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Biochimica et Biophysica Acta 1828 (2013) 2370–2378

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Biochimica et Biophysica Acta

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Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of subtilis

Gabriela Seydlová a,⁎, Radovan Fišer a, Radomír Čabala b, Petr Kozlík b, Jaroslava Svobodová a,⁎, Miroslav Pátek c

a Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Prague 2, Czech Republic b Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 40 Prague 2, Czech Republic c Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic

article info abstract

Article history: Surfactin is a cyclic that disturbs the integrity of the cytoplasmic membrane. In this study, Received 1 March 2013 the role of membrane lipids in the adaptation and possible surfactin tolerance of the surfactin producer Bacillus Received in revised form 21 June 2013 subtilis ATCC 21332 was investigated. During a 1-day cultivation, the of the cell membrane were Accepted 28 June 2013 analyzed at the selected time points, which covered both the early and late stationary phases of growth, when Available online 8 July 2013 surfactin in the medium gradually rose from 2 to 84 μmol·l−1. During this time period, the + Keywords: composition of the surfactin producer's membrane (Sf ) was compared to that of its − fi Surfactin non-producing mutant (Sf ). Substantial modi cations of the polar head group region in response to the presence of surfactin were found, while the fatty content remained unaffected. Simultaneously with Membrane surfactin production, a progressive accumulation up to 22% of the stress phospholipid cardiolipin was Phospholipid determined in the Sf+ membrane, whereas the proportion of phosphatidylethanolamine remained constant. Cardiolipin At 24 h, cardiolipin was found to be the second major phospholipid of the membrane. In parallel, the Laurdan generalized polarization reported an increasing rigidity of the lipid bilayer. We concluded that an enhanced level of cardiolipin is responsible for the membrane rigidification that hinders the fluidizing effect of surfactin. At the same time cardiolipin, due to its negative charge, may also prevent the surfactin-membrane interaction or surfactin pore formation activity. © 2013 Elsevier B.V. All rights reserved.

1. Introduction destabilization [14–18]. It was found that surfactin interacts with the lipid membrane in three different ways: (i) it acts as a mobile cation Surfactin is a lipopeptide antibiotic produced by Bacillus subtilis. It is carrier [19,20], (ii) forms cationic channels [21,22], and (iii) destroys the formed of a cyclic heptapeptide interlinked with a β-hydroxy membrane via a detergent effect [16]. Membrane leakage was observed comprising 12–16 atoms. Surfactin interacts with the membrane at a surfactin concentration of 2 μmol·l−1 [23],i.e.farbelowitscritical and disturbs its integrity [1]. This effect can explain a wide range of concentration (CMC), which ranges from 9 to 50 μmol·l−1, surfactin biological activities such as antibacterial [2–4], antiviral [5], depending on the experimental conditions [24–26]. Channel formation, antifungal [6], antimycoplasma [7], antiparasitic [8] and anti-tumor [9]. membrane or solubilization of the membrane to has been Surfactin as a powerful biosurfactant stimulates biofilm formation by its observed above the CMC of surfactin [21,22,27,28]. Although the molecu- producer strains [10,11]; however, inhibits the biofilm formation of lar mechanism of these complex lipopeptide-membrane interactions has pathogenic [12,13]. been intensively studied, it is not yet fully understood [22,28]. The high demand for new chemotherapeutics driven by the increas- Several studies on model membranes demonstrate the impact of ing drug resistance of pathogens has drawn attention to antibiotic the lipid composition on surfactin-membrane interaction and its that represent potential new antimicrobial agents. A penetration into the bilayer. Both the polar heads and fatty acid number of systematic in vitro studies were therefore devoted to chains play a role in the formation of complexes of surfactin with uncovering the molecular mechanism of surfactin-induced membrane phospholipids [29,30]. Fatty with shorter chains, which have a similar length to the surfactin hydrocarbon chains, interact better with surfactin than fatty acids with longer chains like stearic acid ⁎ Corresponding authors at: Department of Genetics and Microbiology, Faculty of Science, [31]. The polar head group composition profoundly affects the Charles University in Prague, Viničná 5, 128 44 Prague 2, Czech Republic. Tel.: +420 221 951 behavior of surfactin at the phospholipid– interface and its 738; fax: +420 221 951 724. E-mail addresses: [email protected] (G. Seydlová), [email protected] miscibility with phospholipids. Both the electrostatic properties [30] (J. Svobodová). and the volume of the phospholipid head groups modulate these

0005-2736/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamem.2013.06.032 G. Seydlová et al. / Biochimica et Biophysica Acta 1828 (2013) 2370–2378 2371

surfactin–phospholipid interactions [29,32]. Phosphatidylethanolamine medium (0.2% (NH4)2SO4,1.4%K2HPO4,0.6%KH2PO4,0.1%sodiumcitrate, (PE) promotes surfactin stabilization in the model bilayer; presum- 0.02% MgSO4, 0.3% glucose, 0.0005% casamino acids). 0.2 μgofplasmid ably the inverted cone molecular shape of surfactin complements DNA and 1 ml of warm transformation medium were added to 100 μl cone-shaped PE [27,31]. With dipalmitoylphosphatidylserine of the diluted culture, and the tubes were incubated with shaking at (DPPS), surfactin decreases the electrostatic repulsions between 37 °C for 1 h. The tubes were centrifuged for 3 min at 13,000 ×g,the the negatively-charged head groups of DPPS, which results in pellet was resuspended in 300 μl of LB medium and the cells were plated DPPS–surfactin stability in a monolayer [29]. Similarly, the presence on LB agar with chloramphenicol (5 μg·ml−1). of cations that neutralize membrane surface negative charges facilitates the penetration of surfactin into the lipid bilayer [33]. 0 − All antibiotic-producing bacteria ensure their self-resistance by 2.3. Construction of the sfp (Sf ) strain coding for various means of self-defense mechanisms. At the same time, the systematic use of selects resistant variants of Surfactin is produced by nonribosomal synthesis by the surfactin the susceptible target pathogens. As for the resistance mechanism, synthetase complex coded by the srfA operon [38] and sfp gene. The the antibiotic-producing strains are the most convenient source of srfA operon is also found in the chromosome of non-producing strains information [34]. Surprisingly, almost no research has been focused such as B. subtilis 168. However, the sfp gene, which is essential for on the nature of the surfactin resistance of the surfactin producer. It surfactin synthesis, carries a frameshift mutation in B. subtilis 168 is most likely the cytoplasmic membrane which is targeted by yielding a non-functional protein [39]. An isogenic strain defective 0 surfactin both from the cytoplasmic and extracellular side that is surfactin synthesis (sfp mutant) based on the parental strain B. the site of self-resistance against surfactin. In Bacilli, resistance to subtilis ATCC 21332 was constructed as follows. An internal fragment antimicrobial compounds is mediated by the genes controlled by covering 784 bp of the sfp gene carrying the frameshift mutation was fi the extracytoplasmic function sigma factor σw [35]. Nevertheless, ampli ed by PCR using B. subtilis 168 genomic DNA as a template and none of these resistance genes were proven to be engaged in surfactin the primers sfpF1 (TAGAATTCTTGTGGAAGTATGATAGGAT) and sfpR1 fi resistance. The only gene plausibly involved in surfactin resistance is (GAGAATTCAAGATATT GAGCGAGGTG). The ampli ed fragment was swrC [36,37]. It codes for the proton-dependent multidrug efflux cloned in the EcoRI site of the vector pK19cat [40]. The resulting 0 pump belonging to the RND family and contributes to the secretion plasmid pK19cat-sfp was used to transform the B. subtilis ATCC of surfactin. However, surfactin production was observed even in a 21332 strain. The clones with an abolished ability to produce fi swrC-deficient strains that persistently survived at surfactin were identi ed by examination on blood agar according to higher than 10 mmol·l−1 [37]. This finding suggests the existence the absence of erythrocyte lysis. The presence of the frameshift of other additional mechanisms that participate in the surfactin mutation within the sfp gene in the chromosome of these clones fi self-resistance of the producer. was veri ed by sequencing. The parental B. subtilis ATCC 21332 strain In this study, we investigated the modifications of the membrane and its surfactin non-producing derivative are here designated the + − lipid moiety induced by surfactin during its production in order to Sf and Sf strain, respectively. estimate the role of phospholipids in the membrane adaptation resulting in surfactin tolerance. These changes were further corre- 2.4. Surfactin analysis lated with fluidity of the cytoplasmic membrane estimated by means of Laurdan generalized polarization. Samples of the culture were mixed with methanol (1:1, v/v) and centrifuged at 10 000 ×g for 10 min to remove the cell biomass. Surfactin 2. Materials and methods concentration was determined in the cell-free supernatants by reverse-phase C18 HPLC using a HPLC Agilent 1200–Agilent 6460 Triple 2.1. Bacterial strains and growth conditions Quadrupole MS system equipped with a Zorbax Eclipse XDB-C18 column (5 μm, Agilent Technologies). The mobile phase consisted of (a) 0.1% + Bacterial strains used in this study were B. subtilis ATCC 21332 (sfp ) formic acid in acetonitrile and (b) aqueous 0.1% formic acid at a ratio of (wild type, American Type Culture Collection) and B. subtilis strain 0164 a/b 20%:80% (v/v) and the mobile phase flow rate used for the analysis (sfp0) constructed in this work (see Section 2.3). B. subtilis strains were was 1 ml/min. Sample size was 50 μl. Five surfactin isoforms (C12–C16 grown aerobically (120 rpm) in nutrient broth (1 g Lab-Lemco powder, surfactin and sodium adduct ) were detected at m/z 994.6 and 2 g Bacto yeast extract, 5 g Bactopepton, 5 g NaCl per liter) at 30 °C. 1016.6, m/z 1008.6 and 1030.6, m/z 1022.6 and 1044.6, m/z 1036.6 and Culture growth was monitored turbidimetrically at 420 nm. The culture 1058.6, m/z 1050.6 and 1072.6, respectively. Commercial surfactin was inoculated with the overnight inoculum, diluted in the morning (Sigma)servedasastandard.Thedata were analyzed by Agilent with fresh medium and grown to the exponential phase (OD420 of 0.5 MassHunter Workstation Data Acquisition and Agilent MassHunter to 0.6). Finally, the cells were used to inoculate the particular cultivation. Data Analyses. After 3 h, bacterial cultures were harvested by rapid filtration through a Synpor no. 5 filter (Pragochema, Czech Republic). In the stationary phase of growth, the cells were harvested by centrifugation (4300 ×g, 2.5. Membrane isolation 15 min, 4 °C). The number of viable vegetative cells (CFU) was counted by plating diluted cultures on nutrient agar. In parallel, thermoresistant Cytoplasmic membranes were isolated using enzyme digestion, which spores were counted after heat inactivation of the culture (70 °C, enables the membrane fraction to be purified from spore contamination 15 min) and estimated as CFU. [41]. The cells were incubated with lysozyme at the cultivation temper- ature of the initial culture (30 °C) for 30 min with exponential cells or 2.2. Transformation of B. subtilis for 60 min with stationary cells. The cell lysate was centrifuged for 10 min at 3000 ×g and 4 °C to remove the crude cell debris and spores,

A total of 10 ml of pre-transformation medium (0.2% (NH4)2SO4, which remained unaffected by lysozyme. The cell-free supernatant was 1.4% K2HPO4, 0.6% KH2PO4, 0.1% sodium citrate, 0.02% MgSO4, 0.5% then centrifuged (25 000 ×g,25min,4°C)toseparatethemembrane −1 glucose, 5% tryptophane, 0.02% casamino acids, 75 mmol·l CaCl2 vesicles from the cytoplasmic fraction. The membrane sediment was −1 and 2 mmol·l MnSO4) was inoculated with a single colony and used directly for lipid extraction or resuspended in phosphate buffer grown at 37 °C until the culture reached the stationary phase of (100 mmol·l−1,pH6.6)andstoredat−80 °C. Pierce BCA Protein growth. The culture was diluted 10-fold in fresh transformation Assay (Pierce Biotechnology) was used for protein determination. 2372 G. Seydlová et al. / Biochimica et Biophysica Acta 1828 (2013) 2370–2378

2.6. Phospholipid extraction and analysis and mean values of GP were calculated for each image. The graph shows average GP value for all analyzed images ± S.D. Membrane phospholipids (PLs) were extracted from B. subtilis membrane preparations with hexane-isopropanol (3:2, v/v) 2.9. Statistics mixture [42]. After evaporation of the solvent in vaccuo at 40 °C, the PLs were dissolved in chloroform, filtered through glass fiber Student's t test was used to establish differences between two filters (Whatman) and concentrated under a stream of nitrogen. mean values of PL and FA levels, GP and growth parameters. P values The lipid samples were stored at −80°Candusedforpolarhead of b0.05 were considered significant. group or fatty acid analysis. Six PL classes – phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), 3. Results cardiolipin (CL), lysylphosphatidylglycerol (lysylPG), and phospha- tidic acid (PA) – were separated by thin-layer chromatography 3.1. Growth of Sf+ and Sf− B. subtilis strains and surfactin production (TLC, silica gel 60 G plates; Merck) in chloroform-methanol–water (65:25:4, v/v/v) as the mobile phase, collected from the plates and In antibiotic-producing cells, the self-resistance mechanisms are then quantified spectrophotometrically, as described by Rouser et activated simultaneously with the biosynthesis of antibiotics. The al. [43]. The average ± S.D. of results from three independent expression of the defense systems subsequently increases as the experimentsispresented. production progresses. In order to characterize the adaptation mechanisms during surfactin production, the growth characteristics of the wild-type surfactin producer (B. subtilis ATCC 21332, Sf+) and 2.7. Fatty acid analysis its non-producing mutant (B. subtilis 0164, Sf−) were examined during a 1-day cultivation in parallel with the determination of Membrane lipids were transesterified to fatty acid methyl esters surfactin concentration in the growth medium. by incubation in sodium methoxide at room temperature [44]. As shown in Fig. 1, the doubling times of the surfactin producer After neutralization by the addition of methanolic HCl, the and its non-producing mutant were both T = 37 ± 2 min. Both fatty acid methyl esters (FAMEs) were extracted three times with cultures entered the stationary phase of growth (t ) after 4 h of 200 μl of pentane and dried under the flow of nitrogen. Methyl 0 cultivation at log (OD × 1000) = 10.1 ± 0.1. These data indicat- esters were dissolved in 100 μl of heptane and analyzed by gas 2 420 ed that the sfp0 mutation resulting in the Sf− phenotype did not affect chromatography–mass spectrometry (GC–MS) (Shimadzu GC17A– the growth of the non-producing strain in the exponential phase. QP5050A gas chromatograph–mass spectrometer) in a DB-35 However within the stationary phase of growth, while surfactin was column (Agilent Technologies; 32 m by 0.25 mm, film thickness accumulating in the cultivation medium, wild-type B. subtilis ATCC 1 μm). Electron impact spectra were obtained at 70 eV of electron 21332 exhibited a slightly but reproducibly higher viability than the energy. The following operating conditions were used: injection Sf− strain. After 24 h of cultivation (t ), the cell biomass of the Sf+ temperature of 250 °C and initial oven temperature of 50 °C, rising 20 and Sf− strains reached log (OD × 1000) 12.5 ± 0.1 and at 7.5 °C/min to 250 °C, and isothermally for 20 min at 250 °C. The 2 420 12.2 ± 0.2 (P b 0.009), respectively. FAMEs were identified with the aid of FAMEs standards (Sigma) The slight difference in the growth capacity of the two strains and the identity was confirmed using the NIST Mass Spectral Library also resulted in different total vegetative cell counts, which at t (2006). The average ± S.D. of results from three independent 20 reached 4.3 ± 0.2 × 108 ml−1 (Sf+) and 3.7 ± 0.1 × 108 ml−1 experiments is presented below. The percentage of each FA was (Sf−)(P b 0.006), respectively. The proportion of thermoresistant calculated by the ratio of peak area/sum of total identified peak spores was almost the same value in both cultures, 27% (Sf+)and areas. 26% (Sf−), respectively. Based on the course of the growth curves of the two strains, the 2.8. Laurdan generalized polarization time points for sampling and determining the surfactin concentration in the cultivation medium were chosen. As documented in Table 1, Laurdan generalized polarization (GP)measurementswere the presence of surfactin at a concentration of 2.2 mg·l−1 was first carried out using a confocal microscope (Leica TCS SP2). 2- detected after 4.5 h of cultivation (t0.5), i.e. as early as 30 min after Dimethylamino-6-lauroylnaphthalene (Laurdan, Molecular Probes) was entry into the stationary phase, and it gradually accumulated up to −1 −1 added to the membrane samples (protein concentration of 75 μg·ml ) 87 mg·l after 24 h (t20). Four surfactin isoforms with a fatty acid in a phosphate buffer (100 mmol·l−1, pH 7) from a DMSO to chain length of 13 to 16 carbon atoms were identified. The −6 −1 give a final probe concentration of 10 mol·l . After incubation in C15-surfactin isoform was found to be the predominant constituent, the dark at 41 °C for 45 min, the pelleted membranes were placed on a whereas there were only traces of the C16 isoform. The relative coverslip and GP was measured under the confocal microscope at 25 °C proportions of the surfactin isoforms remained stable over the using 405-nm diode laser for excitation. Emission intensities of blue whole cultivation period. No surfactin was detected in the culture fluorescence (415–460 nm) and green fluorescence (460–540 nm) broth of the Sf− strain. were acquired simultaneously into two separated channels using HCX The capacity of the cells to produce surfactin during the increasing PL APO CS 63.0 × 1.40 oil objective, 512 × 512 pixel format with nutritional stress of the stationary phase is illustrated by the amount of 400 Hz scan speed, 4× line averaging and 12-bit picture depth. High surfactin produced per vegetative cell (Table 1). Surfactin production voltage of both photomultiplier tubes was kept constant at 600 V. The steadily rose throughout the early stationary phase of growth (t0–t3). intensity of recorded fluorescence was modified only by laser power. During this period, the amount of surfactin rose from 0.02 to 0.17 pg

Approximately 50 fluorescence photographs were collected for each sam- per cell and reached 0.21 pg per cell at t20. ple and processed using ImageJ software (http://rsbweb.nih.gov/ij/, The data presented above allowed us to select a time-scale for the http://www.uhnresearch.ca/facilities/wcif/)asfollows:separated,green subsequent membrane analyses of cells adapted to their own toxic and blue channels were corrected by subtraction of background intensi- product which covered the whole range of cultivation stages and ties (about 10%) deduced from autofluorescence of unlabeled samples. surfactin concentrations. These were as follows: t−1, when the GP was calculated on a pixel by pixel basis from the blue and green culture grew exponentially and surfactin was not yet synthetized. At channel intensities GP =(Iblue − Igreen)/(Iblue +Igreen). The image about t0.5 the surfactin synthesis was initiated and progressed further areas containing membranes were selected by intensity thresholding (t1.5). We supposed that during this period, the defense mechanism G. Seydlová et al. / Biochimica et Biophysica Acta 1828 (2013) 2370–2378 2373

3.2. Membrane phospholipid profiles

The polar head groups of membrane phospholipids can profoundly modify the surfactin–membrane interaction. To estimate the role of the membrane surface in cell protection against surfactin, the changes in PL composition were monitored in the producing cells throughout the 24-h cultivation. A parallel analysis of the Sf− mutant provided data on the PL modifications that were induced during the stationary phase of growth merely by increasing nutritional and energy depletion, without the effect of surfactin. A detailed analysis of the PL profiles of the Sf+ strain showed that the adaptive response of the cells to surfactin accumulation in the culture medium developed gradually.

In the exponential phase of growth (t−1) in the absence of surfactin, the composition of membrane PLs (Table 2)ofthetwoB. subtilis strains was nearly identical. Phosphatidylglycerol, a principal lipid component of the membrane, amounted to approximately 40% of the total. This barrel-shaped phospholipid that stabilizes the lamellar phase of

membrane bilayer predominated until t20, when the heavy stress of the late stationary growth was combined with the rising concentration of surfactin in the producer strain. At this point, a drop in the PG content from 38 to 16% was observed in the Sf+ strain, while the PG level was reduced to a lesser extent (28%) in the Sf− strain. This 12% difference suggested the involvement of PG in the adaptive response of the surfactin producer, as its decrease correlated with the enhanced level of CL, which is synthesized from two PG . Remarkably, in the Sf+ strain the CL content gradually increased

throughout the stationary phase of growth and peaked at t20 when the surfactin stress culminated. The proportion of CL in the

membrane increased from 13 to 22% (P b 0.01) at t20. Cardiolipin was the second most abundant PL in the membrane of the Sf+ strain in the final stage of CL cultivation. In contrast, in the Sf− strain the

proportion of CL was maintained at a constant level until t20, when it fell to as little as 8%. This opposite tendency indicated that the increase in CL content, similarly to the decrease of PG, was a specific response of the cell to surfactin. Fig. 1. Growth of B. subtilis ATCC 21332 (A) and 0164 (B) strains during 1-day cultivation. During surfactin production the proportion of phosphatidylethanol- Cultures were grown in nutrient broth at 30 °C with shaking. Total bacterial counts were amine, another major representative of B. subtilis membrane PL, was assessed by plating on agar medium and expressed as colony forming units (CFU). The + maintained at a constant level of 22% until t3 in the Sf strain and at t20 proportion of thermoresistant spores in the samples was determined by colony counts the PE content decreased to 11%. In contrast, in the Sf− strain the amount after exposure to a temperature of 70 °C for 15 min. The time point of entry into the of PE rose to 30% at t andthenfellto16%att . Thus, the PE content in the stationary phase of growth is designated as t0. The data represent the means and standard 3 20 + deviations from five (growth) or three (CFU) independent experiments. membrane of the Sf strain was held under control during surfactin pro- duction. The level of the other aminophospholipid, phosphatidylserine,

remained stable throughout the whole cultivation, including t20. At t20, the polar head group composition of the membrane samples against the deleterious effect of surfactin was also being induced. changed sharply, probably as a consequence of the continuing presence

Finally, at t3 and t20, the surfactin concentration was high enough to of the antimicrobial concentration of surfactin combined with the deep be harmful to the producing cells and the resistance response should nutritional exhaustion of the late stationary phase. Accordingly, the therefore have been fully expressed. Since only growing vegetative biosynthesis of PL was strictly reduced and the content of PA, i.e. the cells produce surfactin, the spores present in the culture were common precursor of PL, dramatically rose from trace levels to 36 and separated prior to each analysis in order to exclude biochemical 32% (P b 0.009) in the Sf+ and Sf− strain, respectively. In fact, the fall in contamination of the membranes (see Section 2.5). the proportions of PG + PE in the membranes of the two strains

Table 1 Concentration of surfactin in culture broth of B. subtilis ATCC 21332 during 1-day cultivation.

Time point in stationary phase Surfactin isoforms (mg·l−1)a Total Surfactin (pg) per vegetative cellb

C13 C14 C15 C16

t−1 0.0 0.0 0.0 0.0 0.0 0.0

t0.5 0.2 ± 0.0 0.5 ± 0.0 1.4 ± 0.0 0.1 ± 0.0 2.2 ± 0.0 0.02 ± 0.00

t1.5 0.8 ± 0.0 0.9 ± 0.0 10.6 ± 0.1 0.2 ± 0.0 12.5 ± 0.1 0.08 ± 0.00

t3 3.7 ± 0.1 4.3 ± 0.2 34.5 ± 0.9 0.6 ± 0.0 43.1 ± 1.1 0.17 ± 0.01

t20 8.9 ± 0.2 8.1 ± 0.2 69.1 ± 0.2 1.1 ± 0.0 87.2 ± 0.2 0.21 ± 0.01 Surfactin concentration in the cultivation medium was measured by LC/MS as described in Section 2.4. The data represent the means ± S.D. from three independent measurements performed in duplicate. a Surfactin isoform with fatty acid chain length of 13, 14, 15 and 16 carbon atoms, respectively. b Surfactin production in time expressed as the amount of surfactin (in pg) produced per B. subtilis ATCC 21332 vegetative cell (CFU) during the stationary growth phase. 2374 G. Seydlová et al. / Biochimica et Biophysica Acta 1828 (2013) 2370–2378

Table 2 Phospholipid composition of B. subtilis cytoplasmic membrane.

Time (h) % of total phospholipid content (mean ± S.D.)

t−1 t0.5 t1.5 t3 t20 Strain ATCC 21332 0164 ATCC 21332 0164 ATCC 21332 0164 ATCC 21332 0164 ATCC 21332 0164

LysylPG 3.3 ± 0.3 2.8 ± 0.6 7.6 ± 1.1 5.6 ± 1.3 5.3 ± 1.0 4.5 ± 0.5 3.4 ± 0.6 5.5 ± 0.4 2.8 ± 0.6 1.5 ± 0.1 PS 10.8 ± 1.0 11.1 ± 1.1 13.2 ± 0.0 13.3 ± 1.3 11.5 ± 1.0 11.8 ± 1.1 14.0 ± 0.8 11.6 ± 0.8 13.9 ± 0.3 13.5 ± 0.7 PG 42.6 ± 0.6 43.5 ± 0.4 37.2 ± 0.7 38.7 ± 1.4 39.5 ± 0.6 43.3 ± 1.7 37.9 ± 1.4 36.0 ± 0.6 16.1 ± 1.3 28.3 ± 1.6 PE 25.7 ± 1.5 25.1 ± 1.1 22.1 ± 1.8 21.4 ± 0.7 21.7 ± 1.1 26.5 ± 1.2 22.2 ± 1.1 29.9 ± 1.6 11.0 ± 1.2 16.4 ± 0.8 CL 13.4 ± 0.7 13.8 ± 1.2 12.8 ± 0.7 13.3 ± 0.0 17.9 ± 1.3 13.6 ± 1.0 19.7 ± 0.7 14.1 ± 0.7 21.9 ± 0.5 8.4 ± 1.1 PA 4.2 ± 0.5 3.8 ± 0.8 7.2 ± 1.2 7.8 ± 1.1 4.2 ± 0.8 3.2 ± 0.7 3.0 ± 0.1 2.9 ± 0.2 35.6 ± 0.5 31.9 ± 1.3

Values represent means ± S.D. from three determinations. LysylPG—lysylphosphatidylglycerol, PS—phosphatidylserin, PG—phosphatidylglycerol, PE—phosphatidylethanolamine, CL—cardiolipin, PA—phoshatidic acid. accompanied by the drop in CL in the Sf− strain was quantitatively total iso-branched-chain FAs rose from 36 to 47% in the Sf+ strain, replaced with PA. Nevertheless, despite the energy depletion and ongoing whereas the content of these FAs rose from 39 to 56% of the total in the surfactin production, the pathway leading to CL was still active in the Sf− strain. In the surfactin producer, the decline in the overall level of surfactin-producing cells and CL was the second most abundant anteiso-branched-chain FAs was caused by an 8% drop in the content of membrane PL at the end of the cultivation. This fact indicates its a-15:0 to 33% and by an increase in the proportion of i-15:0 from 17 to importance in the membrane bilayer of the Sf+ strain. 24%. In the surfactin non-producing mutant the observed changes were more profound, as the proportion of a-15:0 fell to 28% and i-15:0 rose to 30%. These changes led to a slight prevalence of the iso-branching 3.3. Membrane fatty acid composition pattern in the Sf− strain and anteiso-branched chain FAs in the Sf+ strain. To a large extent these shifts in FA composition reflected the general late Phospholipid analyses revealed specificmodifications in the polar stationary phase stress, as a similar response was observed in both head groups of membrane phospholipids in the surfactin producer, B. subtilis strains irrespective of the surfactin production. suggesting the impact of the membrane surface composition on the cell GC–MS analyses revealed no substantial differences between the protection against surfactin. Similarly to PL head groups, the fatty acids surfactin producing and non-producing B. subtilis strain in terms of of the membrane, particularly the aliphatic chain length of phospholipids, the presence of acyl chain length profiles, the proportion of straight can also strongly influence the surfactin–membrane interactions. saturated or unsaturated FAs. In contrast to the polar head groups of Therefore, the FA profiles in the different stages of surfactin production membrane PL, the FA composition in the surfactin producer in the Sf+ strain were compared with its non-producing counterpart at developed in a similar manner as in its non-producing counterpart, the same cultivation time points (Table 3). and the slight differences in the levels of iso-FAs are unlikely to be At t−1, similarly to the polar head group of PL, the FA composition of due to the adaptation to surfactin. both strains was the same with almost 90% proportion of the branched-chain FAs. Of these, anteiso- and iso-branching FA accounted for 60% and 30%, respectively. This distribution of FA in the cytoplasmic 3.4. Fluidity of the cytoplasmic membrane membrane was almost unchanged in both strains until t3. + Thelatestationaryphase(t20) was accompanied by alterations in four The fluidity of cytoplasmic membranes isolated from the Sf and major FAs in the membrane. These were branched-chain FAs with Sf− strains was measured by means of Laurdan generalized polariza- aliphatic chain lengths of 15 and 17 carbon atoms. The proportion of tion. The fluorescent membrane probe Laurdan incorporates at the high-melting rigidizing iso-series increased with a corresponding hydrophilic–hydrophobic interface of the phospholipid bilayer. It is decrease in low melting anteiso-branched-chain FAs (Fig. 2). At t20,the known to be sensitive to the polarity of the environment, exhibiting

Table 3 Fatty acid composition of B. subtilis cytoplasmic membrane.

FA Tm (°C) % of total

t−1 t0.5 t1.5 t3 t20 ATCC 21332 0164 ATCC 21332 0164 ATCC 21332 0164 ATCC 21332 0164 ATCC 21332 0164

i-14:0 53 1.4 ± 0.1 2.4 ± 0.1 1.0 ± 0.1 1.4 ± 0.1 0.8 ± 0.0 0.8 ± 0.0 0.9 ± 0.2 1.2 ± 0.0 1.6 ± 0.1 1.3 ± 0.1 14:0 54 0.6 ± 0.1 0.7 ± 0.0 0.4 ± 0.0 0.3 ± 0.4 0.3 ± 0.0 0.4 ± 0.2 0.2 ± 0.1 0.2 ± 0.0 0.4 ± 0.3 0.1 ± 0.0 i-15:0 52 13.0 ± 0.5 12.3 ± 0.4 15.3 ± 1.1 13.4 ± 1.1 15.3 ± 1.6 15.1 ± 1.6 16.9 ± 0.5 19.8 ± 0.7 24.0 ± 0.6 30.2 ± 0.8 a-15:0 23 42.0 ± 1.0 38.3 ± 0.5 41.7 ± 0.7 41.9 ± 0.1 40.5 ± 0.6 39.5 ± 0.5 40.8 ± 1.9 39.7 ± 0.4 32.7 ± 1.4 27.5 ± 0.9 15:0 53 0.2 ± 0.0 0 0.1 ± 0.1 0 0 0 0.1 ± 0.1 0 0 2.1 ± 0.8 i-16:0 62 6.2 ± 0.1 9.9 ± 0.1 4.5 ± 0.3 6.5 ± 0.2 4.0 ± 0.1 5.0 ± 0.2 4.0 ± 0.4 5.3 ± 0.4 6.6 ± 1.2 5.4 ± 0.3 16:1 −0.5 1.2 ± 0.1 1.7 ± 0.4 1.4 ± 0.2 3.4 ± 0.2 1.2 ± 0.0 1.2 ± 0.3 1.2 ± 0.8 1.1 ± 0.0 1.0 ± 1.0 0.5 ± 0.2 16:0 63 5.2 ± 0.8 7.2 ± 0.1 3.9 ± 0.4 3.9 ± 0.2 3.9 ± 0.9 4.8 ± 0.4 3.2 ± 0.2 2.8 ± 0.0 4.6 ± 2.0 2.0 ± 0.2 i-17:0 60 9.3 ± 0.7 9.4 ± 0.3 11.0 ± 0.7 9.9 ± 0.1 11.1 ± 0.0 11.3 ± 0.1 14.3 ± 0.1 13.1 ± 0.6 14.6 ± 1.4 18.3 ± 1.2 a-17:0 37 16.7 ± 1.4 14.6 ± 0.8 17.2 ± 0.7 16.2 ± 1.2 17.2 ± 0.5 17.7 ± 0.1 17.0 ± 0.8 15.6 ± 0.4 11.7 ± 1.9 9.9 ± 0.4 17:0 61 0 0.3 ± 0.2 0.2 ± 0.2 0 2.3 ± 0.0 0 0 0 0 1.6 ± 0.7 18:1 5 1.8 ± 0.2 1.4 ± 0.3 1.1 ± 0.8 0 1.0 ± 0.2 0.9 ± 0.3 0.4 ± 0.1 0.5 ± 0.2 1.0 ± 1.4 0.2 ± 0.1 18:0 71 2.1 ± 0.8 1.7 ± 0.1 1.9 ± 0.5 3.1 ± 0.2 2.4 ± 0.9 3.4 ± 0.3 1.0 ± 0.1 0.8 ± 0.0 1.8 ± 0.8 0.8 ± 0.6

Ratios Iso/anteiso 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 0.6 ± 0.1 0.7 ± 0.0 1.1 ± 0.0 1.5 ± 0.0 Branched/straight 7.8 ± 0.8 6.6 ± 0.7 9.9 ± 1.6 8.3 ± 0.5 8.0 ± 0.9 8.4 ± 0.5 16.9 ± 1.1 17.6 ± 0.8 14.6 ± 1.4 16.2 ± 1.4

Values represent means ± S.D. from three determinations. Iso and Anteiso stand for the respective branching pattern for iso- and anteiso-branched fatty acids, respectively. G. Seydlová et al. / Biochimica et Biophysica Acta 1828 (2013) 2370–2378 2375

the cytoplasmic membrane rigidity significantly increased concomitantly with the accumulation of surfactin.

4. Discussion

The aim of this study was to investigate the role of membrane lipids in adaptation of B. subtilis ATCC 21332 to its own toxic product surfactin. Surfactin is synthesized in cytoplasm and is transported through the membrane by an unknown mechanism. This implies that it can interact with the membrane both from the cytoplasmic and extracellular side. However, surfactin clearly did not impair its producer, since the multiplication of bacteria continued in the culture in parallel with surfactin accumulation for almost 20 h. Within this period the cell number increased almost fivefold while the surfactin concentration increased from 2 to 84 μmol·l−1. Furthermore, the

most potent C15 surfactin isoform [17] was also the most abundant one, amounting to 80% of the surfactin present. Surprisingly, during + Fig. 2. Proportions of iso- (circles) and anteiso-branched FAs (squares) in B. subtilis the production period, the Sf culture grew even faster than the − cytoplasmic membrane during 24-h cultivation. The time point of entry into the stationary control Sf population. This stimulatory effect of surfactin could phase of growth is designated as t0. The data represent averages and S.D. probably stem from its quorum-sensing role which includes the triggering of cannibalism, as observed recently during the biofilm a 50 nm red shift in emission spectrum over the gel to liquid-crystalline development of B. subtilis [45]. The producer cells survived phase transition. Laurdan spectral shifts are usually quantified in the long-term exposure to surfactin concentrations that can perturb the form of generalized polarization (GP), which is inversely proportional to barrier properties in model membranes [23,28]. This is even more membrane fluidity (see Section 2.8). We used this technique in order to striking when we consider that the minimum inhibitory concentrations find how the changes in the composition of the polar head groups of of surfactin determined for Salmonella enteritidis, Proteus vulgaris, membrane PLs changed the respective biophysical characteristics of Enterobacter cloacae, Bacillus pumilus and Escherichia coli are all within − membranes isolated from B. subtilis. the range 6–30 μmol·l 1 [3,4,46]. These data suggested that bacteria From the data in Fig. 3, it is apparent that in the exponential phase of producing surfactin are equipped with an efficient self-protective growth (t−1) cytoplasmic membranes derived from both B. subtilis mechanism. In searching for this mechanism, our primary attention was strains showed the same values of Laurdan GP in line with the same focused on membrane phospholipids, which are a well-documented

PL composition. As the PL profile remained almost unchanged at t0.5, surfactin target [16,29,33]. the same GP values were also obtained in the early stationary phase, The interaction of surfactin with membrane PLs is hypothesized to and this tendency continued until t1.5 despite the subsequent changes initiate as the insertion of individual surfactin molecules. This step in the polar head group composition. However, at t3 and t20,i.e.the does not highly disorganize the PL bilayer, however after the time points, at which major changes in lipid composition of the Sf+ insertion of several other surfactin molecules in the membrane, strain were detected, GP values increased from about −0.02 to 0.02 pores can be formed and the mixed micelles of surfactin with PLs (P b 0.02) and 0.06 (P b 0.002), respectively. The increasing GP param- could lead to bilayer solubilization [17,22,32]. This model was also eter of the Sf+ membranes thus indicated a substantial rigidization of supported by the study of Nazari et al. [47] where surfactin was the membrane of the surfactin producer compared to the Sf− strain. thought to segregate within the membrane into surfactin-rich

At t20, this trend was much more profound than at t3. On the other clusters that disrupted the membrane locally. In our study, the hand, the membrane fluidity of the Sf− strain remained almost amount of surfactin molecules in the medium per B. subtilis cell of + 7 8 unchanged despite the observed PL modifications. This indicates that the Sf strain ranged from 1.1 × 10 at t0.5 to 1.2 × 10 at t20. Therefore, during 1-day cultivation, we should also expect all of the above-mentioned modes of surfactin action in the cells of the surfactin producer, which however obviously resisted the adverse effect of surfactin. In the membrane of the surfactin producer, two specificfeatures found in the polar head group composition indicated their possible role in the tolerance to surfactin. First, cardiolipin was the only PL to progressively accumulate over the whole 24-h cultivation (Table 2).

At t20, the CL concentration reached 22% of the total and CL became the second most abundant PL. The significance of this preferential synthesis was underlined by the parallel decline of CL content down to 8% in the Sf− strain in the same time point. Second, in contrast to CL, the level of PE was maintained strictly constant in the producer's membrane, perhaps due to PE having a stabilizing effect on surfactin in the membrane [31]. This might be unfavorable as surfactin can aggregate in the membrane [47] and tilts the acyl chains of lipids [48]. On the other hand, in the non-producing strain the proportion of PE steadily increased, which is typical of the stationary phase of growth of B. subtilis [49]. In contrast to the polar head groups of membrane PL, almost no Fig. 3. GP parameter during different cultivation periods measured in B. subtilis ATCC changes in the fatty acid composition were found. The observed 21332 (full symbols) and 0164 (open symbols) membranes. The time point of entry tendency of the lower content of iso-branched-chain FAs in the Sf+ into the stationary phase of growth is designated as t0. The data represent averages and S.D. strain (Fig. 3) was unlikely to be due to an adaptive response to 2376 G. Seydlová et al. / Biochimica et Biophysica Acta 1828 (2013) 2370–2378 surfactin. As an explanation of this effect we suggest a competition head and aliphatic chain bilayer moieties (data not shown). On the for and between branched-chain FA synthesis otherhand,theobserveddecreaseofmembranefluidity was apparently and biosynthesis of surfactin. Leu and Val are precursors of not high enough to impair cell growth as the number of viable cells iso-branched-chain FAs [50], whereas surfactin contains 4 Leu increased and surfactin production progressed throughout the stationary and 1 Val residues in its heptapeptide cycle [51].Suchaweak phase of the Sf+ strain cultivation. adaptive response at the level of FAs found in the surfactin At t20, the key lipid component that contributed to the substantial rise producer was also published in the recent study on B. subtilis of the rigidity in producer membrane was most likely CL, along with a resistance to lipopeptide daptomycin, where the major determinant of severe decrease in PG, which is one of the PLs that has a fluidizing effect DapR was the composition of the polar head groups [52]. [75]. Accumulation of PA, the common precursor of PLs up to one third Cardiolipin is an unusual anionic lipid carrying four acyl chains and of the total suggested an imbalance in PA de novo synthesis and its two phosphatidyl moieties. Its consists of a large hydrophobic conversion into other lipid species [76] in the membranes of both strains, region and strongly charged relatively small polar head group, which perhaps as a consequence of the energy depletion of cells. This notion was imply that CL favors negative curvature and forms both lamellar and supported by the substantial exhaustion of the PG pool for direct CL inverted non-lamellar lipid phases [53]. At the same time, the mobility synthesis, which surprisingly remained active in the Sf+ cells despite and conformational flexibility of CL should be severely restricted [54], the double surfactin and nutritional stress at t20 and indicated the specific which probably enhances the structural rigidity of CL-containing need for this PL. membranes [55]. In bacteria, CL is regarded as a stress phospholipid The presence of the CL head group in the lipid membrane can [56]. Enhanced levels of it were observed in different bacteria in response result in an increase in the order and tighter packing even in lipid to various adverse conditions such as high salinity [57–59], alkaline pH acyl chains [77]. Hence, the increase in rss DPH in the PL aliphatic [60] or the presence of chloramphenicol and tetracycline [61].CLalso chain region that we observed in the Sf+ strain can also be ascribed specifically interacts with key protein components of the cell cycle to CL, although the FA composition only exhibited minor alterations. [62,63]. Together with PE, it is involved in forming a mosaic of The rigidizing effect of CL on the membrane bilayer has been microdomains in polar and septal membranes, i.e. in regions of increased documented both in computational simulations and in experimen- membrane curvature [64,65]. Recently, a coordinated regulation of CL and tal studies of model membranes using a wide variety of techniques PE levels was discovered in the inner mitochondrial membrane of yeast [77–80]. The presence of CL in the membrane may also enhance the cells [66]. order of the lipid bilayer [81]. The accumulation of CL with the Different roles of increased CL content in the membrane of surfactin concomitant decrease in the proportion of PE is crucial for the producer could be thus hypothesized. Since CL is an anionic phospholipid long-term adaptation of Pseudomonas putida to the presence of bearing two negative charges, elevated concentration of CL may increase toluene. These parallel changes in CL and PE lead to increased cell the net negative charge of the membrane [62]. As the cycle of membrane rigidity and should be regarded as physical mecha- surfactin also bears two negative charges, an electrostatic repulsion nisms that prevent solvent penetration [82,83]. Thus, increased could hinder the interaction of surfactin with the phospholipid head CL content might be a general cell response due to membrane groups [67]. The opposite coulombic mechanism is involved in the action adjustment [57]. From this point of view, the rigidization of the of cationic antimicrobial , which preferentially bind to negatively membrane bilayer brought about by CL can be considered not charged phospholipid membranes and thus increase their permeability only as a side-effect of surfactin repulsion but also as a compensa- [68]. Consequently, resistance of cells to these peptides is mediated by tory response of the cell preventing fluidization [27,84],disorderof enhancing the net positive charge [69]. This mechanism has also been its barrier properties and solubilization [18] caused by surfactin. described in B. subtilis resistant to positively charged lipopeptide Finally, one more function of CL in the adaptation of B. subtilis daptomycin, where reduced level of anionic phospholipid PG reduced ATCC 21332 to surfactin could be assumed from the complementary thenetnegativesurfacechargeandweakenedtheinteractionwith shapes of CL and surfactin molecules. The cone-shaped CL can result daptomycin [52]. in the formation of aggregates with negative spontaneous curvature Another possible mechanism of the membrane protection against [55,85] which can counteract the positive curvature stress introduced surfactin can be deduced from the rigidizing effect of CL [55] on the by the inverted cone shape of surfactin [17,27]. CL was recently membrane. In vitro surfactin induces fluidization of the phospholipid considered to force inverse cone-shaped lipids away from pores, bilayer [27]. Therefore, it could be expected that an adaptation thus stabilizing the bilayer [85,86]. An analogous CL mode of action strategy of the surfactin producer would include the increase of the against surfactin pore expansion and rupture could be proposed as rigidity of its membrane. When we observed the parallel control of an additional mechanism increasing the membrane resistance of CL and PE during surfactin production, we decided to study the surfactin producer. changes in the fluidity of the upper layer of the membrane as a Despite systematic experimental effort being focused on CL, its primary target of the interaction with surfactin. A membrane unique physical and chemical characteristics in cell membranes are fluorescence probe Laurdan was used for this. Both the fluorescent not well understood [55]. Moreover, a novel role of CL as a specific moiety of Laurdan and surfactin are located at the glycerol backbone regulator of fundamental processes occurring at biomembranes has of the PL head groups [25,70]. It has been shown that the Laurdan emerged. P. putida defective in CL synthesis exhibited a compromised fluorescence spectrum is sensitive to the polarity [71] and phase functioning of the RND efflux pumps that were involved in antibiotic state of the phospholipid bilayers [72].Fromt1.5 onwards, our results and solvent resistance [61]. In mitochondria, the CL molecule can showed an increase in the general polarization (GP) of Laurdan during disrupt the supramolecular complex of the voltage-dependent anion surfactin production, which documented an intense progressive increase channel that plays a central role in apoptosis [87]. in rigidity of the membrane (Fig. 3). This kinetics followed the In this study, both the changes in the phospholipid composition development of the polar head group, which reflected the response to and physical properties of the membrane showed that an adaptive the gradually rising surfactin stress in the producer cells. The Laurdan response was induced during surfactin production in B. subtilis ATCC GP data were also confirmed by steady-state fluorescence anisotropy, 21332. We conclude that in this process, the enhanced level of rss, using TMA-DPH and DPH probe molecules that monitor the polar cardiolipin was the key factor which possibly brought about the [73] and hydrophobic [74] regions of the lipid bilayer. The resulting values membrane protection. Our preliminary data obtained with model of rss TMA-DPH and rss DPH were significantly higher in the membranes membranes show that the presence of cardiolipin increases of the Sf+ strain than those determined in the Sf− membranes, and membrane stability after surfactin challenge (data not shown). indicated a higher rigidity of the producer membranes, both in the polar However, the precise mechanism of the effect of cardiolipin on G. 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