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.

Surfactin and Other Lipopeptides from Bacillus spp.

“…For the second approach (Bumpus et al 2009), the authors took advantage of the size of the NRPS enzymes and the presence of unique marker ions derived from the common phosphopantetheinyl cofactor to adapt mass spectrometry-based proteomics to selectively detect NRPS and PKS gene clusters in microbial proteomes without requiring genome sequence information. In both cases, authors discovered in Bacillus thuringiensis (Abderrahmani et al 2010) and Bacillus cereus the genes involved in the biosynthesis of the kurstakin. The exponential growth of genome sequences and the development of such interesting tools to detect or predict potentially novel NRPs highlight the interest of a database, such as NORINE, which collects all the known structures of NRPs and provides software for efficient structure comparison (Caboche et al 2009).…”

“…The L and D forms or the amino acid residues are not yet characterised. However, the recent identification of the genes involved in the biosynthesis of such or similar compounds indicate that amino acids in 1 and 6 positions could be in the D-form (Bumpus et al 2009;Abderrahmani et al 2010) (see Sect. 3.5).…”

“…These compounds display antifungal activity against Stachybotrys charatum and are mainly adsorbed to the spore surfaces. Additionally, the potential operon encoding kurstakin synthetases was recently discovered (Bumpus et al 2009;Abderrahmani et al 2010). …”

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

“…Such an approach could be designed for LP-producers due to the following: the high homology of NRPS operons in different species, the number of already designed primers in the literature and the possibility of utilizing different databases and platforms, like BLAST, NRPS predictor or AntiSMASH as examples. [39][40][41][42][43][44] In our opinion, this may be the most costeffective and efficient way to analyze LP/BS produced by microorganisms in different environments.…”

Antibiotic Compounds from <i>Bacillus</i>: Why are they so Amazing?

“…With emergence of powerful bioinformatics and molecular tools, identifying new metabolites by genome mining has become a reality (Ansari et al, 2004;Lanen and Shen, 2006). For instance, a bioinformatics analysis of the genome of B. thuringiensis led to the prediction of an NRPS constituted of seven modules that could be involved in the synthesis of a heptalipopeptide similar to kurstakin (Abderrahmani et al, 2011). The discovery of new natural products by genome mining is an encouraging sign, suggesting that this methodology could lead to the isolation of novel molecules of pharmacological interest.…”