APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Feb. 1977,
p. 221-226
Copyright X) 1977 American Society for Microbiology
Vol. 33, No. 2 Printed in U.S.A.
Fatty Acid Fingerprints of Streptococcus mutans NCTC 10832 Grown at Various Temperatures D. B. DRUCKER* AND F. J. VEAZEY Department of Bacteriology and Virology, University of Manchester, Manchester M13 9PL, Great Britain
Received for publication 19 August 1976
Fatty acid fingerprints were determined gas chromatographically for Streptococcus mutans NCTC 10832, which had been grown in batch brain heart infusion at a series of nine temperatures ranging from 29.0 to 40.0°C. The major acids at all temperatures were n-palmitic and octadecenoic acids. Other acids detected at all temperatures included n-myristic, palmitoleic, n-stearic, and eicosenoic acids. An increase in temperature resulted in a decrease in the proportion of unsaturated to saturated fatty acids, indicating the importance of accurate temperature control in such gas-liquid chromatographic-chemotaxonomic studies. 36, 37, 39, or 40°C. In every case, stationary phase was reached. Cells were harvested by centrifugation at 6,000 x g for 30 min, washed with pH 7.4 0.1 M Sorensen buffer, and then with distilled water, at 10°C, and freeze-dried for 18 h. Triplicate cultures were not pooled. Chemical extraction of cells. After organisms grown at each of the specified temperatures were freeze-dried, 10-mg aliquots of each triplicate lyophilized preparation were weighed into three separate ampoules, which were sealed after addition of 1 ml of methylating reagent, consisting of 15% (wt/vol) boron trifluoride in anhydrous methanol (British Drug Houses; 18), and evacuation. After heating the ampoules to 100°C for 1 h and allowing to cool, the contents were added to 10 ml of water and extracted twice with 1 volume of heptane (British Drug Houses) by shaking for 2 min in a mechanical shaker (Baird & Tatlock). The aqueous phases were discarded, and the two heptane phases were pooled and stored at - 18°C. The heptane phase included methyl esters of carboxylic acids. Separation of enoate classes. Portions of the heptane extract were used for preparation of acetoxymercurimethoxy derivatives of the enoate methyl MATERIALS AND METHODS esters. The technique used, based on that described Bacterial strain. The organism studied was S. by Stahl (22), was as follows: 10 ml of heptane exmutans NCTC 10832 (4), a cariogenic streptococcus tract was evaporated to 0.1 ml by evacuation in a of human origin used in a number of previous chem- Thunberg tube, with a sparsely greased stopper. A 1-ml portion of reagent (14 g of mercuric acetate, 2.5 otaxonomic studies. Growth and harvesting of cultures. Before and ml of water, 1.0 ml of glacial acetic acid, 250 ml of after growth, the organism was checked for purity methanol) was added. Air in the Thunberg tube was by Gram morphology, appearance on plates of 5% removed by evacuation and replaced by N2. After horse blood-brain heart infusion (Oxoid) and mitis allowing the reaction to proceed for 48 h at 22°C in salivarius agar (Oxoid), and reactions in sugar se- the dark, excess methanol was removed by evacuarum-peptone media and 5% sucrose-brain heart in- tion and the residue was dissolved in 0.5 ml of chlofusion. Starter cultures, consisting of 1-ml aliquots roform. Surplus reagent was removed by washing. of brain heart infusion, were inoculated into tripli- The chloroform phase was evaporated to 0.1 ml, and a spot was applied repeatedly to a thin layer of Silica cate 100-ml volumes of brain heart infusion. The Gel G (Merck) along with acetoxy-mercurimethoxy cultures were incubated in a shaking water bath (Hearson) for 48 h, without aeration, at one of nine derivatives of standard methyl enoate esters. The specified temperatures, namely, 29, 32, 33, 34, 35, thin-layer chromatography plate was run for 18 cm 221
The feasibility of classifying microorganisms by the chemical analytical method of gas chromatography was originally suggested by Abel et al. (1). Subsequently, their approach has been applied to a wide variety of microorganisms, including blue-green algae (15), marine and estuarine bacteria (20), viruses (16), mycobacteria (23), clostridia (19), micrococci (11), bacilli (12), pseudomonads (3), and streptococci (2, 8, 21). The bacterial components most often studied, structural fatty acids, are subject to variation with changing experimental parameters. Bacterial fatty acid profiles have been shown to vary with culture age (13), substrate (10), temperature (14), dilution rate in continuous culture (5), aeration (9), concentration of micronutrients and vitamins (6), and pH (7). In the present study, the effect of temperature on the fatty acid profile of Streptococcus mutans is examined.
222
DRUCKER AND VEAZEY
in petroleum spirit-diethyl ether (80:20), dried, and rerun for 14 cm in a polar solvent consisting of propan-1-ol-glacial acetic acid (100:1). After drying, enoate derivatives could be visualized by spraying with 0.1% S-diphenylcarbazone in 96% alcohol. Recovery of enoate classes for gas-liquid chromatographic analysis was possible by scraping off the thin layer and eluting with 1 ml of methanol-hydrochloric acid (10:1) and re-extracting with heptane. Gas-chromatographic analysis. Heptane extracts of methyl carboxylic esters were evaporated to small volume, and 2-,ul aliquots were analyzed three times on columns (5 feet by 0.25 inch, outer diameter [ca. 152.4 by 0.64 cm]) of 10% (wt/wt) polyethylene glycol adipate (PEGA) on 100- to 120-mesh diatomite C at 190°C in a PYE 104 chromatograph, using a N2 carrier-gas flow rate of 45 ml/min, with a detector temperature of 200°C; an injection point heater was also used. For each growth temperature, nine analyses were performed. Peaks were detected by a flame ionization detector; the detector signal was amplified by a paramatic amplifier and fed via a Spectraphysics computing integrator to a 10-mV chart recorder. The integrator printed out retention times and peak areas. Tentative identification of peaks and normalization of data was achieved by a Data General NOVA computer. This was programmed to ignore early solvent peaks and calculated the following: retention times relative to methyl palmitate, equivalent carbon number (ECN), and percent peak area, for each of the nine analyses per growth condition. Identical peaks in different analyses were averaged, and artefacts were excluded. Finally, mean percent peak areas for esters of mean ECN were printed out. Whereas absolute identity of peaks is never known in gas-liquid chromatography-chemotaxonomy application, corroboration of peak identity was sought in the present study by (i) co-chromatography with standard mixtures of methyl carboxylic esters (Applied Science) containing the methyl esters of straight-chain C,4, C,5, C,6, C,7, C,8, C19, C20, C21, and C22, iso-branched i-C,4, i-C,,6, i-C,8, and iC20, anteiso-branched ai-Cl5, ai-C,7, ai-C,9, and aiC21, and monoenoic C,8,l and C20:1, dienoic C18:2, and trienoic C,8:3; (ii) reanalysis on a nonpolar Apiezon L column; (iii) gas chromatography of separated enoate classes on PEGA; and (iv) gas chromatography with on-line mass spectrometry. On nonpolar columns the methyl esters of unsaturated acids have smaller retention times than their saturated analogues, whereas on polar columns, e.g., PEGA, their retention times are greater. Samples (2 ,l) were rerun on a glass column (5 feet by 0.25 inch [outer diameter]) of 10% Apiezon L on Celite 545 AW DMCS of 100 to 120 mesh at 225°C with an N2 carrier flow rate of 45 ml/min. Data were computed as described above. Mass spectrometry offered information on the molecular weight of esters and the mass of molecular fragments formed by electron bombardment, which in turn provided structural data. For mass spectrometry, a column of PEGA (3 feet by 0.25 inch, outer diameter [ca. 91.4 by 0.64 cm]) was run at 165°C, in a PYE 104 gas chromatograph, which temperature much reduced
APPL. ENVIRON. MICROBIOL. column bleed. Instead of using a "splitter," the entire eluate from the column was fed to an AEI MS30 mass spectrometer, and a gas chromatograph recorded in terms of ion current. A scan time of 10 s was used.
RESULTS
The strain used grew well at all the test temperatures, although above and below this range of temperatures the growth rate is very rapidly decreased. The fatty acid profiles of S. mutans NCTC 10832 obtained on PEGA, after growth at various temperatures, are shown in Table 1. Peaks observed in all samples had the following ECN values and retention characteristics on PEGA: 14.00 (n-myristate), 15.55 (iso-palmitate), 16.00 (n-palmitate), 16.30 (palmitoleate), 18.00 (nstearate), 18.40 (octadecenoate), 20.40 (eicosenoate). Other peaks occurring in more than trace amounts (>0.1% total peak area) had the following ECN values and retention characteristics on PEGA: 15.30 (pentadecenoate), 16.71 (anteiso-heptadecanoate), 17.00 (n-heptadecanoate), 17.35 (heptadecenoate), 17.60 (iso-stearate), 19.00 (n-nonadecanoate), 19.40 (nonadecenoate), 19.70 (anteiso-eicosanoate), 20.00 (n-arachidate), 20.80, 21.20. The acid of ECN 20.80 may have been the CG,:2 diunsaturated acid. Figure 1 shows a thin-layer chromatographic separation of the acetoxy-mercurimethoxy derivatives of fatty acid methyl esters of S. mutans NCTC 10832, in which spots corresponding to derivatives of monoenoates can be seen. Elution, re-extraction, and gas chromatography of the separated derivatives confirmed the presence of palmitoleate, and octadecenoate, methyl esters. Co-chromatography with standards and separate analysis of standards led to the computation of accurate ECN values, which enabled tentative identifications to be made (vide supra). Analysis of standard and sample methyl esters on nonpolar Apiezon L showed that the retention times of the unsaturated acids' esters decreased relative to saturated acids' esters. A typical sample chromatographed on polar and nonpolar columns is shown in Fig. 2. The movement of palmitoleate relative to n-palmitate, and octadecenoate relative to n-stearate, is clearly seen, whereas the relative retentions of iso- and n-palmitate remain the same. If ECN data for PEGA and Apiezon L columns are plotted against one another, an interesting two-dimensional representation is obtained (Fig. 3), analogous to the two-dimensional chromatogram familiar in thin-layer chromatography. Conjunction of two points, one of which represents a known compound, in such a repre-
VOL. 33, 1977 )
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sentation is strong evidence of the absolute identity of an unknown peak. In Fig. 3 two unknown peaks are identified as probably being methyl stearate and methyl octadecenoate. A typical result of combined gas chromatography-mass spectrometry is shown in Fig. 4; this shows re-drawn mass spectra of an unknown peak and a standard compound, methyl myristate. Both compounds have a molecular ion (M+) of mie = 242 and fragments indicating loss of methoxy (M-31) and acetyl (M-43). Both spectra have a large methyl ester peak of m/e = 74. Other fragments showing close similarities are also obvious. Difficulty was experienced in obtaining reliable mass spectral data for the minor peaks as a result of low amounts present, contamination by column bleed, and contamination by major peaks of similar retention volume.
The data for the effect of temperature on the fatty acid profile (Table 1) indicate that temper-
'-1X
224
APPL. ENVIRON. MICROBIOL.
DRUCKER AND VEAZEY
perior integration equipment now available commercially. The general change in proportions of unsaturated-saturated fatty acid with temperature is not only relevant to S. mutans, but also agrees with the data currently availa9. _._ . ...t__.'-'.. '- . . . . ble for gram-negative organisms for only two or three test temperatures for Serratia marcesb... ...-n -C16:0 cens (9, 14) or two temperatures for Vibrio marinus (20), where temperature had a less marked effect, and for eight test temperatures C1 :0 in the case of Escherichia coli (17). In all cases, increase in temperature has resulted in a decrease in the ratio of unsaturated-saturated = ,,n C16:0 fatty acid. It is quite probable that other grampositive organisms, if tested, would display a similar temperature effect, which probably reflects an attempt by the microorganism to conchrmaogapic.epraio.o FG2.Gslqi trol the changing viscosity of membrane lipid after changing temperature. Provided that time is allowed for stationary phase to be reached at all growth temperatures, there is no question of differing profiles being attributable to the organism's being at different phases of growth. The risk of misclassification of microorgaFI. 2.. Ga-iqi choaorpi of, seaato nisms grown with inadequate temperature control is fortunately not too great: the measure of association between profiles of S. mutans NCTC 10832 grown at 34 and 37°C is 0.963, calculated as the coefficient of linear correlation. This value is similar to that obtained for strains of the same species, grown identically. In view of the likelihood of fatty acid profiles methyl carboxylic esters of S. mutans on (a) polar being temperature affected for a wide range columns of PEGA and (b) nonpolar columns of Apiezon L. On a polar column, peaks due to methyl palmitoleate and methyl octadecenoate immediately fol191 low, those ofpalmitate (n-Cj;:)) and stearate (n-C, :,), whereas on a nonpolar column they elute before the
a
saturated acid esters.
ature can alter the fatty acid profile of a microorganism, that the alteration is progressive, and that the proportion of unsaturated fatty acid falls as the temperature rises. This is best seen graphically (Fig. 5), when an increase in from C results in a fall of C 39c temperature 10m in the ratio of unsaturated-saturated acids of C16 and C18 from almost unity to .
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35 GROWTH TEMPERATURE
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of microorganisms, incubation temperatures should be carefully maintained at a universally acceptable temperature such as 37°C, wherever possible, in gas-liquid chromatographic-chemotaxonomic studies, so that meaningful data may be obtained. ACKNOWLEDGMENT We are gratefully indebted to the Department of Organic Chemistry at Manchester University for providing mass spectrometry facilities. LITERATURE CITED 1. Abel, K., H. de Schmertzing, and J. I. Peterson. 1963. Classification of microorganisms by analysis of chemical composition. 1. Feasibility of utilizing gas chromatography. J. Bacteriol. 85:1039-1044. 2. Amstein, C. F., and P. A. Hartman. 1973. Differentiation of some enterococci by gas chromatography. J. Bacteriol. 113:38-41. 3. Brooks, J. B., R. E. Weaver, H. W. Tatum, and S. A. Billingsley. 1972. Differentiation between Pseudomonas testeroni and P. acidovorans by gas chromatography. Can. J. Microbiol. 18:1477-1482. 4. Drucker, D. B., R. M. Green, and D. K. Blackmore. 1972. Production of buccal and lingual caries in three weeks by a streptococcus isolated from man. J. Dent. Res. 50:1510.
5. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1973. Fatty acid fingerprints of Streptococcus mutans grown in a chemostat. Microbios 7:17-23. 6. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1974. The influence of vitamin and magnesium limitations on fatty acid fingerprints of chemostat grown Streptococcus sp. SS. Microbios 10:183-185. 7. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1975. The effect of changing pH on fatty acid profiles of Streptococcus mutans NCTC 10832. Microbios 13:99103. 8. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1976. Fatty acid fingerprints of some chemostat-grown streptococci with computerized data analysis. Microbios Lett. 1:31-34. 9. Drucker, D. B., and I. Owen. 1973. Chemotaxonomic fatty acid fingerprints of bacteria grown with, and without, aeration. Can. J. Microbiol. 19:247-250. 10. Farshtchi, D., and N. M. McClung. 1970. Effect of substrate on fatty acid production in Nocardia asteroides. Can. J. Microbiol. 16:213-217. 11. Jantzen, E., T. Bergan, and K. Bovre. 1974. Fatty acid composition of strains within Micrococcaceae. Acta Pathol. Microbiol. Scand. Sect. B 82:785-798. 12. Kaneda, T. 1967. Fatty acids in the genus Bacillus. I. Iso- and anteiso fatty acids as characteristic constituents of lipids in 10 species of Bacillus. J. Bacteriol. 93:894-903. 13. Kates, M., G. A. Adams, and S. M. Martin. 1964. Lipids of Serratia marcescens. Can. J. Biochem. 42:461-479. 14. Kates, M., and P.-O. Hagen. 1964. Influence of temperature on fatty acid composition of psychrophilic and mesophilic Serratia species. Can. J. Biochem. 42:481488. 15. Kenyon, C. N. 1972. Fatty acid composition of unicellular strains of blue-green algae. J. Bacteriol. 109:827834. 16. Levanon, A., Y. Klibansky, and A. Kohn. 1973. Detection of encephalomyocarditis virus infection in animal cells by gas-liquid chromatography. Experientia 29:1305-1307. 17. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol. 84:1260-1267. 18. Metcalfe, L. D., and A. A. Schmitz. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33:363-364. 19. Moss, C. W., and V. J. Lewis. 1967. Characterisation of clostridia by gas chromatography. I. Differentiation of species by cellular fatty acids. Appl. Microbiol. 15:390-397.
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20. Oliver, J. D., and R. R. Colwell. 1973. Extractable lipids of gram-negative marine bacteria: fatty acid composition. Int. J. Syst. Bacteriol. 23:442-458. 21. Shiori-Nakano, K., and I. Tadokoro. 1968. Sugar and fatty acid composition of hemolytic Streptococcus, p. 383-386. In H. Ishizuka and T. Hesegawa (ed.), Culture collections of microorganisms; proceeding of the international conference on culture collections. Uni-
APPL. ENVIRON. MICROBIOL. versity Park Press, Tokyo. 22. Stahl, E. 1965. Thin layer chromatography. SpringerVerlag, Berlin. 23. Thoen, C. O., A. G. Karlson, and R. D. Ellefson. 1972. Differentiation between Mycobacterium kanasasii and Mycobacterium marinum by gas-liquid chromatographic analysis of cellular fatty acids. Appl. Microbiol. 24:1009-1010.
AND
ENVIRONMENTAL MICROBIOLOGY, Feb. 1977,
p. 221-226
Copyright X) 1977 American Society for Microbiology
Vol. 33, No. 2 Printed in U.S.A.
Fatty Acid Fingerprints of Streptococcus mutans NCTC 10832 Grown at Various Temperatures D. B. DRUCKER* AND F. J. VEAZEY Department of Bacteriology and Virology, University of Manchester, Manchester M13 9PL, Great Britain
Received for publication 19 August 1976
Fatty acid fingerprints were determined gas chromatographically for Streptococcus mutans NCTC 10832, which had been grown in batch brain heart infusion at a series of nine temperatures ranging from 29.0 to 40.0°C. The major acids at all temperatures were n-palmitic and octadecenoic acids. Other acids detected at all temperatures included n-myristic, palmitoleic, n-stearic, and eicosenoic acids. An increase in temperature resulted in a decrease in the proportion of unsaturated to saturated fatty acids, indicating the importance of accurate temperature control in such gas-liquid chromatographic-chemotaxonomic studies. 36, 37, 39, or 40°C. In every case, stationary phase was reached. Cells were harvested by centrifugation at 6,000 x g for 30 min, washed with pH 7.4 0.1 M Sorensen buffer, and then with distilled water, at 10°C, and freeze-dried for 18 h. Triplicate cultures were not pooled. Chemical extraction of cells. After organisms grown at each of the specified temperatures were freeze-dried, 10-mg aliquots of each triplicate lyophilized preparation were weighed into three separate ampoules, which were sealed after addition of 1 ml of methylating reagent, consisting of 15% (wt/vol) boron trifluoride in anhydrous methanol (British Drug Houses; 18), and evacuation. After heating the ampoules to 100°C for 1 h and allowing to cool, the contents were added to 10 ml of water and extracted twice with 1 volume of heptane (British Drug Houses) by shaking for 2 min in a mechanical shaker (Baird & Tatlock). The aqueous phases were discarded, and the two heptane phases were pooled and stored at - 18°C. The heptane phase included methyl esters of carboxylic acids. Separation of enoate classes. Portions of the heptane extract were used for preparation of acetoxymercurimethoxy derivatives of the enoate methyl MATERIALS AND METHODS esters. The technique used, based on that described Bacterial strain. The organism studied was S. by Stahl (22), was as follows: 10 ml of heptane exmutans NCTC 10832 (4), a cariogenic streptococcus tract was evaporated to 0.1 ml by evacuation in a of human origin used in a number of previous chem- Thunberg tube, with a sparsely greased stopper. A 1-ml portion of reagent (14 g of mercuric acetate, 2.5 otaxonomic studies. Growth and harvesting of cultures. Before and ml of water, 1.0 ml of glacial acetic acid, 250 ml of after growth, the organism was checked for purity methanol) was added. Air in the Thunberg tube was by Gram morphology, appearance on plates of 5% removed by evacuation and replaced by N2. After horse blood-brain heart infusion (Oxoid) and mitis allowing the reaction to proceed for 48 h at 22°C in salivarius agar (Oxoid), and reactions in sugar se- the dark, excess methanol was removed by evacuarum-peptone media and 5% sucrose-brain heart in- tion and the residue was dissolved in 0.5 ml of chlofusion. Starter cultures, consisting of 1-ml aliquots roform. Surplus reagent was removed by washing. of brain heart infusion, were inoculated into tripli- The chloroform phase was evaporated to 0.1 ml, and a spot was applied repeatedly to a thin layer of Silica cate 100-ml volumes of brain heart infusion. The Gel G (Merck) along with acetoxy-mercurimethoxy cultures were incubated in a shaking water bath (Hearson) for 48 h, without aeration, at one of nine derivatives of standard methyl enoate esters. The specified temperatures, namely, 29, 32, 33, 34, 35, thin-layer chromatography plate was run for 18 cm 221
The feasibility of classifying microorganisms by the chemical analytical method of gas chromatography was originally suggested by Abel et al. (1). Subsequently, their approach has been applied to a wide variety of microorganisms, including blue-green algae (15), marine and estuarine bacteria (20), viruses (16), mycobacteria (23), clostridia (19), micrococci (11), bacilli (12), pseudomonads (3), and streptococci (2, 8, 21). The bacterial components most often studied, structural fatty acids, are subject to variation with changing experimental parameters. Bacterial fatty acid profiles have been shown to vary with culture age (13), substrate (10), temperature (14), dilution rate in continuous culture (5), aeration (9), concentration of micronutrients and vitamins (6), and pH (7). In the present study, the effect of temperature on the fatty acid profile of Streptococcus mutans is examined.
222
DRUCKER AND VEAZEY
in petroleum spirit-diethyl ether (80:20), dried, and rerun for 14 cm in a polar solvent consisting of propan-1-ol-glacial acetic acid (100:1). After drying, enoate derivatives could be visualized by spraying with 0.1% S-diphenylcarbazone in 96% alcohol. Recovery of enoate classes for gas-liquid chromatographic analysis was possible by scraping off the thin layer and eluting with 1 ml of methanol-hydrochloric acid (10:1) and re-extracting with heptane. Gas-chromatographic analysis. Heptane extracts of methyl carboxylic esters were evaporated to small volume, and 2-,ul aliquots were analyzed three times on columns (5 feet by 0.25 inch, outer diameter [ca. 152.4 by 0.64 cm]) of 10% (wt/wt) polyethylene glycol adipate (PEGA) on 100- to 120-mesh diatomite C at 190°C in a PYE 104 chromatograph, using a N2 carrier-gas flow rate of 45 ml/min, with a detector temperature of 200°C; an injection point heater was also used. For each growth temperature, nine analyses were performed. Peaks were detected by a flame ionization detector; the detector signal was amplified by a paramatic amplifier and fed via a Spectraphysics computing integrator to a 10-mV chart recorder. The integrator printed out retention times and peak areas. Tentative identification of peaks and normalization of data was achieved by a Data General NOVA computer. This was programmed to ignore early solvent peaks and calculated the following: retention times relative to methyl palmitate, equivalent carbon number (ECN), and percent peak area, for each of the nine analyses per growth condition. Identical peaks in different analyses were averaged, and artefacts were excluded. Finally, mean percent peak areas for esters of mean ECN were printed out. Whereas absolute identity of peaks is never known in gas-liquid chromatography-chemotaxonomy application, corroboration of peak identity was sought in the present study by (i) co-chromatography with standard mixtures of methyl carboxylic esters (Applied Science) containing the methyl esters of straight-chain C,4, C,5, C,6, C,7, C,8, C19, C20, C21, and C22, iso-branched i-C,4, i-C,,6, i-C,8, and iC20, anteiso-branched ai-Cl5, ai-C,7, ai-C,9, and aiC21, and monoenoic C,8,l and C20:1, dienoic C18:2, and trienoic C,8:3; (ii) reanalysis on a nonpolar Apiezon L column; (iii) gas chromatography of separated enoate classes on PEGA; and (iv) gas chromatography with on-line mass spectrometry. On nonpolar columns the methyl esters of unsaturated acids have smaller retention times than their saturated analogues, whereas on polar columns, e.g., PEGA, their retention times are greater. Samples (2 ,l) were rerun on a glass column (5 feet by 0.25 inch [outer diameter]) of 10% Apiezon L on Celite 545 AW DMCS of 100 to 120 mesh at 225°C with an N2 carrier flow rate of 45 ml/min. Data were computed as described above. Mass spectrometry offered information on the molecular weight of esters and the mass of molecular fragments formed by electron bombardment, which in turn provided structural data. For mass spectrometry, a column of PEGA (3 feet by 0.25 inch, outer diameter [ca. 91.4 by 0.64 cm]) was run at 165°C, in a PYE 104 gas chromatograph, which temperature much reduced
APPL. ENVIRON. MICROBIOL. column bleed. Instead of using a "splitter," the entire eluate from the column was fed to an AEI MS30 mass spectrometer, and a gas chromatograph recorded in terms of ion current. A scan time of 10 s was used.
RESULTS
The strain used grew well at all the test temperatures, although above and below this range of temperatures the growth rate is very rapidly decreased. The fatty acid profiles of S. mutans NCTC 10832 obtained on PEGA, after growth at various temperatures, are shown in Table 1. Peaks observed in all samples had the following ECN values and retention characteristics on PEGA: 14.00 (n-myristate), 15.55 (iso-palmitate), 16.00 (n-palmitate), 16.30 (palmitoleate), 18.00 (nstearate), 18.40 (octadecenoate), 20.40 (eicosenoate). Other peaks occurring in more than trace amounts (>0.1% total peak area) had the following ECN values and retention characteristics on PEGA: 15.30 (pentadecenoate), 16.71 (anteiso-heptadecanoate), 17.00 (n-heptadecanoate), 17.35 (heptadecenoate), 17.60 (iso-stearate), 19.00 (n-nonadecanoate), 19.40 (nonadecenoate), 19.70 (anteiso-eicosanoate), 20.00 (n-arachidate), 20.80, 21.20. The acid of ECN 20.80 may have been the CG,:2 diunsaturated acid. Figure 1 shows a thin-layer chromatographic separation of the acetoxy-mercurimethoxy derivatives of fatty acid methyl esters of S. mutans NCTC 10832, in which spots corresponding to derivatives of monoenoates can be seen. Elution, re-extraction, and gas chromatography of the separated derivatives confirmed the presence of palmitoleate, and octadecenoate, methyl esters. Co-chromatography with standards and separate analysis of standards led to the computation of accurate ECN values, which enabled tentative identifications to be made (vide supra). Analysis of standard and sample methyl esters on nonpolar Apiezon L showed that the retention times of the unsaturated acids' esters decreased relative to saturated acids' esters. A typical sample chromatographed on polar and nonpolar columns is shown in Fig. 2. The movement of palmitoleate relative to n-palmitate, and octadecenoate relative to n-stearate, is clearly seen, whereas the relative retentions of iso- and n-palmitate remain the same. If ECN data for PEGA and Apiezon L columns are plotted against one another, an interesting two-dimensional representation is obtained (Fig. 3), analogous to the two-dimensional chromatogram familiar in thin-layer chromatography. Conjunction of two points, one of which represents a known compound, in such a repre-
VOL. 33, 1977 )
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sentation is strong evidence of the absolute identity of an unknown peak. In Fig. 3 two unknown peaks are identified as probably being methyl stearate and methyl octadecenoate. A typical result of combined gas chromatography-mass spectrometry is shown in Fig. 4; this shows re-drawn mass spectra of an unknown peak and a standard compound, methyl myristate. Both compounds have a molecular ion (M+) of mie = 242 and fragments indicating loss of methoxy (M-31) and acetyl (M-43). Both spectra have a large methyl ester peak of m/e = 74. Other fragments showing close similarities are also obvious. Difficulty was experienced in obtaining reliable mass spectral data for the minor peaks as a result of low amounts present, contamination by column bleed, and contamination by major peaks of similar retention volume.
The data for the effect of temperature on the fatty acid profile (Table 1) indicate that temper-
'-1X
224
APPL. ENVIRON. MICROBIOL.
DRUCKER AND VEAZEY
perior integration equipment now available commercially. The general change in proportions of unsaturated-saturated fatty acid with temperature is not only relevant to S. mutans, but also agrees with the data currently availa9. _._ . ...t__.'-'.. '- . . . . ble for gram-negative organisms for only two or three test temperatures for Serratia marcesb... ...-n -C16:0 cens (9, 14) or two temperatures for Vibrio marinus (20), where temperature had a less marked effect, and for eight test temperatures C1 :0 in the case of Escherichia coli (17). In all cases, increase in temperature has resulted in a decrease in the ratio of unsaturated-saturated = ,,n C16:0 fatty acid. It is quite probable that other grampositive organisms, if tested, would display a similar temperature effect, which probably reflects an attempt by the microorganism to conchrmaogapic.epraio.o FG2.Gslqi trol the changing viscosity of membrane lipid after changing temperature. Provided that time is allowed for stationary phase to be reached at all growth temperatures, there is no question of differing profiles being attributable to the organism's being at different phases of growth. The risk of misclassification of microorgaFI. 2.. Ga-iqi choaorpi of, seaato nisms grown with inadequate temperature control is fortunately not too great: the measure of association between profiles of S. mutans NCTC 10832 grown at 34 and 37°C is 0.963, calculated as the coefficient of linear correlation. This value is similar to that obtained for strains of the same species, grown identically. In view of the likelihood of fatty acid profiles methyl carboxylic esters of S. mutans on (a) polar being temperature affected for a wide range columns of PEGA and (b) nonpolar columns of Apiezon L. On a polar column, peaks due to methyl palmitoleate and methyl octadecenoate immediately fol191 low, those ofpalmitate (n-Cj;:)) and stearate (n-C, :,), whereas on a nonpolar column they elute before the
a
saturated acid esters.
ature can alter the fatty acid profile of a microorganism, that the alteration is progressive, and that the proportion of unsaturated fatty acid falls as the temperature rises. This is best seen graphically (Fig. 5), when an increase in from C results in a fall of C 39c temperature 10m in the ratio of unsaturated-saturated acids of C16 and C18 from almost unity to .
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35 GROWTH TEMPERATURE
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of microorganisms, incubation temperatures should be carefully maintained at a universally acceptable temperature such as 37°C, wherever possible, in gas-liquid chromatographic-chemotaxonomic studies, so that meaningful data may be obtained. ACKNOWLEDGMENT We are gratefully indebted to the Department of Organic Chemistry at Manchester University for providing mass spectrometry facilities. LITERATURE CITED 1. Abel, K., H. de Schmertzing, and J. I. Peterson. 1963. Classification of microorganisms by analysis of chemical composition. 1. Feasibility of utilizing gas chromatography. J. Bacteriol. 85:1039-1044. 2. Amstein, C. F., and P. A. Hartman. 1973. Differentiation of some enterococci by gas chromatography. J. Bacteriol. 113:38-41. 3. Brooks, J. B., R. E. Weaver, H. W. Tatum, and S. A. Billingsley. 1972. Differentiation between Pseudomonas testeroni and P. acidovorans by gas chromatography. Can. J. Microbiol. 18:1477-1482. 4. Drucker, D. B., R. M. Green, and D. K. Blackmore. 1972. Production of buccal and lingual caries in three weeks by a streptococcus isolated from man. J. Dent. Res. 50:1510.
5. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1973. Fatty acid fingerprints of Streptococcus mutans grown in a chemostat. Microbios 7:17-23. 6. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1974. The influence of vitamin and magnesium limitations on fatty acid fingerprints of chemostat grown Streptococcus sp. SS. Microbios 10:183-185. 7. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1975. The effect of changing pH on fatty acid profiles of Streptococcus mutans NCTC 10832. Microbios 13:99103. 8. Drucker, D. B., C. J. Griffith, and T. H. Melville. 1976. Fatty acid fingerprints of some chemostat-grown streptococci with computerized data analysis. Microbios Lett. 1:31-34. 9. Drucker, D. B., and I. Owen. 1973. Chemotaxonomic fatty acid fingerprints of bacteria grown with, and without, aeration. Can. J. Microbiol. 19:247-250. 10. Farshtchi, D., and N. M. McClung. 1970. Effect of substrate on fatty acid production in Nocardia asteroides. Can. J. Microbiol. 16:213-217. 11. Jantzen, E., T. Bergan, and K. Bovre. 1974. Fatty acid composition of strains within Micrococcaceae. Acta Pathol. Microbiol. Scand. Sect. B 82:785-798. 12. Kaneda, T. 1967. Fatty acids in the genus Bacillus. I. Iso- and anteiso fatty acids as characteristic constituents of lipids in 10 species of Bacillus. J. Bacteriol. 93:894-903. 13. Kates, M., G. A. Adams, and S. M. Martin. 1964. Lipids of Serratia marcescens. Can. J. Biochem. 42:461-479. 14. Kates, M., and P.-O. Hagen. 1964. Influence of temperature on fatty acid composition of psychrophilic and mesophilic Serratia species. Can. J. Biochem. 42:481488. 15. Kenyon, C. N. 1972. Fatty acid composition of unicellular strains of blue-green algae. J. Bacteriol. 109:827834. 16. Levanon, A., Y. Klibansky, and A. Kohn. 1973. Detection of encephalomyocarditis virus infection in animal cells by gas-liquid chromatography. Experientia 29:1305-1307. 17. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol. 84:1260-1267. 18. Metcalfe, L. D., and A. A. Schmitz. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33:363-364. 19. Moss, C. W., and V. J. Lewis. 1967. Characterisation of clostridia by gas chromatography. I. Differentiation of species by cellular fatty acids. Appl. Microbiol. 15:390-397.
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20. Oliver, J. D., and R. R. Colwell. 1973. Extractable lipids of gram-negative marine bacteria: fatty acid composition. Int. J. Syst. Bacteriol. 23:442-458. 21. Shiori-Nakano, K., and I. Tadokoro. 1968. Sugar and fatty acid composition of hemolytic Streptococcus, p. 383-386. In H. Ishizuka and T. Hesegawa (ed.), Culture collections of microorganisms; proceeding of the international conference on culture collections. Uni-
APPL. ENVIRON. MICROBIOL. versity Park Press, Tokyo. 22. Stahl, E. 1965. Thin layer chromatography. SpringerVerlag, Berlin. 23. Thoen, C. O., A. G. Karlson, and R. D. Ellefson. 1972. Differentiation between Mycobacterium kanasasii and Mycobacterium marinum by gas-liquid chromatographic analysis of cellular fatty acids. Appl. Microbiol. 24:1009-1010.
