Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria

Abstract

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A lipopeptides (LPs) C14 and C15 are potent biosurfactants synthetized by the Bacillus subtilis I'1a strain. The biological activity of extracted LPs was confirmed by examining extracts from I'1a cultures against uropathogenic bacteria that had been isolated from biofilms on urinary catheters. Compared with cultures of DSM 3257, which produce surfactin at a relatively low level, the extract obtained from strain I'1a exhibited a greater inhibitory effect against both planktonic and sessile forms of Escherichia coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and Enterococcus faecalis. Moreover, cyclic LP biosurfactants may disturb the integrity of cytoplasmic membranes; therefore, we investigated the effects of synthetized LPs on fatty acids and phospholipids of B. subtilis. LPs and lipids were analyzed using GC-MS, LC-MS/MS and MALDI-TOF/TOF techniques. Compared with B. subtilis DSM 3257, membranes of the I'1a strain were characterized by an increased amount of anteiso fatty acids and a ten-fold higher ratio of phosphatidylglycerol (PG)-to-phosphatidylethanolamine (PE). Interestingly, in cultures of B. subtilis DSM 3257 supplemented with LP extracts of the I'1a strain, the PG-to-PE ratio was fourfold higher, and the amount of anteiso fatty acids was also increased.

Keywords: Bacillus subtilis; Iturin; Lipidomics; Lipopeptides; Phospholipids; Uropathogens.

Fig. 1

Growth of B. subtilis species during 72-h cultivation in LB medium. Error bars represent standard deviation (n = 3)

Fig. 2

MRM chromatograms and MALDI-TOF spectra of lipopeptides isolated from B. subtilis DSM 3257 (a, c) and I′1a (b, d). All extracts were obtained from a similar culture volume. The RT for surfactin, iturun A and fengycin homologues was 2.8, 2.53 and 3.2, respectively

Fig. 3

MALDI-MS/MS spectrum of [M + H]⁺ ion at m/z 1057.5 from B. subtilis I′1a

Fig. 4

Relative percentage of phospholipid classes measured in B. subtilis during 72 h of culture. PA phosphatidic acid, PE phosphatidylethanolamine, PG phosphatidylglycerol, LPG lysyl-phosphatidylglycerol, Cl cardiolipin. Error bars indicate standard deviation (n = 3)

Fig. 5

Comparison of phospholipids extracted from 48-h-old B. subtilis strains. PA phosphatidic acid, PE phosphatidylethanolamine, PG phosphatidylglycerol, LPG lysyl-phosphatidylglycerol, Cl cardiolipin. Data indicate mean ± SD (n = 3)

Fig. 6

Relative abundance of phospholipids classes (a) and species (b) in B. subtilis DSM 3257 incubated with I′1a LPs extract. PA phosphatidic acid, PE phosphatidylethanolamine, PG phosphatidylglycerol, LPG lysyl-phosphatidylglycerol, Cl cardiolipin. Data represent mean ± SD (n = 3)