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De nombreuses bactéries et de nombreux champignons utilisent la voie non ribosomale pour produire des peptides ou d'autres petites molécules. Cette voie, nommée NRPS pour " non-ribosomal peptide synthesis ", fait appel à des mégaenzymes modulaires qui catalyse nt l'assemblage de peptides à partir d'acides aminés protéinogéniques ou non protéinogéniques. Les petites molécules synthétisées par cette voie peuvent intervenir dans des processus très variés tels que "adaptation aux environnements défavorables, la communication ou la compétition avec d'autres microorganismes dans leur habitat naturel, ou encore l'interaction avec un hôte. Xanthomonas albilineans est l'agent causal de "échaudure des feuilles, une des principales maladies bactériennes de la canne à sucre. Le séquençage de son génome a conduit à la découverte d'un nouveau locus NRPS, appelé META-B, qui est également présent dans le génome de souches appartenant à Xanthomonas oryzae ou Xanthomonas /rans/ucens. Les analyses in silico indiquent que ce locus ne ressemble à aucun autre locus décrit à ce jour et que chaque souche séquencée produit un peptide META-B différent. La présence du locus META-B chez trois espèces de Xanthomonas phylogénétiquement distantes et associées à des plantes monocotylédones suggère que la nouvelle famille de petites molécules produites par ce locus pourrait jouer un rôle dans les interactions plante-bactérie. Un protocole permettant de surproduire et d' isoler le peptide META-B chez la souche GPE PC73 de X. abilineans a été développé. Deux approches sont actuellement mises en oeuvre pour trouver la fonction de ce peptide META-B. La première est basée sur l'étude du peptide META-B purifié (tests in vitro pour évaluer les activités antibactériennes ou antifongiques ou pour mettre en évidence un rôle dans les interactions avec la canne à sucre). La seconde est basée sur l'étude de mutants affectés dans l'expression de gènes du locus META-B (expérimentations sur canne à sucre). (Texte intégral)

International audience; Various bacteria use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. These molecules exhibit broad structural diversity and display biological activities that range from adaptation to unfavorable environments, communication or competition with other microorganisms in their natural habitat, or even to action as virulence factors. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in siIico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. To date, the only known small molecule synthesized by NRPS in the genus Xanthomonas is albicidin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald-a lethal disease of sugarcane. In silica analysis of available genomic sequences of Xanthomonas strains led to the discovery of a novel NRPS gene cluster called META-B which doesn't resemble to any gene cluster de- scribed to date. This NRPS gene cluster occurs in (i) X. albilineans, (ii) two pathovars of Xanthomonas oryzae which are the causal agents of two agronomically important diseases of rice (bacterial leaf blight caused by X. oryzae pv. oryzae and bacterial leaf streak caused by X. oryzae pv. oryzicola), and (iii) Xanthomonas translucens , the causal agent of the bacterial leaf streak of wheat. Interestingly, the NRPS gene cluster META-B seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement in plant-bacteria interactions. (Résumé d'auteur)

Background: To date, the only known small molecule synthesized by non-ribosomal peptide synthesis (NRPS) in the genus Xanthomonas is the phytotoxin albicidin produced by Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Recent analyses of available genomic sequences indicated that X. albilineans, as well as Xanthomonas oryzae and Xanthomonas translucens, possess a novel NRPS gene cluster called META-B whose architecture is strainspecific (each sequenced strain is predicted to synthesize a different peptide) and doesn´t resemble to any NRPS gene cluster described to date. Because the META-B NRPS gene cluster is specific to three phylogenetically distant species of Xanthomonas associated with monocotyledonous plants, we assume a putative involvement of the new family of small molecules produced by this gene cluster in plant-bacteria interactions. Objectives: We aimed at isolating and characterizing the small molecule encoded by the META-B NRPS gene cluster of X. albilineans in order to study its putative involvement in plant-bacteria interactions. Methods: We developed a META-B overproducing X. albilineans strain by over-expressing the AraC transcriptional regulator which is present within the META-B NRPS gene cluster. HPLC profiles obtained from cultures of wild type vs the AraC over-expressing strain were compared. Peaks with significantly increased amplitude in the HPLC profile from the AraC over-expressing strain were collected and their content analyzed by mass spectrometry. Conclusions: We identified a peptide with a nominal molecular mass > 2 kDa and with an amino acid sequence (MS/MS experiments) which excellently matches the one predicted by A-domain specificities of META-B NRPS genes. (Texte intégral)

Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by non-ribosomal peptide synthesis (NRPS) in this genus is albicidin produced by Xanthomonas albilineans. The DNA gyrase inhibitor albicidin is not only an important virulence factor but also a possible lead structure for novel antibiotics. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas. We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. The most unexpected result of these analyses is that these two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters. Furthermore, these Xanthomonas spp. strains share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or for the secretion of peptides. In silico prediction of peptide structures according to the NRPS architecture accounts for eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas transluscens and X. oryzae pv. oryzicola. Further genome mining revealed (i) novel NRPS genes shared by Xanthomonas spp. strains GPE PC73 and XaS3 with the plant-associated bacterium Bradyrhizobium spp. strain BTAi and (ii) novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari. This study revealed the significant potential of the genus Xanthomonas of producing new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of new non-ribosomally synthesized peptides in plant-bacteria interactions.