Bacillus amyloliquefaciens - ABiTEP GmbH

VOL. 186, 2004 BACILLUS LIPOPEPTIDES 1095
to the phages, the IS elements present in B. amyloliquefaciens
FZB42 may also be involved in events of horizontal gene transfer.
The most striking property of the genome of FZB42 is that
a significant part of the genome (�7.5%, 306 kb, organized in
six operons) is devoted to the biosynthesis of polyketides and
peptides, enabling this bacterium to cope with competing organisms
within the plant rhizosphere. Two gene clusters encoding
PKS and one large gene cluster involved in nonribosomal
peptide synthesis (bmy) have been identified. The
impressive genetic capacity of environmental FZB42 for the
production of secondary metabolites, such as lipopeptides and
polyketides, exceeds twice that of the laboratory model organisms
B. subtilis 168 (37) and Streptomyces coelicolor (4) and has
been until now comparable only to Streptomyces avermitilis,
which is well known for its production of a wide range of
secondary metabolites and in which 6.4% of the entire genome
is devoted to the production of secondary metabolites (27).
Strain FZB42 is naturally competent for DNA uptake and
homologous recombination ideally suitable for genetic approaches
in analyzing its metabolic capacity e.g., by targeted
construction of mutants impaired in the synthesis of cyclic
lipopeptides. Here we demonstrated that disruption of one of
the bmy, fen, and srf genes prevented production of the respective
lipopeptides, providing evidence that these gene clusters
are involved in their biosynthesis. A double mutant that was
unable to produce bacillomycin D and fengycin retained its
antibacterial activity directed against Streptomyces spp. but did
not develop antifungal activity, suggesting that both lipopeptides
might act synergistically in order to intensify suppression
of fungal growth. A phenomenon until now only described in
actinomycetes. Microcosm experiments with wild-type and mutant
strains are necessary to clarify role of the cyclic lipopeptides
in biocontrol function within plant rhizosphere.
The present study characterized the production of the three
cyclic lipopeptides surfactin, fengycin, and bacillomycin D by
B. amyloliquefaciens FZB42. The sequence obtained for the
first time for a gene cluster involved in bacillomycin D biosynthesis
reflects perfectly colinearity in nonribosomal PKS domain
order and its peptide synthesis function. Adenylation
domains specifying amino acids different from other iturin-like
peptides displayed a high degree of variability, but their functional
amino acids lining the substrate binding pocket matched
perfectly with the known selectivity code of the respective
amino acids compiled for NRPS (4, 31). The description of the
putative polyketide products directed by the three gene clusters
pks1, pks2, and pks3 awaits further investigation.
ACKNOWLEDGMENTS
This study was done within the GenoMik program of the BMBF, the
German ministry for education and research.
We thank the Göttingen Genomics Laboratory and especially G.
Gottschalk for continuous support of this project. We are very grateful
to M. Meixner, B. Krebs, and B. Hoeding from FZB Berlin for advice
and support in performing the SSH experiments and biological activity
tests. We are indebted to Nicolas Grammel and Ariane Zwintscher of
the ActinoDrug GmbH for the intensive cooperation in MALDI-
TOF-MS analysis. We also thank Steffen Porwollik, San Diego, Calif.,
and the unknown referees for improving the manuscript by many
corrections and suggestions.
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