Phospholipids of Lactobacillus spp.

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1 JOURNAL OF BACTERIOLOGY, Nov. 1995, p Vol. 177, No /95/$ Copyright 1995, American Society for Microbiology Phospholipids of Lactobacillus spp. D. B. DRUCKER, 1,2 * G. MEGSON, 2 D. W. S. HARTY, 3 I. RIBA, 4 AND S. J. GASKELL 4 School of Biological Sciences, University of Manchester, Manchester M15 6FH, 1 Microbiology Department, Hope Hospital, Salford M6 8HD, 2 and Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester M60 1QD, 4 United Kingdom, and Institute of Dental Research, University of Sydney, Sydney, Australia 3 Received 10 March 1995/Accepted 30 July 1995 Lactobacillus phospholipid molecular species were analyzed by mass spectrometry. Prominent anions were consistent with presence of the phosphatidylglycerols PG(37:2), PG(36:2), PG(35:1), PG(34:1), and PG(33:1). Diglycosyldiacylglycerol molecular species were also observed, although nitrogen-containing phospholipids were absent. An anion of m/z 759 was derived from an apparently novel type of lipid. Numerous studies have investigated the lipid composition of Lactobacillus spp. (8, 19, 21). Lactobacilli typically have n-c 16:0, n-c 18:1, and cyc-c 19 as the major carboxylate constituents, with lesser amounts of n-c 14:0, n-c 16:1, and n-c 18:0 and traces of odd-carbon-number acids in some species (6). Such fatty acid profiles are atypical of gram-positive groups but common in gram-positive bacteria (17). Other studies have analyzed polar lipids of lactobacilli and discovered that the vast majority of polar lipid is comprised of phosphatidylglycerol (PG) (8, 11), with smaller amounts of phosphatidic acid, diphosphatidylglycerol (cardiolipin), lysylphosphatidylglycerol (8, 10), phosphoglycolipids (10), and diglycosyldiacylglycerol (DGDG) (18). Virtually nothing is known of the distribution of individual molecular species within each type of phospholipid. In other groups of organisms, such an analysis has required expenditure of considerable time and effort to purify individual molecular species by chromatographic separation and then to analyze each molecular species in turn (2). An alternative approach, recently applied to the study of bacterial phospholipids (1, 3), is fast atom bombardment (FAB) mass spectrometry (MS) in which analysis of mixtures of molecular species is facilitated by the generation of ions retaining the intact molecular structure. FAB MS data for a single strain of Lactobacillus rhamnosus with respect to phospholipid molecular species have been published (7). The aim of this study was to determine the distribution of individual molecular species of phospholipids in a collection of Lactobacillus isolates. Fifteen Lactobacillus strains available from culture collections were analyzed (Table 1). All strains have been described previously (12, 13), and their identities were determined by using the API 50CHL test kit (API System, Vercieu, France) and the IDENTIFY computer program (5). Recent changes in the species assignments of some strains, not covered by the API 50CHL test kit for the identification of L. rhamnosus and L. paracasei subsp. paracasei (4), were incorporated into the computer program. For growth and harvesting, all strains were grown in 200-ml aliquots of MRS broth (Oxoid) for 18 to 24 h * Corresponding author. Mailing address: Microbiology Laboratory, School of Biological Sciences, University of Manchester Dental School, Manchester M15 6FH, United Kingdom. Phone: Fax: at 37 C as static batch cultures. Cultures were harvested by centrifugation and washed once in phosphate-buffered saline (ph 7.3). They were then washed in deionized water and freeze-dried. Phospholipid analysis was as follows. The methanol-chloroform extraction procedure, which used mechanical shaking at room temperature, was as described in a previous study (1). Phospholipid molecular species were separated and analyzed by conventional FAB MS analysis by our usual method (1). Putative identifications were confirmed by tandem MS analyses of individual molecular species, performed with a VG 7070Q hybrid instrument (Fisons Instruments, Manchester, United Kingdom). A typical negative-ion FAB MS spectrum for L. rhamnosus (L14) is shown in Fig. 1. The m/z (mass-to-charge) range 200 to 350 includes peaks consistent with assignment as carboxylate anions. These include m/z 295, 281, 267, 255, 253, 227, and 211, attributable to cyc-c 19,C 18:1, cyc-c 17,C 16:0,C 16:1,C 14:0, and C 13:1 carboxylate anions, respectively, which include anions formed from phospholipids. The m/z range 500 to 1,000 (Fig. 1) includes peaks of m/z 733, 759, 761, 787, and 773 consistent with assignment to the following phospholipid anions: PG(33:1), PG(35:2) (however, see below), PG(35:1), PG(37:2), and PG(36:2). Less intense peaks, e.g., of m/z 919 and 931, were observed, consistent with the expected presence of DGDG molecular species, specifically DGDG(34:0) and DGDG(35:1). Peaks in the mid-range, e.g., m/z 409, 431, 483, and 505, arise by molecular fragmentation (Table 2). Identification of phospholipids on the basis of mass/ charge ratio alone, however, is necessarily tentative. Substantiation of these assignments was therefore sought via tandem MS whereby anions of one molecular species were separated in one instrument and fragment ions so formed were scanned with a second mass spectrometer to yield a product ion spectrum. Thus, these analyses established unequivocal connectivities between selected precursor and observed product ions. Figure 2 shows the product ion spectrum obtained by collisional activation of negative ions of m/z 761; assignments are summarized in Table 2. These data collectively are consistent with an assignment of a PG structure with C 16:0 and cyc-c 19 acyl substituents to the ion with m/z 761. Similar tandem MS analyses of other molecular species are shown in Table 3. Tandem MS analysis of m/z 759 (Table 3) suggests that this may represent a novel structure because the backbone of the molecule clearly has an unsaturation. Phospholipid compositions of species examined are shown in Table 1; Table 4 lists the major anions, consistent with data for phospholipids, of 6304

2 VOL. 177, 1995 NOTES 6305 TABLE 1. Phospholipids a of Lactobacillus isolates b Peak m/z Phospholipid Content (% of total) L.a. (n 1) L.f. (n 2) L.o. (n 2) L.pa. (n 3) L.pl. (n 2) L.r. (n 3) L.sv. (n 1) L.sn. (n 1) 505 PG(19:1) PG(30:4) PG(30:3) PG(32:2) PG(32:1) PG(32:0) PG(33:3) PG(33:2) PS(32:1) PG(33:1) PS(32:0) PG(34:3) PG(34:2) PS(33:1) PG(34:1) PS(33:0) PG(34:0) PG(35:3) PS(34:2) PX(34:1) c PS(34:1) PG(35:1) PS(34:0) PG(35:0) PG(36:3) PS(35:2) PG(36:2) PS(35:1) PG(36:1) PS(35:0) PG(36:0) PG(37:3) PS(36:1) PG(37:2) PS(36:0) PG(37:1) PG(38:2) DGDG(34:0) PS(39:0) DGDG(35:1) PS(41:2) a Tentative identification unless confirmed by tandem MS (see Tables 2 and 3). b Lactobacillus spp., with strains and in parentheses, study numbers: L.a., L. acidophilus II, ATCC 4356/NCDO 1748/DSM (L26); L.f., L. fermentum NCTC 6691 (L10), ATCC (L13); L.o., L. oris NCFB 2160 (L33), NCFB 2161 (L34); L.pa., L. paracasei subsp. paracasei NCFB 151 (L42), ATCC (L45), A198/89 (L51); L.pl., L. plantarum ATCC 14917/NCFB 1752/DSM (L12), NCIB 7220 (L11); L.r., L. rhamnosus ATCC 7469 (L14), NCTC (L4), A64/87 (L52); L.sv., L. salivarius subsp. salivarius ATCC (L5); L.sn., L. salivarius subsp. salicinus ATCC (L8). Strains prefixed by A are held at the Central Public Health Laboratories, Colindale, London, United Kingdom. c PX, unknown type of phospholipid.

3 6306 NOTES J. BACTERIOL. FIG. 1. FAB spectra for polar lipids of L. rhamnosus. See tables for peak identifications. strains tested and of single examples of three other grampositive species for comparative purposes. Except for a single strain of L. rhamnosus, which has a characteristic profile of phospholipid molecular species different from those of other genera (7), previous studies have never separated individual phospholipid molecular species of Lactobacillus. However, overall fatty acid composition is known (19 22). Similarly, the major type of phospholipid is known to be PG (8, 10). The analytical procedure used here extracts phospholipid and glycophospholipid (18) but not necessarily all polar lipids. Detection of phospholipids is favored because (i) lipids are extracted by the hydrophobic solvent and (ii) FAB selectively desorbs surface-active molecules such as polar lipids, though not all types equally well. The technique is applicable to mixtures because molecular structure is largely preserved (14, 15). The tandem MS analyses performed in this study provided invaluable information for correct structure assignments. For example, the peak at m/z 759 has the expected value for PG(35:2), yet tandem MS reveals that the fatty acyl substituents are definitely C 18:1 and C 16:0. Thus, it cannot be an anion of PG or FIG. 2. Product ion spectrum of m/z 761 anion recorded during tandem MS analysis of polar lipids of L. rhamnosus.

4 VOL. 177, 1995 NOTES 6307 m/z TABLE 2. Product ion spectrum of m/z 761 (negative ions; see Fig. 2) Assignment [M-H] Loss of C 15 H 30 CO Loss of C 15 H 31 COOH Loss of C 18 H 34 CO Loss of C 18 H 35 COOH Loss of CH 2 (OH)CH CH&C 15 H 31 COOH {} O Loss of CH2(OH)CH CH and C 18 H 34 CO {} O C 18 H 35 COO C 15 H 31 COO CH 2 (OH)CH(OH)CH 2 OPO 3 H CH-CHCH 2 OPO 3 H another expected phospholipid. This is because PG with C 18:1 and C 16:0 would have a molecular weight of and yield an anion of nominal m/z 747 and not 759 as actually observed. Therefore, the anion m/z 759 must be due to a novel type of polar lipid. Further work is in progress to characterize it. In the case of m/z 761 product ions, and on the basis of previous generalizations of the relative abundances of carboxylate anions derived from the sn-1 and sn-2 positions (3), the saturated C 16 acyl group is assigned to sn-2. The literature (8, 11, 14 16) suggests that the amino acid-containing phospholipid, lysyl PG, might be expected as a minor polar lipid. In this study, data consistent with lysyl PG were not observed; possibly only trace amounts were present because FAB MS is capable of detecting lysyl PG (14, 15). The phospholipid identifications were supported by the carboxylate anions present and by previously published data from gas chromatographic analyses (19 22). The absence of phosphatidylethanolamine or its methyl-substituted derivatives was confirmed. There are no previously published data on phospholipids of L. oris, L. rhamnosus, orl. salivarius with which the present findings may be compared. Within the group, there are quantitative, and minor qualitative, differences in molecular species of phospholipids between strains, suggesting that FAB MS could provide a rapid means of typing strains. TABLE 3. Major anion peaks of Lactobacillus spp. for which tandem MS data were recorded presenting data consistent with phospholipids Precursor anion m/z Fatty acyl substituent sn-1 sn-2 Backbone of molecule a 673 C 18:1 C 16:0 PA 719 C 18:1 C 14:0 PG 747 C 18:1 C 16:0 PG 759 C 18:1 C 16:0 PS 761 C 19:1 C 16:0 PG 771 C 18:2 C 18:1 PG 773 C 18:1 C 18:1 PG 787 C 18:1 b C 19:1 PG a PA, phosphatidic acid; PG, phosphatidylglycerol; PS, phosphatidylserine in which an unsaturation exists in the backbone. b C 19:1 is most probably cyc-c 19, and its precise position on glycerol in the anion of m/z 787 has yet to be confirmed. TABLE 4. Major phospholipid anions a of Lactobacillus spp. and representatives of other genera Strain (study no. or name) m/z values of major phospholipid anions L L / /762 L L L L /787 L L L L L L L L L Bacillus subtilis b Micrococcus luteus b Streptococcus mutans b , a Data consistent with those for phospholipid anions and shown in order of descending relative peak intensity. For identities of ions and of strain study numbers, see Table 1. b Published data (6). We thank V. Boote (Mass-Spectrometry Laboratory, Chemistry Department, University of Manchester) for skilled technical assistance. REFERENCES 1. Aluyi, H. S., V. Boote, D. B. Drucker, and J. M. Wilson Fast atom bombardment-mass spectrometry for bacterial chemotaxonomy: influence of culture age, growth temperature, gaseous environment and extraction techniques. J. Appl. Bacteriol. 72: Batley, M., N. H. Packer, and J. W. Redmond Molecular analysis of the phospholipids of Escherichia coli K12. Biochim. Biophys. Acta 710: Cole, M. J., and C. G. Enke Direct determination of phospholipid structures in microorganisms by fast atom bombardment triple quadrupole mass spectrometry. Anal. Chem. 63: Collins, M. D., B. A. Phillips, and P. Zanoni Deoxyribonucleic acid homology studies of Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. tolerans, and Lactobacillus rhamnosus sp. nov., comb. nov. Int. J. Syst. Bacteriol. 39: Cox, R. P., and J. K. Thomsen Computer-aided identification of lactic acid bacteria using the API 50CHL system. Lett. Appl. Bacteriol. 10: Drucker, D. B Microbiological applications of gas chromatography. Cambridge University Press, Cambridge. 7. Drucker, D. B Fast atom bombardment mass spectrometry of phospholipids for bacterial chemotaxonomy, p In C. Fenselau (ed.), Mass spectrometry for the characterization of microorganisms. American Chemical Society, Washington, D.C. 8. Exterkate, F. A., B. J. Otten, H. W. Wassenberg, and J. H. Veerkamp Comparison of the phospholipid composition of Bifidobacterium and Lactobacillus strains. J. Bacteriol. 106: Fenselau, C Mass spectrometry for characterization of microorganisms. An overview, p In C. Fenselau (ed.), Mass spectrometry for the characterization of microorganisms. American Chemical Society, Washington, D.C. 10. Fischer, W Bacterial phosphoglycolipids and lipoteichoic acids, p In M. Kates (ed.), Handbook of lipid research, vol. 6. Plenum Press, New York. 11. Fischer, W., R. A. Laine, and M. Nakano On the relationship between glycerophosphoglycolipids and teichoic acids in Gram-positive bacteria. II. Structures of glycerophosphoglycolipids. Biochim. Biophys. Acta 528: Harty, D. W. S., and K. W. Knox An in vitro study of adhesion of various Lactobacillus species. Microb. Ecol. Health Dis. 4: Harty, D. W. S., M. Patrikakis, and K. W. Knox Identification of Lactobacillus strains isolated from patients with infective endocarditis and comparison of their surface associated properties with those of other strains from the same species. Microb. Ecol. Health Dis. 4: Heller, D. N., R. J. Cotter, C. Fenselau, and O. M. Uy Profiling of

5 6308 NOTES J. BACTERIOL. bacteria by fast atom bombardment mass spectrometry. Anal. Chem. 59: Heller, D. N., C. M. Murphy, R. J. Cotter, C. Fenselau, and O. M. Uy Constant neutral loss scanning for the characterisation of bacterial phospholipids desorbed by fast atom bombardment. Anal. Chem. 60: Ikawa, M Nature of the lipids of some lactic acid bacteria. J. Bacteriol. 85: O Leary, W. M., and S. G. Wilkinson Gram-positive bacteria, p In C. Ratledge and S. G. Wilkinson (ed.), Microbial lipids, vol. 1. Academic Press, London. 18. Shaw, N Bacterial glycolipids. Bacteriol. Rev. 34: Thorne, K. J. I The phospholipids of Lactobacillus casei. Biochim. Biophys. Acta 84: Uchida, K., and K. Mogi Cellular fatty acid spectra of Pediococcus species in relation to their taxonomy. J. Gen. Appl. Microbiol. 18: Uchida, K., and K. Mogi Cellular fatty acid spectra of Hiochi bacteria, acid-tolerant lactobacilli, and their separation. J. Gen. Appl. Microbiol. 19: Veerkamp, J. H Fatty acid composition of Bifidobacterium and Lactobacillus strains. J. Bacteriol. 108:

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