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.

FIG. 1.

Colinear scaffold of the B. amyloliquefaciens FZB42 genome over the B. subtilis 168 genome. DNA sequences from FZB42 were compared by TBLASTN with the proteins of B. subtilis 168. The locations of NRPS and PKS gene clusters are indicated by purple bars. Insertions of transposon (red) and phage (blue) sequences into FZB42 are also indicated. The outer circle shows FZB42 genes homologous and colinear with B. subtilis 168 genes. The region adjacent to the bidirectional origin of replication from 0 to 195 kb displayed highest homology to B. subtilis 168. Second circle (gray) shows B. subtilis 168 genes without orthologs in FZB42 (cutoff, <30% amino acid identity). Two hotspots of missing genes are close to the terminator region, which is indicated by a sudden change in the direction of genes and at a region corresponding to 265 to 2.77 Mb of B. subtilis 168. These three areas are mainly occupied by phage-like sequences with unknown function in B. subtilis 168. The third circle shows conserved genes that have been rearranged in FZB42. The color code indicates amino acid identities of >90% (blue), 80% (green), 70% (yellow), 60% (orange), and 50% (red). The inner circle, with coordinates in base pairs, shows the B. subtilis 168 genome, enlarged by the 306-kb sequence containing additional NRPS and PKS operons, identified in the FZB42 genome.

FIG. 2.

MALDI-TOF-MS analysis of lipopeptides from B. amyloliquefaciens FZB42 and the mutant strains. (A) Detection of surfactin and bacillomycin D mass peaks in extracts prepared from the lyophilysate of the culture filtrate of FZB42 wild-type cells grown in the ACS medium. Panels B to F show mass spectra from intact whole cells grown on agar plates by using Landy medium. (B) Detection of surfactin and bacillomycin D mass peaks in FZB42 wild-type cells; (C) detection of surfactin mass peaks but not of bacillomycin D in mutant AK1 (ΔbmyA::Em^r); (D) detection of fengycin mass peaks in FZB42 wild-type cells; (E) AK2 (ΔfenA::Cm^r) was deficient in production of fengycin; (F) mutant CH1 (srfA::Em^r) was deficient in production of surfactin, but mass peaks indicating bacillomycin D production are still remaining. This sample was analyzed by using a Voyager DE-Pro instrument (Applied Biosystems/Applera Deutschland GmbH, Darmstadt, Germany). For peak identifications, refer to Table 3. The same patterns were detected in samples prepared from culture filtrates from cells growing in Landy and ACS medium (data not shown).

FIG. 3.

In situ structural analysis of the lipopeptide product of B. amyloliquefaciens FZB42 with an m/z of 1,031.5 by PSD-MALDI-TOF-MS of whole cells of B. amyloliquefaciens FZB42. The structure was derived from a series of N- and C-terminal fragments [b_n and Y_n(−H₂O) ions, as well as proline-directed P_n fragments]. FA, fatty acid.

FIG. 4.

ORF organization of the bacillomycin and fengycin operon in B. amyloliquefaciens FZB42 (AJ575417). The intersecting dotted lines indicate events of insertion and rearrangement in FZB42 compared to the respective B. subtilis 168 genome region. The organization and positions of the homologous gene clusters of B. subtilis 168 (fengycin biosyntheses [pps]), B. subtilis RB14 (iturin A biosynthesis [itu]), and B. subtilis ATCC 6633 (mycosubtilin biosynthesis [myc]) were drawn by referring to references and .

FIG. 5.

Schematic representation of the bacillomycin D operon of FZB42 comprising the ORFs bmyD (malonyl coenzyme A transacylase), bmyA, bmyB, and bmyC. The deduced domain organizations of the different proteins specified by the operon are indicated. The module numbers are given in parentheses. The region which is highly similar to the iturin operon of RB14 is shaded. AL, acyl coenzyme A ligase domain; ACP, acyl carrier protein domain; KS, β-ketoacyl synthetase domain; AMT, aminotransferase domain.

FIG. 6.

Biological activity of supernatants drawn from FZB42 and the mutants impaired in the biosynthesis of surfactin (CH1), bacillomycin D (AK1), fengycin (AK2), or both bacillomycin D and fengycin (AK3). A volume of 2 μl of a 20-h culture of FZB42 or the respective mutant strains grown in Landy medium was dropped onto Waksman agar plates with regularly arranged actively growing F. oxysporum f. sp. cucumerinum DSMZ 62313 cultures. (A and B) The plates were incubated for 3 days at 27°C. (C) Inhibition of S. coelicolor DSMZ 40233 by FZB42 and mutant cultures grown for 20 h in Landy medium. The S. coelicolor indicator strain was mixed with LB soft agar (0.3%) and poured onto LB agar dishes. Supernatants (300 μl) obtained from the respective Bacillus strains were applied and incubated overnight at 37°C.