A Flavor Lactone Mimicking AHL Quorum-Sensing Signals Exploits the Broad Affinity of the QsdR Regulator to Stimulate Transcription of the Rhodococcal qsd Operon Involved in Quorum-Quenching and Biocontrol Activities

In many Gram-negative bacteria, virulence, and social behavior are controlled by quorum-sensing (QS) systems based on the synthesis and perception of N-acyl homoserine lactones (AHLs). Quorum-quenching (QQ) is currently used to disrupt bacterial communication, as a biocontrol strategy for plant crop protection. In this context, the Gram-positive bacterium Rhodococcus erythropolis uses a catabolic pathway to control the virulence of soft-rot pathogens by degrading their AHL signals. This QS signal degradation pathway requires the expression of the qsd operon, encoding the key enzyme QsdA, an intracellular lactonase that can hydrolyze a wide range of substrates. QsdR, a TetR-like family regulator, represses the expression of the qsd operon. During AHL degradation, this repression is released by the binding of the γ-butyrolactone ring of the pathogen signaling molecules to QsdR. We show here that a lactone designed to mimic quorum signals, γ-caprolactone, can act as an effector ligand of QsdR, triggering the synthesis of qsd operon-encoded enzymes. Interaction between γ-caprolactone and QsdR was demonstrated indirectly, by quantitative RT-PCR, molecular docking and transcriptional fusion approaches, and directly, in an electrophoretic mobility shift assay. This broad-affinity regulatory system demonstrates that preventive or curative quenching therapies could be triggered artificially and/or managed in a sustainable way by the addition of γ-caprolactone, a compound better known as cheap food additive. The biostimulation of QQ activity could therefore be used to counteract the lack of consistency observed in some large-scale biocontrol assays

N-Acyl Homoserine Lactones in Diverse Pectobacterium and Dickeya Plant Pathogens: Diversity, Abundance, and Involvement in Virulence

Soft-rot bacteria Pectobacterium and Dickeya use N-acyl homoserine lactones (NAHSLs) as diffusible signals for coordinating quorum sensing communication. The production of NAHSLs was investigated in a set of reference strains and recently-collected isolates, which belong to six species and share the ability to infect the potato host plant. All the pathogens produced different NAHSLs, among which the 3-oxo-hexanoyl- and the 3-oxo-octanoyl-l-homoserine lactones represent at least 90% of total produced NAHSL-amounts. The level of NAHSLs varied from 0.6 to 2 pg/cfu. The involvement of NAHSLs in tuber maceration was investigated by electroporating a quorum quenching vector in each of the bacterial pathogen strains. All the NAHSL-lactonase expressing strains produced a lower amount of NAHSLs as compared to those harboring the empty vector. Moreover, all except Dickeya dadantii 3937 induced a lower level of symptoms in potato tuber assay. Noticeably, aggressiveness appeared to be independent of both nature and amount of produced signals. This work highlights that quorum sensing similarly contributed to virulence in most of the tested Pectobacterium and Dickeya, even the strains had been isolated recently or during the past decades. Thus, these key regulatory-molecules appear as credible targets for developing anti-virulence strategies against these plant pathogens

Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

International audienceBACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates

Stabilité et persistance de l’activité d’un agent de biocontrôle formulé contre la jambe noire

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Stabilité et persistance de l’activité d’un agent de biocontrôle formulé contre la jambe noire

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An increasing opine carbon bias in artificial exudation systems and genetically modified plant rhizospheres leads to an increasing reshaping of bacterial populations

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Core genome and plasmidome of the quorum-quenching bacterium Rhodococcus erythropolis.

International audienceRhodococcus erythropolis is a worldwide-distributed actinobacterium that exhibits a remarkable metabolic versatility illustrated by its ability to degrade complex compounds, such as quorum-sensing signals N-acylhomoserine lactones (NAHLs), phenols, sterols and fuel derivatives. Because of its catabolic properties, R. erythropolis strains are proposed as anti-biofouling agents against NAHL-dependent biofilms, biocontrol agents against NAHL-emitting plant pathogens, and bioremediation agents in contaminated waters and soils. Here, we used the PacBio technology to resolve the complete genome sequence of the biocontrol strain R. erythropolis R138. Its genome consisted in a circular chromosome (6,236,862 bp), a linear plasmid pLRE138 (477,915 bp) and a circular plasmid pCRE138 (91,729 bp). In addition, draft genomes of five R. erythropolis strains were determined by Illumina technology and compared with the other five R. erythropolis genomes that are available in public databases: 5,825 common CDSs were present in all of the eleven analyzed genomes and represented up to 87 % of those identified in R. erythropolis R138. This study highlighted the high proportion of core-genome genes in R. erythropolis, but a high variability of the plasmid content. Key-metabolic pathways which are involved in the degradation of complex molecules, such as NAHLs and phenol, catechol and sterol derivatives are coded by the R. erythropolis core-genome

Draft Genome Sequences of Pseudomonas fluorescens Strains PA4C2 and PA3G8 and Pseudomonas putida PA14H7, Three Biocontrol Bacteria against Dickeya Phytopathogens.

International audiencePseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya

A metagenomic study highlights phylogenetic proximity of quorum-quenching and xenobiotic-degrading amidases of the AS-family.

Quorum-sensing (QS) signals of the N-acylhomoserine lactone (NAHL) class are cleaved by quorum-quenching enzymes, collectively named NAHLases. Here, functional metagenomics allowed the discovery of a novel bacterial NAHLase in a rhizosphere that was treated with γ-caprolactone. As revealed by rrs-DGGE and rrs-pyrosequencing, this treatment increased the percentage of the NAHL-degrading bacteria and strongly biased the structure of the bacterial community, among which Azospirillum dominated. Among the 29 760 fosmids of the metagenomic library, a single one was detected that expressed the qsdB gene conferring NAHL-degradation upon E. coli and decreased QS-regulated virulence in Pectobacterium. Phylogenetic analysis of the 34 orfs of the fosmid suggested that it would belong to an unknown Proteobacterium - probably a γ-proteobacterium. qPCR quantification of the NAHLase-encoding genes attM, qsdA, and qsdB revealed their higher abundance in the γ-caprolactone-treated rhizosphere as compared to an untreated control. The purified QsdB enzyme exhibited amidase activity. QsdB is the first amidase signature (AS) family member exhibiting NAHLase-activity. Point mutations in the AS-family catalytic triad K-S-S abolished the NAHLase activity of QsdB. This study extends the diversity of NAHLases and highlights a common phylogenic origin of AS-family enzymes involved in the degradation of natural compounds, such as NAHLs, and xenobiotics, such as nylon and linuron

Of the interest to listen and disrupt the dialogue of pathogens: details of soft-rot biocontrol by the rhodococcal quorum quenching

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