Structural and Functional Characterization of Gene Clusters Directing Nonribosomal Synthesis of Bioactive Cyclic Lipopeptides in Bacillus amyloliquefaciens Strain FZB42

Abstract

The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid 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 whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were 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 corresponding secondary 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 ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.The rhizosphere colonizing Bacillus amyloliquefaciens strain FZB42 is distinguished from the related model organism Bacillus subtilis 168 by its ability to stimulate plant growth and to suppress plant pathogenic organisms (12,14). However, the basis for successful mutualistic colonization of plant rhizosphere by some Bacillus strains is still unknown. We assume that rhizosphere competence and biocontrol function in bacilli are partly caused by nonribosomally produced cyclic lipopeptides acting against phytopathogenic viruses, bacteria, fungi, and nematodes. These lipopeptides are synthesized at modular multienzymatic templates (33) and consist of a ␤-amino or ␤-hydroxy fatty acid component that is integrated into a peptide moiety.Some of these lipopeptides have been studied in greater detail, including surfactin, fengycins, and several iturins. Surfactin is a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a ␤-hydroxy fatty acid with 13 to 15 carbon atoms. Surfactin exerts its antimicrobial and antiviral effect by altering membrane integrity (30). Fengycin and the closely related plipastatin are cyclic lipodecapeptides containing a ␤-hydroxy fatty acid with a side chain length of 16 to 19 carbon atoms. Four D-amino acids and ornithine (a nonproteinogenic residue) have been identified in the peptide portion of fengycin. It is specifically active against filamentous fungi and inhibits phospholipase A 2 (26). Members of the iturin famil...

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

“…Fusaricidins are protonated preferentially at the guanidine group. Therefore, precursor ions for mass spectrometric sequencing are predominantly available in protonated form in contrast to many other cyclic lipopeptides, such as the surfactins, fengycins, and iturins [37,38] with a neutral fatty acid side chain, which mainly appear as the alkali adducts when investigated by MALDI-TOF MS. The intensities of the protonated precursor ions of fusaricidins are 10-to 100-fold higher than their corresponding alkali counterparts.…”

“…This finding is in contrast to many other lipopeptides reported in the literature, where the fatty acid part is a major target of structural variation. Prominent examples are the surfactins, iturins, and fengycins produced by numerous B. subtilis and B. amyloliquefaciens strains [37,38]. From our mass spectrometric sequence analysis, we classified the fusaricidins into three main families (series 1-3 in Table 2).…”

Intact-cell MALDI-TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea: can molecular phylogeny of species predict peptaibol structures?

“…Comparing the genomes of Bacillus subtilis 168 and the closely related Bacillus amyloliquefaciens FZB 42, three and six NRPS/PKS clusters were found, respectively (Koumoutsi et al, 2004). Two NRPS clusters and operons were positionally conserved with respect to B. subtilis, and several transposase sequences are contained in it.…”

Nonribosomal cyclic peptides: specific markers of fungal infections

“…For instance, six large gene clusters encoding NRPS and polyketide synthases occupy 7.5% of the whole Bacillus amyloliquifaciens genome. 103 In addition to the typical molecular weight of nonribosomal cyclic peptides, several examples of larger (2-3 kDa) cyclic molecules originating in the ribosomal pathway have been reported in plants 104 or enterobacteria. 105 In vitro preparation of small cyclic peptides by retroviruses has been reported recently.…”