In situ detection of the intermediates in the biosynthesis of surfactin, a lipoheptapeptide from Bacillus subtilis OKB 105, by whole‐cell cell matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry in combination with mutant analysis

Abstract

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.

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

“…Because surfactin is an amphiphilic molecule, its accumulation at gas-liquid interfaces can lead to foam production4041. The biosynthesis of surfactin is controlled by a non-ribosomal peptide synthase enzyme (SrfC)42 and a thioesterase/acyltransferase (SrfD)43.…”

“…The biosynthesis of surfactin is controlled by a non-ribosomal peptide synthase enzyme (SrfC)42 and a thioesterase/acyltransferase (SrfD)43. The internal thoiesterase domain of srfC, which is a one-module enzyme, controls the conversion of a linear lipoheptapeptide to its cyclic form, as well as the release of surfactin41. B. subtilis ATCC 6051a produces a large amount of foam during fermentation, which has an extreme affect on fermentation process control and may lead to contamination.…”

Direct surfactin–gramicidin S antagonism supports detoxification in mixed producer cultures of Bacillus subtilis and Aneurinibacillus migulanus

“…1). Although Srf production is linked to swarming (Kinsinger et al, 2003;Kearns & Losick, 2003;Julkowska et al, 2005), B. subtilis OKB120, producing lipopeptides with a partial Srf sequence (Vater et al, 2009), and the non-producer B. subtilis 168 also showed some swarming behaviour (Fig. 1a-d).…”

“…The bacterial samples were bombarded with 5000-20 000 shots and a laser intensity of 40 %. The mass peaks obtained were compared against MS data of Srf produced by Bacillus strains and GS produced by A. migulanus ATCC 9999 (Leenders et al, 1999;Vater et al, 2002Vater et al, , 2009). …”

“…Peptides (10.0 ml) were then diluted to 10.0 mM in water (final volume was 2.00 ml, ,0.2 % ethanol) before measurement. The Gram-positive; strain OKB105 mutant with a mutation between modules 4 and 5 in the srf operon (Nakano et al, 1988(Nakano et al, , 1991Vollenbroich et al, 1994;Vater et al, 2009) Tetrapeptide and shorter Srf fragments Mixed culture assays. To assess the survival in mixed cultures, cultures of B. subtilis OKB120 and M. luteus NCTC8340 were prepared from subcultured preparations and mixed in equal proportions according to c.f.u.…”