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Paenibacillus polymyxa strains are qualified for agro-biotechnological uses such as plant growth promotion and for biocontrol strategies against deleterious phytopathogenic competitors in the soil depending on their attractive arsenal of bioactive compounds. Moreover, they are potent producers of antibiotics for medical applications. To identify new products of such organisms, genome mining strategies in combination with mass spectrometry are the methods of choice. Herein, we performed such studies with the Paenibacillus strain E681. Bioinformatic evaluation of its genome sequence revealed four gene clusters A-D encoding nonribosomal peptide synthetases (NRPSs). Accordingly, four lipopeptide families were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Clusters A and D codify the well known fusaricidins and polymyxins. A yet-unknown lipoheptapeptide was discovered and structurally characterized by de novo sequencing by using MALDI-LIFT-TOF/TOF MS. It was designated as paenilipoheptin. From structure predictions we infer that the production of this agent is encoded by gene cluster C. Gene cluster B encodes the synthesis of tridecaptins, a family of open-chain lipotridecapeptides. Strain E681 produces new subspecies of such compounds (tridecaptins E) showing variations both in their fatty-acid part as well as in their peptide part., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P. polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P. polymyxa strain M-1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed-phase (rp) HPLC. Their structure was determined by MALDI-LIFT-TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m/z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino-3-hydroxyhepta- and nonadecanoic acid as fatty acid constituents. Apparently, the end-standing guanidine group is not modified as shown by direct infusion nano-electrospray ionization mass spectrometry (nano-ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Vesicles of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) are known to disintegrate upon treatment with surfactin, a lipoheptapeptide biosurfactant from Bacillus subtilis OKB 105, as was observed by static light scattering (SLS) and cryo-transmission electron microscopy (cryo-TEM) recently. The lysis of DMPC bilayers occurs strongly dependent on the surfactin concentration according to a three-stage model. Unilamellar DMPC vesicles are disrupted to form sheet-like lamellar intermediates at a moderate surfactant concentration, but undergo a transition towards smaller particles of unknown structure at a higher surfactant concentration according to earlier neutron scattering experiments. Here we present direct structural evidence from cryo-electron tomography data that thread-like micelles with a uniform diameter of 6.5 nm are organized into loops of different sizes at a surfactin concentration of > 15 mol%., (2010 Elsevier B.V. All rights reserved.)

An innovative technique to investigate the intermediates involved in the biosynthesis of the lipoheptapeptide surfactin from Bacillus subtilis OKB105 combining whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with targeted generation of knock-out mutants was demonstrated. This method allows efficient, sensitive detection of biosynthetic intermediates in a minimum of time directly at the outer surface of microbial cells picked from agar plates or in surface extracts prepared thereof. Biosynthesis of surfactin is encoded by the srf-operon which is organized into four open reading frames which have been attributed to three multifunctional NRPS enzymes (SrfA-C) and a thioesterase/acyltransferase enzyme SrfD. For the wild-type strain OKB 105 only the end product surfactin was found mass spectrometrically. For the detection of lipopeptide intermediates three plasmid- and transposon-insertion mutants were generated interrupting the surfactin assembly line at defined positions. Strain LAB 327 was mutated in the spacer region between enzymes SrfA and B. Here only SrfA was active with the lipotripeptide beta-OH-acyl-L-Glu-L-Leu-D-Leu as the end product. Mutant OKB 120 bears a transposon mutation in SrfB between the first and second amino acid activating modules SrfB1 and SrfB2. It showed all intermediates from the lipodi- until to the lipotetrapeptide beta-OH-acyl-L-Glu-L-Leu-D-Leu-L-Val. In LAB 223 SrfC was knocked out by a transposon mutation. It produced the lipohexapeptide beta-OH-acyl-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu. Our work highlights the applicability and the potential of whole-cell MALDI-TOFMS as an innovative efficient tool for the analysis of intermediate steps of biosynthetic pathways., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Abstract: The genome of plant-associated Bacillus amyloliquefaciens FZB42 harbors an array of giant gene clusters involved in synthesis of lipopeptides and polyketides with antifungal, antibacterial and nematocidal activity. Five gene clusters, srf, bmy, fen, nrs, dhb, covering altogether 137kb, were shown to direct synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide, and the iron-siderophore bacillibactin. In addition, one gene cluster encoding enzymes involved in synthesis and export of the antibacterial dipeptide bacilysin is also functional in FZB42. Three gene clusters, mln, bae, and dfn, with a total size of 199kb were shown to direct synthesis of the antibacterial acting polyketides macrolactin, bacillaene, and difficidin. In total, FZB42 dedicates about 340kb, corresponding to 8.5% of its total genetic capacity, to synthesis of secondary metabolites. On the contrary, genes involved in ribosome-dependent synthesis of lantibiotics and other peptides are scarce. Apart from two incomplete gene clusters directing immunity against mersacidin and subtilin, only one peptide-like compound has been detected in the culture fluid that inhibits the growth of B. subtilis lacking the alternative sigma factor W. [Copyright &y& Elsevier]