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 srfA operon of Bacillus subtilis functions in the biosynthesis of the lipopeptide antibiotic surfactin. On the basis of nucleotide sequence and genetic analysis, it is believed to encode three enzymes (E1A, E1B, and E2) that catalyze the incorporation of the surfactin substrate amino acids. Insertion, deletion, and amino acid substitution mutations of srfA were analyzed for subunit composition and activity as determined by assays of both amino acid-dependent ATP-PPi exchange and aminoacyl thioester formation. Insertion mutations in srfAA (encoding E1A, the subunit that incorporates Glu, Leu, and D-Leu) eliminated production and activity of all three enzymes. Deletions within srfAA and extending from srfAA to srfAB (encoding E1B, which incorporates Val, Asp, and D-Leu) abolished the activity and production of all three enzymes. Insertions between srfAA and srfAB and within srfAB eliminate the production and activity of E1B and E2. An insertion mutation in srfAC (encoding E2, which incorporates Leu) abolished the activity of E2 only. Mutations of the active serine in the putative 4'-phosphopantetheine-binding motif of the second and third domains of E1A eliminated thioester formation and severely reduced the ATP-PPi exchange activity of the two domains. However, the same mutation in the first domain of E1B had little effect on Val-dependent ATP-PPi exchange activity but abolished thioester formation. These results indicate that the coding assignments of the srfA genes are srfAA (E1A), srfAB (E1B), and srfAC (E2).

No abstract

No abstract