Molecular characterization of the phenolic acid metabolism in the lactic acid bacteria Lactobacillus plantarum

The lactic acid bacteria Lactobacillus plantarumdisplays substrate-inducible decar- boxylase activities on p-coumaric, caffeic and ferulic acids. Purification of the p-coumaric acid decarboxylase (PDC) was performed. Sequence of the N-terminal part of the PDC led to the cloning of the corresponding pdc gene. Expression of this gene in Escherichia colirevealed that PDC displayed a weak activity on ferulic acid, detectable in vitro in the presence of ammonium sulfate. Transcrip- tional studies of this gene in L. plantarum demonstrated that the pdc transcription is phenolic acid- dependent. A mutant deficient in the PDC activity, designated LPD1, was constructed to study phe- nolic acid altern…

Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator.

ABSTRACTPediococcus pentosaceusdisplays a substrate-inducible phenolic acid decarboxylase (PAD) activity onp-coumaric acid. Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of theLactobacillus plantarum pdcgene was used to screen aP. pentosaceusgenomic library in order to clone the corresponding gene of this bacteria. One clone detected with this probe displayed a low PAD activity. Subcloning of this plasmid insertion allowed us to determine the part of the insert which contains a 534-bp open reading frame (ORF) coding for a 178-amino-acid protein presenting 81.5% of identity withL. plantarumPDC enzyme. This ORF was identified as thepadAgene. A second O…

DIAGSOL Development of a new functional marker for B-triketone herbicides exposure in agricultural soils

International audience; The-triketone herbicides are maize selective herbicides that have been largely applied in replacement of atrazine, banned in Europe in 2004. Their mode of action lays on the inhi- bition of the p-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme of the carotenoid biosynthesis.In recent studies, we showed that within the soil bacterial community, many microorganisms possess a functional HPPD enzyme involved in tyrosine metabolism. These ”non-target or- ganisms” harbor the target of the-triketone herbicides and consequently may be affected in response to its exposure. From this point of view, the bacterial community harboring the hppd gene might be a relevant mark…

Phenolic Acid-Mediated Regulation of the padC Gene, Encoding the Phenolic Acid Decarboxylase of Bacillus subtilis

ABSTRACT In Bacillus subtilis , several phenolic acids specifically induce expression of padC , encoding a phenolic acid decarboxylase that converts these antimicrobial compounds into vinyl derivatives. padC forms an operon with a putative coding sequence of unknown function, yveFG , and this coding sequence does not appear to be involved in the phenolic acid stress response (PASR). To identify putative regulators involved in the PASR, random transposon mutagenesis, combined with two different screens, was performed. PadR, a negative transcriptional regulator of padC expression, was identified. padR is not located in the vicinity of padC , and the expression of padR is low and appears const…

Development of a new functional marker for β-triketone herbicides exposure in agricultural soils

The β-triketone herbicides are maize selective herbicides that have been largely applied in replacement of atrazine, banned in Europe in 2003. Their mode of action lays on the inhibition of the p-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme of the carotenoid biosynthesis. In recent studies, we showed that within the soil bacterial community, many microorganisms possess a functional HPPD enzyme involved in tyrosine metabolism. These “non-target organisms” harbor the target of the β-triketone herbicides and consequently may be affected in response to its exposure. Within this context, the objective of our work is to check for the interest of hppd bacterial community as a marker of e…

Inactivation of PadR, the repressor of the phenolic acid stress response, by molecular interaction with Usp1, a universal stress protein from Lactobacillus plantarum, in Escherichia coli

ABSTRACT The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum , the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1 , which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promo…

A new tool to assess the ecotoxicological impact of β-triketone herbicides on soil microbial communities

International audience; The β-triketone herbicides are post-emergence maize selective herbicides that have beenintroduced on the market, in replacement of atrazine, banned in Europe in 2004. Qualified as “eco-friendly”, since they are based on natural phytotoxin properties, these herbicides target an enzymeinvolved in carotenoid biosynthesis called 4-hydroxyphenylpyruvate dioxygenase (HPPD) encoded bythe hppd gene. The inhibition of this enzyme provokes bleaching symptoms, necrosis and death ofweeds.The hppd gene is not only find in eukaryotes such as plants, animals and humans but also inprokaryotes such as fungi, yeasts and bacteria. In recent studies, we showed that, within the soil bact…

Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization

By using degenerate primers designed from the first 19 N-terminal amino acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC), a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank. Of the 2,880 clones in the genomic bank, one was isolated by colony hybridization and contained a 519-bp open reading frame (pdc gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic transcriptional unit, which is transcribed from promoter sequences homologous to Lactococcus promoter sequences. No mRNA from pdc and no PDC activity were detected in uninduced cell extracts, indicating that the…

DIAGSOL : Développement d’un outil microbien pour évaluer l’exposition des sols agricoles aux herbicides β-tricétones

International audience; En 2011, en France, plus de 2 millions d’hectares de champs de maïs ont été traités avec desherbicides β-tricétones. Qualifiés de « respectueux de l’environnement » en raison d’une efficacité àfaible dose, de récentes études ont démontré un effet transitoire des β-tricétones sur la diversitébactérienne des sols (Romdhane et al., 2016). L’utilisation récurrente des β-tricétones pourrait, àterme, perturber la diversité bactérienne et les fonctions écosystémiques des sols qu’elle soutient.Il est essentiel d’évaluer l’exposition des microorganismes des sols aux β-tricétones.L’enzyme 4-hydroxyphénylpyruvate dioxygénase (4-HPPD), cible de ces herbicides chez les plantesadv…

Response of soil bacterial and hppd communities to tembotrione herbicide

Herbicides used in agriculture aim to prevent weed growth but are known to end up in contactwith soil microorganisms, thus defined as non-target organisms. Tembotrione, a recentlymarketed β-triketone herbicide, is known to inhibit the 4-HydroxyPhenylPyruvateDioxygenase(4-HPPD) in weeds. This enzyme is also found in numerous soil microorganisms, such as somePGPR and symbiotic bacteria, that play a key role in maintenance of ecosystem services.In this study, one of the major concerns is to assess whether tembotrione could have toxiceffects on soil microorganisms and could disturb soil microbial community dynamic andstructure. To investigate the possible impacts of this herbicide on these comm…

Kinetics and Intensity of the Expression of Genes Involved in the Stress Response Tightly Induced by Phenolic Acids in <i>Lactobacillus plantarum</i>

In <i>Lactobacillus plantarum</i>, PadR, the negative transcriptional regulator of <i>padA </i>encoding the phenolic acid decarboxylase, is divergently oriented from <i>padA. </i>Moreover, it forms an operonic structure with <i>usp1,</i> a genewhose products display homology with proteins belonging to the UspA family of universal stress proteins. PadR is inactivated by the addition of <i>p-</i>coumaric, ferulic or caffeic acid to the culture medium. In order to better characterize the stress response of this bacterium to phenolic acids, we report here the kinetics and quantitative expression by qRT-PCR of the 3 genes from the <i…

Bacterial hppd: a biomarker of exposure of soils to beta-triketone herbicides?

National audience; β-triketone herbicides are among the most used herbicides in corn crop to control broadleaf weeds.These herbicides inhibit the 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) and lead to bleaching anddeath of weeds. This enzyme is not only found in plants but in all living organisms, includingmicroorganisms where it takes part to the tyrosine degradation pathway. Thus, microorganismsclassified as “non-target organisms” by current EU regulation for pesticide authorization, might beimpacted by β-triketones, with possible domino effect on microbial functions supporting soilecosystem services (Thiour-Mauprivez et al. 2019). Since microorganisms have been proposed by EFSAas key-d…

Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum.

ABSTRACTLactobacillus plantarumdisplays a substrate-induciblepadAgene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate. The putative regulator ofpadAwas located in thepadAlocus based on its 52% identity with PadR, thepadAgene transcriptional regulator ofPediococcus pentosaceus(L. Barthelmebs, B. Lecomte, C. Diviès, and J.-F. Cavin, J. Bacteriol.182:6724-6731, 2000). Deletion of theL. plantarum padRgene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently orientedpadA. ThepadRgene is cotranscribed with a downstream open reading frame (ORF1), the product of which m…

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Biodegradation of synthetic β-triketone herbicide

Expression in Escherichia coli of Native and Chimeric Phenolic Acid Decarboxylases with Modified Enzymatic Activities and Method for Screening Recombinant E. coli Strains Expressing These Enzymes

ABSTRACT Four bacterial phenolic acid decarboxylases (PAD) from Lactobacillus plantarum , Pediococcus pentosaceus , Bacillus subtilis , and Bacillus pumilus were expressed in Escherichia coli , and their activities on p -coumaric, ferulic, and caffeic acids were compared. Although these four enzymes displayed 61% amino acid sequence identity, they exhibit different activities for ferulic and caffeic acid metabolism. To elucidate the domain(s) that determines these differences, chimeric PAD proteins were constructed and expressed in E. coli by exchanging their individual carboxy-terminal portions. Analysis of the chimeric enzyme activities suggests that the C-terminal region may be involved …

Purification and characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum

Abstract Lactobacillus plantarum cells displayed substrate-inducible decarboxylase activities on p-coumaric and ferulic acids of 0.6 and 0.01 μmol min−1 mg−1, respectively. Activity in uninduced cells or corresponding cell extracts was undetectable (

Fate, biodegradation and ecotoxicological impact of the bioherbicide leptospermone on soil bacterial community

International audience

Impact of Leptospermone, a Natural β-Triketone Herbicide, on the Fungal Composition and Diversity of Two Arable Soils

Impact of leptospermone, a β-triketone bioherbicide, was investigated on the fungal community which supports important soil ecological functions such as decomposition of organic matter and nutrients recycling. This study was done in a microcosm experiment using two French soils, Perpignan (P) and Saint-Jean-de-Fos (SJF), differing in their physicochemical properties and history treatment with synthetic β-triketones. Soil microcosms were treated with leptospermone at recommended dose and incubated under controlled conditions for 45 days. Untreated microcosms were used as control. Illumina MiSeq sequencing of the internal transcribed spacer region of the fungal rRNA revealed significant chang…

Impact of Leptospermone, a Natural β-Triketone Herbicide, on the Fungal Composition and Diversity of Two Arable Soils

International audience; Impact of leptospermone, a β-triketone bioherbicide, was investigated on the fungal community which supports important soil ecological functions such as decomposition of organic matter and nutrients recycling. This study was done in a microcosm experiment using two French soils, Perpignan (P) and Saint-Jean-de-Fos (SJF), differing in their physicochemical properties and history treatment with synthetic β-triketones. Soil microcosms were treated with leptospermone at recommended dose and incubated under controlled conditions for 45 days. Untreated microcosms were used as control. Illumina MiSeq sequencing of the internal transcribed spacer region of the fungal rRNA re…

Knockout of thep-Coumarate Decarboxylase Gene fromLactobacillus plantarumReveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

ABSTRACTLactobacillus plantarumNC8 contains apdcgene coding forp-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomalpdcgene was replaced with the deletedpdcgene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolizep-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown thatL. plantarumhas a second acid phenol decarboxylase enzyme, better induced with ferulic acid than withp-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when gl…