title: Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.
abstract: Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.
authors.affiliation: Laboratoire Littoral Environnement et Sociétés, LIENSs - UMR 7266 - CNRS, Université de La Rochelle, Avenue Michel Crépeau, 17042, La Rochelle Cedex 1, France.
authors.forename: Fatoumata
authors.initials: F
authors.lastname: Tambadou
authors.forename: Thibault
authors.initials: T
authors.lastname: Caradec
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Gagez
authors.forename: Antoine
authors.initials: A
authors.lastname: Bonnet
authors.forename: Valérie
authors.initials: V
authors.lastname: Sopéna
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bridiau
authors.forename: Valérie
authors.initials: V
authors.lastname: Thiéry
authors.forename: Sandrine
authors.initials: S
authors.lastname: Didelot
authors.forename: Cyrille
authors.initials: C
authors.lastname: Barthélémy
authors.forename: Romain
authors.initials: R
authors.lastname: Chevrot
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D003091:Colistin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008826:Microbial Sensitivity Tests; D005810:Multigene Family / Q000235:genetics; D056507:Paenibacillus / Q000235:genetics / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D003091:Colistin