Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix‐assisted laser desorption/ionization mass spectrometry of intact cells

Abstract

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) has been used for rapid and efficient typing of Bacillus subtilis strains according to their secondary metabolite products using whole intact cells as targets. Cells were grown either in liquid media or on agar plates, respectively. Strains were classified by their unique spectra of bioactive peptides in the m/z range from 500 to 5000. The lipopeptide products surfactin, fengycin and the members of the iturin family were structurally characterized in situ by post-source decay (PSD) analysis. The potential of MALDI-MS for taxonomic studies, as well as for the investigation of the regulation and compartimentalization of secondary metabolites in the bacterial cell, is demonstrated. These achievements qualify this methodology for rapid screening of microorganisms to exploit bioactive compounds for biotechnological and pharmaceutical applications. Figure 4. MALDI-TOF post-source decay mass spectrometric analysis of surfactin from intact cells of B. subtilis 21332. The parent ion at m/z 1061.1 corresponds to the Na adduct of surfactin with a C-15 fatty acid side chain. The b 1 -b 5 ions of the protonated fragments and the b* 1 -b* 6 ions of the Na-fragments are indicated in the spectrum.The insert in the spectrum shows the corresponding structure of surfactin. It is not possible to distinguish Ile and Leu by this technique.

Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciensS499

“…Conversely, little is known about the impact of biofilm formation on lipopeptide production. Some previous studies have reported the consequences of growing on solid support on cLP synthesis by Bacillus and it is clear that lipopeptide production by biofilm-aggregated cells developing on gelified media may be qualitatively and quantitatively modified compared to cells living freely in liquid cultures (Leenders et al, 1999;Nihorimbere et al, 2009). In another approach, Gancel et al (2009) have also demonstrated an enhancement of surfactin production upon cell immobilization on polymer particles.…”

Whole‐cell matrix‐assisted laser desorption/ionization mass spectrometry for rapid identification of bacteriocin/lantibiotic‐producing bacteria

“…Whole-cell MALDI-TOFMS for secondary metabolite profiling of microorganisms was demonstrated for a large diversity of bioactive peptides formed by cyanobacteria 33,34 and Bacillus lipopeptide antibiotics. 5,7,8,11 However, reports on bacteriocin/lantibiotic detection by whole-cell MALDI-MS are rare. Published linear mode MALDI-TOF spectra were of very low resolution, and the variance in the determined mass numbers made unambiguous assignments impossible.…”

“…Published linear mode MALDI-TOF spectra were of very low resolution, and the variance in the determined mass numbers made unambiguous assignments impossible. 7,35,36 The methodology introduced in this communication can be generally applied for efficient screening of bacteria to identify novel bacteriocins and lantibiotics. The sample preparation -whole cells embedded into the DHBs-matrix crystals -exhibited a remarkable stability, which demonstrates the robustness of the technique.…”

Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat

“…The molecular mass and identities of each fraction, as determined by MALDI-TOF mass spectra analysis, are summarized in Table 3. Comparing the mass data obtained for individual fractions and the mass numbers reported for the lipopeptide complexes from other B. subtilis strains [19,[26][27][28][29][30][31][32], the major lipopeptide products of B. subtilis DM-03 could be identified as iturins (C 16 -C 19 ) and surfactins (C 13 -C 15, ) whereas the major lipopeptide isoforms of B. subtilis DM-04 were iturins (C 17 -C 18 ) and surfactins (C 13 -C 15 ). The isoforms of these CLPs were resolved on the basis of their hydrophobicities, which in turn were determined mainly by the length of the fatty acid chains of lipopeptides [19]; therefore iturin isoforms were eluted first from the C 18 -l Nova pak RP-HPLC column followed by surfactin isoforms.…”