrifuged and resuspended in minimal medium for 2 h prior to extract preparation. As another approach, a culture in minimal medium was exposed to 10 mM glyphosate to cause starvation for aromatic amino acids (9). Although growth inhibition was observed, derepression of AroA II did not occur.…”

Natural tolerance of cyanobacteria to the herbicide glyphosate

“…Both Synechocystis 6803 and A. variabilis showed tolerance towards the free acid of glyphosate, with little or no inhibition of growth even at 20 mM, compared with total inhibition of growth by 5 mM free acid found for other bacteria (Shulz et al., 1984;Fischer et al, 1986;Pipke et al, 1987), The two species of cyanobacteria studied showed some tolerance to the isopropylamine salt of glyphosate although this compound was more toxic than the free acid. Roundup was the most toxic formulation though it was far less toxic towards Synechocystis 6803 than towards A. variabilis.…”

“…Growth of plant tissue cultures is inhibited by < 1 mM glyphosate (Rubin, Gaines and Jensen, 1984;Smarter a/., 1985;Steinrucken et al,, 1986), while bacteria are generally resistant to between 2 mM and 5 mM glypbosate (Schulz, Sost & Amrhein, 1984;Fischer et al 1986;Pipke et al, 1987).…”

Transformação genética e aplicação de glifosato na microbiota do solo, fixação biológica de nitrogênio, qualidade e segurança de grãos de soja geneticamente modificada

“…2.5.1.19, CP4 EPSPS), com peptídio sinal, da via do chiquimato, resistente ao glifosato, mantendo ativa a via biossintética de aminoácidos aromáticos nas plantas. Essa via de biossíntese é inibida em genótipos de soja não modificada (NM), sem a isoforma CP4 EPSPS (BUSSE et al, 2001), tendo em vista que o herbicida glifosato [(N-(fosfonometil) glicina, Roundup (Monsanto, St. Loius, MO)], inibe a síntese de aminoácidos aromáticos (fenilalanina, triptofano e tirosina) em plantas (FISHER et al, 1986). O mecanismo de ação do glifosato compreende a inibição da EPSPS, que catalisa a condensação do ácido chiquímico e fosfofenolpiruvato, dando origem à bioconversão de aminoácidos aromáticos.…”

“…A inibição da via de shiquimato pelo glifosato resulta na acumulação do ácido chiquímico e/ou ácidos hidroxibenzoicos, bem como de ácidos protocatequímicos e/ou ácidos gálicos em espécies de plantas sensíveis (DUKE, 1988) e em Bradyrizobium japonicum (ZABLOTOVICZ & REDDY, 2004). Efeitos tóxicos do glifosato podem ser atribuídos à inibição da síntese de aminoácidos aromáticos, à exaustão energética resultante do consumo de adenosina trifosfato (ATP) e fosfofenolpiruvato na síntese do chiquimato, ao 3-deoxi-D-arabino-heptulose-7-fosfato, aos ácidos hidroxibenzóicos e gálico e à toxidade resultante do acúmulo de moléculas intermediárias na via do shiquimato (FISHER et al, 1986). Desse modo, é possível que haja alterações no metabolismo secundário, incluindo a síntese de compostos fenólicos, que inclui esses metabólitos (REDDY et al, 2000).…”