Search

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)

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. The final biosynthetic step towards plantazolicin (PZN) comprises Nα,Nα-arginyl methyltransferase (PznL) mediated N-terminal bismethylation. We show that PznL processes truncated desmethyl-plantazolicin analogues, but only those with an N-terminal guanidine side chain derived from arginine. PznL specificity, which is narrow, depends on the side chain of the N-terminal amino acid linked to an azole, and not so much on the number of azoles.

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.

Xanthomonas albilineans is a xylem-invading plant pathogen that causes leaf scald disease of sugarcane. Unlike most plant pathogenic bacteria, X. albilineans does not possess a Hrp-Type Three Secretion System. Pathogenicity of this bacterial species must therefore rely on other virulence factors. X. albilineans produces albicidin, a toxin and potent inhibitor of DNA gyrase which inhibits proplastic DNA replication. Consequently, chloroplast differentiation is blocked and disease symptoms develop. Albicidin is also bactericidal at nanomolar concentrations against a range of Gram-positive and Gram-negative bacteria. This potent and novel antibiotic is especially of interest because of its activity against Escherichia coli, a species causing nosocomial diseases. Sequencing and annotation of the entire genome of X. albilineans recently revealed that X. albilineans possesses 12 large genes encoding nonribosomal peptide synthetases (NRPSs) which are located in four gene clusters covering 4% of the genome (1). One of these NRPS clusters corresponds to the previously identified albicidin biosynthesis gene cluster. The mode of action of this antibiotic was extensively studied but its structure remains unknown. Characterization of albicidin is the main bottleneck which slows development of its therapeutic application. X. albilineans is a slow growing bacterium and production yields of albicidin are low, i.e. it turned out to be extremely tedious to obtain sufficient amounts for structure elucidation. To overcome this problem, we successfully considered heterologous overproduction by transferring all albicidin biosynthesis genes into the fast growing bacterium Xanthomonas axonopodis pv. vesicatoria (2). Production of albicidin in this heterologous system already allowed us to obtain several milligrams of pure compound and promising preliminary results regarding the structural characterization of albicidin. As an example, 1H-NMR analyses showed the presence of a number of para-substituted aromates, putatively tyrosine or 4-hydroxyphenylglycines. However, the number of amide protons found in 1H-NMR spectra is lower than the number expected from the biosynthesis assembly lines, suggesting the involvement of tailoring steps during or post-NRPS biosynthesis. In silico analysis of the three other NRPS gene clusters resulted in partial prediction of the sequences of the precursor peptides synthesized by these clusters which do not resemble any peptide described to date. One of these NRPS gene clusters encodes a complete machinery predicted to be required for secretion and tailoring of small molecules: ABC transporter, MbtH like protein, isomerase, aminotransferase, acyltransferase, enoyl-CoA hydratase. The two other NRPS gene clusters encode only NRPSs which are thought to trans-act with the first one. Interestingly, the NRPSs encoded by X. albilineans share characteristics with NRPSs encoded by the root and stem-nodulating Bradyrhizobium sp. BTAi1, suggesting structural similarities between small molecules produced by these two plant interacting species. In X. albilineans, functional analyses of the phosphopantheteinyl transferase gene (which is required for activation of NRPSs) showed that these non-albicidin NRPS gene clusters are most likely involved in the biosynthesis of at least one new virulence factor. Future work will focus on characterization of the full structure of albicidin as well as the isolation and characterization of other small molecules assembled by NRPS. These compounds will be chromatographically isolated and characterized by high-resolving FTICR mass spectrometry, 2D-NMR spectroscopy and X-ray crystallography. Isolation and structural characterization of these new bioactive molecules will facilitate the annotation of biosynthesis gene clusters and the study of their role during interactions between sugarcane and X. albilineans. On a biochemical level, deciphering their role in pathogenicity should result in the identific

L'albicidine est une molécule à activité phytotoxique et antibiotique produite par la bactérie Xanthomonas albilineans, l'agent causal de la maladie de l'échaudure des feuilles de la canne à sucre. L'albicidine, qui est un composant-clé de la pathogénie de X. albilineans, est synthétisée par un système hybride PKS/NRPS ("PolyKetide Synthase/NonRibosomal Peptide Synthetase"). Il s'agit d'un puissant inhibiteur de l'ADN gyrase de plantes et de bactéries. Son mode d'action est original car, au contraire de celui d'autres antibiotiques connus, l'albicidine bloque la partie C-terminale du site de fixation à l'ADN de la gyrase A. La caractérisation de l'albicidine a jusqu'à présent été ralentie à cause des faibles rendements de production de cette molécule par X. albilineans. C'est pourquoi, l'ensemble des gènes nécessaires à la biosynthèse de l'albicidine ont été transférés dans la bactérie à croissance rapide X. axonopodis pv. vesicatoria. Ce transfert de gènes a conduit à une production d'albicidine beaucoup plus élevée. Une combinaison de méthodes biochimiques et chromatographiques a permis la purification de l'albicidine. De plus, plusieurs techniques de spectrométrie de masse (ESI-MALDI-TOF, FTICR-MS) ont permis (i) de déterminer la masse exacte de l'albicidine (842.2 Da) et (ii) de proposer un nombre limité de formules chimiques compatibles avec la masse de la molécule. Enfin, plusieurs caractéristiques chimiques de l'albicidine ont pu être déterminées grâce à des analyses préliminaires en RMN. En conclusion, l'augmentation des rendements de production de l'albicidine en système hétérologue, conjuguée à une plus grande efficacité de sa purification, ont permis de réaliser des progrès dans la caractérisation structurale de l'albicidine. De plus grandes quantités d'albicidine purifiée sont en cours de préparation et celles-ci seront prochainement analysées grâce à des techniques de caractérisation structurale performantes. (Texte intégral)