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Structural and Functional Characterization of Gene Clusters Directing Nonribosomal Synthesis of Bioactive Cyclic Lipopeptides in Bacillus amyloliquefaciens Strain FZB42. Alexandra Koumoutsi, 2004.The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere . We sampled sequenced the genome of FZB42and identified 2,947 genes with >50% identity on the aminoacid level to the corresponding genes of Bacillus subtilis 168.Six large gene clusters encoding nonribosomal peptide synthetases[NRPS] and polyketide synthases [PKS] occupied 7.5% of the wholegenome . Two of the PKS and one of the NRPS encoding gene clusterswere unique insertions in the FZB42 genome and are not presentin B . subtilis 168 . Matrix-assisted laser desorption ionization-timeof flight mass spectrometry analysis revealed expression ofthe antibiotic lipopeptide products surfactin, fengycin, andbacillomycin D . The fengycin [fen] and the surfactin [srf] operonswere organized and located as in B . subtilis 168 . A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D . The bmy island was found inserted close to the fen operon . The responsibility of the bmy, fen, and srf gene clusters for the production of the correspondingsecondary metabolites was demonstrated by cassette mutagenesis,which led to the loss of the ability to produce these peptides.Although these single mutants still largely retained their abilityto control fungal spread, a double mutant lacking both bacillomycinD and fengycin was heavily impaired in its ability to inhibitgrowth of phytopathogenic fungi, suggesting that both lipopeptidesact in a synergistic manner. De Novo Design of Potent Antimicrobial Peptides. V. Frecer, 2004.Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome . A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues . The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock . The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic ß-hairpin-like structures when they bind to LPS or LA . They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs . Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity . On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide . On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed . The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed .
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