"Whole cell"--matrix-assisted laser desorption ionization-time of flight-mass spectrometry, an emerging technique for efficient screening of biocombinatorial libraries of natural compounds-present state of research.

Whole Cell-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) is an emerging sensitive technique for rapid typing of microorganisms, efficient screening of biocombinatorial libraries of natural compounds and the analysis of complex biological samples, as whole cells, subcellular particles, cell extracts and culture filtrates. It is unique to detect metabolites in-situ without the need to isolate and purify the investigated compounds. In favourite cases it enables in-situ structure analysis on the basis of the fragment pattern generated by postsource MALDI-TOF-mass spectrometry. The state of research of this methodology which has mainly been obtained by investigation of lipopeptides from bacilli and the large spectrum of bioactive peptides produced by cyanobacteria is reviewed. The potential of this innovative technique is demonstrated for the lipopeptides produced by various Bacillus subtilis strains.

Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42.

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp(0)), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide. The GenBank accession numbers for gene clusters pks1(bae), pks2, and pks3(dif) are AJ 634060.2, AJ 6340601.2, and AJ 6340602.2, respectively.

Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and in situ structure analysis of streptocidins, a family of tyrocidine-like cyclic peptides.

Streptocidins, a family of tyrocidine-like cyclic decapeptides, are an ideal demonstration object for the detection and in situ structure analysis of natural compounds directly in microbial cells using whole cell matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOFMS), an emerging technique that can be used for rapid sensitive metabolic profiling of microorganisms. Five main members of the streptocidin family (A-E) were detected in Brevibacillus cells picked from agar plates and identified by in situ structure analysis with post-source decay MALDI-TOFMS. This efficient modern method allows the precise detection of metabolites within minutes without the need to isolate and purify the target compounds. The generated mass spectra are of similar quality to those obtained for the purified peptides. In addition, surface extracts were prepared by treating Brevibacillus cells with 70% acetonitrile in the presence of 0.1% trifluoroacetic acid and fractionated by high-resolution reversed-phase high-performance liquid chromatography (HPLC). In this way ten minor streptocidins were detected demonstrating the full biosynthetic variety of streptocidin production on the cellular level. The streptocidins differ from the well-known tyrocidines essentially in position 3 of the decapeptide chain by replacement of the aromatic amino acid (F/W) found in tyrocidines by L-leucine or L-valine.

Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42.

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.