Etude structurale et biologique de petites molécules impliquées dans la pathogénie de Xanthomonas albilineans, une bactérie pathogène de la canne à sucre – ALBILINEANS

Xanthomonas albilineans (Xa) is a plant pathogen bacterium that causes leaf scald disease of sugarcane. Xa produces a potent inhibitor of DNA gyrase called albicidin which inhibits proplastic DNA replication and consequently blocks chloroplast differentiation, resulting in disease symptoms. 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. 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. Xa 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, UMR BGPI considered successfully heterologous overproduction by transferring all albicidin biosynthesis genes into a fast growing bacterium. Large production of albicidin in this heterologous system already allowed us to obtain promising preliminary results on the characterization of albicidin. The entire genome of Xa was recently sequenced at Genoscope (Evry, France). A higly prominent genomic feature of Xa is its distinctive 12 large genes encoding nonribosomal peptide synthetases (NRPSs) that cover 4 % of the genome. The NRPS genes identified in the genome of Xa are clustered into four NRPS gene clusters. The first one corresponds to the previously identified albicidin biosynthesis gene cluster. 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, that do not resemble any peptide described to date. Functional analyses showed that these three new NRPS gene clusters are probably involved in the biosynthesis of at least three virulence factors, called Metacols. The first part of this project will be devoted to the full structure characterization of albicidin, and the evaluation as a lead structure for use as an antibacterial drug, particularly for Gram-negative bacteria. The second part will be devoted to the isolation and characterization of other secondary metabolites assembled by NRPS and involved in pathogenicity of Xa. These compounds, called Metacols, will be isolated at UMR BGPI (Montpellier, France) and will be characterized at the Technical University of Berlin by high-resolving FTICR mass spectrometry, 2D-NMR spectroscopy and X-ray crystallography. Isolation and structural characterization of Metacols will facilitate the annotation of biosynthesis gene clusters and allow studying their role in the interaction of Xa with sugarcane. On a biochemical level, deciphering their role in pathogenicity should reveal (a) new mechanism(s) involved in the colonization of the xylem by bacterial plants pathogens and finally give important insights for crop protection. To isolate these new small molecules, we propose three complementary approaches. The first approach is the functional analysis (gene inactivation mutagenesis and assessment of pathogenicity of Xa mutants) of (i) all three NRPS gene clusters required for biosynthesis of Metacols, and (ii) selected genes coding for putative tailoring enzymes required for transformation of the precursor peptides to bioactive molecules. The second approach is to support the functional analysis by MALDI-TOF and HPLC-ESI-orbitrap mass spectrometry in order to detect changes in the metabolites production profiles between Xa wild-type and Xa mutants. The third approach is the heterologous expression of the NRPS gene clusters in a fast growing bacterial host. This latter approach, which already has been efficient for albicidin, will allow overproduction of Metacols, and will consequently facilitate their isolation. This cooperative research project submitted to the ANR-DFG 2009 call will structure a new collaboration between French and German scientists who work in academic institutions. This project proposes to isolate and characterize new bioactive or antibacterial compounds. Compounds will be supplied by the French scientists from UMR BGPI which have a long-lasting experience with overproduction and isolation of albicidin and with molecular analysis of the pathogenicity of Xa. Compounds will be characterized by professor Süssmuth from the TU Berlin which has an outstanding experience with the structure elucidation of antibiotics derived from fungi and bacteria. The research topic of this project is related to two major critical issues: the control of plant diseases through the knowledge of mechanisms of plant-microbe interactions and the development of antibiotics.