An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.Biosurfactants are a structurally diverse group of surfaceactive molecules synthesized by microorganisms (5,7,9,10,32,36). They have unique amphipathic properties derived from their complex structures, which include a hydrophilic moiety and a hydrophobic portion. The most efficient biosurfactants reduce the surface tension of water from 72 dynes/cm to values in the range of 25 to 30 dynes/cm. Biosurfactant production can be determined by measuring the change in surface tension of cell-free culture broth.Microbial surfactants have commonly been classified into the following categories: (i) glycolipids, (ii) lipopeptides, (iii) fatty acids, neutral lipids, and phospholipids, (iv) polymeric surfactants, and (v) particulate biosurfactants (5,7,9,10,32,36). The lipopeptides are an interesting class of microbial surfactants (36) because of their manifold attractive properties. Members of this group often possess antibiotic activity as well.Bacillus subtilis strains produce a broad spectrum of bioactive peptides with great potential for biotechnological and biopharmaceutical applications. A well-known class of such compounds includes the lipopeptides surfactin (1, 13, 14, 17, 18), fengycin (35), and the iturin compounds (3) (iturins [26], mycosubtilins [25], and bacillomycins [27]), which are amphiphilic membrane-active biosurfactants and peptide antibiotics with potent antimicrobial activities. All these agents occur as families of closely related isoforms which differ in the length and branching of the fatty acid side chains and in the amino acid substitutions in the peptide rings (20,36). The surfactin and iturin compounds are cyclic lipoheptapeptides which contain a -hydroxy fatty acid and a -amino fatty acid, respectively, as lipophilic co...
The aim of this study was to isolate bacteria that are resistant to the strong antimicrobial metabolites characteristic of Aplysina aerophoba. For this purpose, bacterial isolation was performed on agar plates to which sponge tissue extract had been added. Following screening for antifungal and antimicrobial activities, 5 strains were chosen for more detailed analyses. 16S ribosomal DNA sequencing revealed that all isolates belonged to the genus Bacillus, specifically B. subtilis and B. pumilus. Using a combination of matrix-assisted laser desorption/ ionization mass spectrometry typing of whole cells and antimicrobial bioassays against selected reference strains, the bioactive metabolites were identified as lipopeptides.
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.
Metabolic diversity is being studied intensively by evolutionary biologists, but so far there has been no comparison of biosynthetic pathways leading to a particular secondary metabolite in both prokaryotes and eukaryotes. We have detected the bioactive anthraquinone chrysophanol, which serves as a chemical defense in diverse eukaryotic organisms, in a bacterial Nocardia strain, thereby permitting the first comparative biosynthetic study. Two basic modes of folding a polyketide chain to fused-ring aromatic structures have so far been described: mode F (referring to fungi) and mode S (from Streptomyces). We have demonstrated that in eukaryotes (fungi, higher plants and insects), chrysophanol is formed via folding mode F. In actinomycetes, by contrast, the cyclization follows mode S. Thus, chrysophanol is the first polyketide synthase product that is built up by more than one polyketide folding mode.
In this paper, the initiation reactions in surfactin biosynthesis by Bacillus subtilis OKB 105 were investigated. Evidence for a specific role of the SrfD protein, the external thioesterase enzyme in surfactin biosynthesis, was obtained for the first time. The action of SrfD was investigated both with the native, but only partially purified, enzyme and the highly purified, His-tagged protein overexpressed in Escherichia coli. Surfactin can be formed by the interaction of the three amino acid activating components of surfactin synthetase SrfA, B and C alone. This process is stimulated by SrfD. In the initiation reactions, the beta-hydroxy fatty acid substrate is transferred from beta-hydroxymyristoyl-coenzyme A to the start enzyme SrfA followed by formation of beta-hydroxymyristoyl-glutamate. The same reactions were also observed with the recombinant L-Glu-activating module of surfactin synthetase. Lipopeptide formation can be initiated by these function units alone, but SrfD efficiently supports and stimulates the formation of initiation products. From these results, we infer that SrfD functions as the thioesterase/acyltransferase enzyme in the initiation process previously postulated by Menkhaus et al. [Menkhaus et al. (1993) J. Biol. Chem. 268, 7678-7684], thus enhancing surfactin formation.
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