title: Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from Bacillus thuringiensis.
abstract: Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.
authors.affiliation: Laboratoire des Procédés Biologiques, Génie Enzymatique et Microbien, ProBioGEM, UPRES-EA 1026, Polytech'Lille/IUT A, Université Lille Nord de France-Sciences et Technologies, USTL, Avenue Paul Langevin, 59655, Villeneuve d'Ascq Cedex, France.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abderrahmani
authors.forename: Arthur
authors.initials: A
authors.lastname: Tapi
authors.forename: Farida
authors.initials: F
authors.lastname: Nateche
authors.forename: Marlène
authors.initials: M
authors.lastname: Chollet
authors.forename: Valérie
authors.initials: V
authors.lastname: Leclère
authors.forename: Bernard
authors.initials: B
authors.lastname: Wathelet
authors.forename: Hocine
authors.initials: H
authors.lastname: Hacene
authors.forename: Philippe
authors.initials: P
authors.lastname: Jacques
mesh_terms: D000462:Algeria; D001409:Bacillus cereus / Q000235:genetics; D001413:Bacillus thuringiensis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D017931:DNA Primers; D015723:Gene Library; D055666:Lipopeptides / Q000096:biosynthesis*; D009876:Operon; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D055666:Lipopeptides; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase