Biochimico Elsevier
BBALIP
et Biophysics
17
Acta, 1045 (1990) 17-20
53426
Composition of polar lipid acyl chains of Bacillus stearothermophilus as affected by temperature and calcium Ligia 0. Martins,
Amhlia S. Jurado
and Vitor M.C. Madeira
Centro de Biologia Celular, Universidade de Coimhra, Coimbra (Portugal)
Key words:
Fatty acid; Growth
(Received
27 December
temperature;
Calcium;
1989)
Polar acyl chain;
(Thermophilic
bacteria)
Bacillus stearothemophilus was grown within the temperature range of 48 to 68°C in a complex medium and in the range of 45 to 72” C in the presence of 2.5 mM Ca *+. The main fatty acids of lipid extracts contain 15 to 17 carbon atoms, mostly branched-chain species. The most prevalent saturated straight-chain fatty acid is n-C16. The total amount of branched-chain species decreases with increasing temperature of growth from 48 to 68” C, whereas the straight-chain species increase. Thus, n-C,, almost doubles while i-C ,6, i-C,, and a-C,, decrease by 41.2, 28.9 and 41.9%, respectively. In the presence of Ca”, the lipid metabolism favours the biosynthesis of straight-chain fatty acids with depression of branched-chain species, especially at lower temperatures. At high temperatures, Ca” has a less pronounced effect in the lipid biosynthesis. However, above 68”C, a significant decrease is observed among the branched-chain fatty acids i-C,,, i-C,, and a-C,, with a consequent increase in n-C,,. Furthermore, a remarkable increase is observed in oleic acid (from 2.7% at 68°C to 11.5% at the extreme 72“C).
Introduction Attempting to explain the molecular basis of thermophily, Brock [l] suggested that the plasma membrane would be one of the most critical and vulnerable cellular components. It must possess an inherent stability, since it is the structure most directly exposed to the hot external environment. Thermophilic as well as mesophilic bacteria alter the fatty acid composition of membrane lipids upon changing the growth temperature [2-91. Such a change on fatty acid composition has been taken as a prerequesite for maintaining a stable fluidity of membrane lipids over the entire growth temperature range, a process termed ‘ homeoviscous adaptation’ [lo], with a consequent improvement of membrane thermostability. Most of research on the lipids of thermophilic bacteria have been concerned with fatty acid composition and it has been shown that iso and anteiso branched-chain fatty acids predominate in the thermophilic eubacteria as compared with the mesophilic counterparts [2,3,11,12]. Increasing the temperature of growth, the proportion of the relatively high melting point fatty acids (saturated straight-chain and iso acids)
Correspondent: V.M.C. Madeira, Centro de Biologia versidade de Coimbra, 3049 Coimbra Codex, Portugal.
OOOS-2760/90/$03.50
0 1990 Elsevier Science Publishers
Celular,
Uni-
B.V. (Biomedical
increases. Conversely, the proportion of the lower melting point fatty acids (unsaturated and anteiso acids) decreases [2-5,8,9,12]. Ions, specially divalent cations, also have a role in thermostability [13-161. Calcium extends the range of maximal temperature for growth in B. stearothermophilus, including higher specific growth rates, higher bacterial yields and shorter lag phases [17]. Mosley et al. [16] have suggested that calcium increases the cell stability by binding to acidic membrane phospholipids forming cationic bridges, rather than to cellular proteins as proposed by Ljunger [13]. Several studies have shown that different cations induce temperature shifts of the lipid phase transition to higher values, thus stabilizing the membrane against the perturbation of order induced by heat excess [18-201. This study describes the temperatureand calciumdependent changes on fatty acid composition of membrane polar lipids from the thermopmlic eubacteria B. stearothermophilus. Materials and Methods A description of the organism and conditions for its maintenance and culture have been reported previously [17]. Cultures were grown in one liter Erlenmeyer flasks containing 200 ml of medium, shaken at 110 t-pm in a New Brunswick Scientific water bath shaker; growth Division)
18 was measured by turbidimetry at 610 nm in a Bausch & Lomb Spectronic 21 instrument.
linear relationship within an homologous the logarithm of the relative retention number of carbon atoms.
series between times and the
Preparation of fatty acid methyl esters Growth was stopped at the beginning of the stationary phase and cells were immediately collected by centrifugation and washed once with 0.9% NaCl. The lipids of B. stearothermophilus were extracted by the method of Bligh and Dyer [21]. The amount of lipid was determined by phosphate quantification in the extracts, after solvent evaporation. The lipid hydrolysis was carried out by the procedure described by Botcher et al. [22] and phosphate was determined by the Bartlett method [23]. The fatty acid methyl esters were prepared by heating the lipid with 3 ml of BF,-methanol complex (14%, w/v) at 100°C for 2-3 min. After addition an equal volume of water, the methyl esters were extracted in hexane and the extract was dried over anhydrous Na,SO,. After evaporation of solvent, the methyl esters residues were dissolved in a small volume of CS,.
B. stearothermophilus exhibited a maximum growth temperature at 68 o C and no growth below 48 o C, under the present culture conditions. Calcium had stimulatory effect on growth, extending the Tmin and 7&, for growth to 45 and 72” C, respectively. In the presence of 2.5 mM Ca2+, cultures showed shorter lag phases, higher specific growth rates and higher amounts of growth (Fig. 1).
Identification of fatty acids The fatty acid methyl esters were analysed with a gas-liquid chromatograph Varian Aerograph (series 2700) equipped with a flame ionization detector. Nitrogen was used as the carrier gas at a flow rate of 16.7 ml per min. The injector and detector temperatures were set at 210 and 230 o C, respectively. Chromatography was routinely carried out on a polar column, 4 m x l/8”, packed with 10% SP-2340 (75% cyanopropylsilicone) on Chromosorb WAW 100/120). A non-polar open tubular glass column coated with methylsilicone (OV-1) 50 m x 0.75 mm was also used to confirm identifications made on the polar column. The analysis were carried out isothermally at oven temperatures of 160°C and 180°C. Fatty acid esters were identified by comparing their relative retention times (with n-C,, as reference) with those of standard fatty acid methyl esters and by the
Effect of temperature on fatty acid composition The fatty acid composition of B. stearothermophilus at different growth temperatures is shown in Table I. The dominating fatty acids representing between 84 to 92.5% of the total were 13-methyltetradecanoic (i-c,,), 12-methyltetradecanoic (a-C,,), 14-methylpentadecanoic (i-c,,), palmitic (n-C,,), 15-methylhexadecanoic (i-C,,) and 14-methylhexadecanoic acid (a-C,,). Branched-chain species were the predominating (Table II), with i-C,, and a-C,, in significant amounts, 38.7 to 49.2% of the total fatty acids. The most prevalent saturated straight-chain acid was palmitic (n-C,,). As the temperature of growth increases to 68 o C, the total of branched-chain fatty acids decreases by 18% (relative value) and the amount of linear fatty acids doubles (Table II). The major shifts are the decrease in the relative contents of a-C,, (41.9%), i-C,, (41.2%) and i-C,, (28.9%) and the increase in the amount of n-C,,.
1
Quantitative determination of fatty acids The relative amount (mol%) of the fatty acids present in the lipid extracts were calculated by three different methods: (1) triangulation; (2) product of the peak height by the relative retention time; (3) weighing the recorded peak after cutting it from the chart. The mean of the values was taken as representative. Results and Discussion
A
0.5 i
a01
1,
0
1
2 TI ME
3 I HOURS
L I
5
6
7
8
L5
50
55 GROWTH
60 TEMP
65
L
70
75
(~Cl
Fig. 1. Effect of Ca*+ addition on the growth of B. stearofhermophilur. (A) 6S” C in control medium (Q) and medium containing 2.5 mM Ca*+. (0); (B) over the entire range of growth temperatures in control medium, (k); and medium containing 2.5 mM Ca*+. (0).
19 TABLE I Fatty acid composition of B. stearothermophilw Fatty acids
i-C,,
n-Cl4 i-C,,
a-% n-C,, i-C,, a-C,, n-C,, i-C,, a-C,, n-C,7 n-G 18 : lcis a-% 19: ltrans
lipids as affected by temperature 60°C
72OC
45OC
48°C
Ca2+
Ca*+
control
Ca*+
control
Ca*+
control
Ca*+
0.9 1.9 19.7 7.3 0.6 13.0 1.0 16.0 14.0 21.5 0.6 1.5 1.4 _
1.2 2.8 20.7 7.7 1.2 12.0 _
0.6 1.4 23.0 8.1 0.8 8.5
0.8 3.6 25.2 6.7 1.4 7.4
0.7 3.3 22.9 6.5 1.3 7.5
1.5 2.9 22.4 8.9 1.6 5.0
_
_
0.7 3.6 21.6 7.2 1.6 5.3 _
0.5 3.2 17.7 6.0 1.6 4.6 _
17.1 13.8 18.9 0.5 1.1 2.3 0.4 0.5
10.7 15.9 26.2 0.8 1.5 2.7
19.5 14.9 15.9 0.6 1.2 1.8 0.5 0.4
21.7 15.2 15.8 0.7 1.5 1.7 0.6 0.3
24.5 12.5 16.6 0.8 2.3 2.7
20.7 11.3 15.2 0.5 3.4 5.7 _
26.8 9.2 11.3 0.3 4.3 11.5 0.7 1.8
_
68°C
which almost doubles, whereas the contents of branched C,, fatty acids remains relatively constant (Table I). In general, our findings are in agreement with data reported by several workers [2-5,7,11,12,24]. A predominance of branched-chain fatty acids at lower temperatures suggests the presence of lipid complexes with decreased phase transition temperatures. Conversely, the presence of n-C,, acid in relatively large amounts in cells grown at T,,, indicates lipids with higher phase transition temperatures. Therefore, these results are consistent with the idea that B. stearothermophilus controls the lipidic composition of the membrane, in order to compensate the destabilizing effect of excess heat, and, therefore, preserves a relatively constant order compatible with a functional biomembrane.
_ 0.4
1.1
At 48°C (Tmi, in Ca*+ absence), the proportion of linear fatty acids increases with a consequent decrease in the proportion of branched-chain species (Table II). Since linear fatty acids induce membrane order and, therefore, increased membrane stability, these results are, in principle, strange and puzzling. However, as it becomes clear from the work of several researchers [25-331, Ca*+ may exert destabilizing effects by inducing higher proportions of H,, phase in some lipid species. Cardiolipin is particularly prone to undergo cone shapes on interaction with Ca*+, thus inducing Hi, phases [25-271. Therefore, synthesis of linear fatty acids may oppose the destabilizing effect of Ca*+ by increasing the proportion of cylindrical versus conical shapes. Below 48 o C, where growth can only proceed in the presence of Ca*+, the changes observed may serve the purpose of counteracting the ordering effect of temperature upon the aliphatic chains of the membrane lipids. Additionally, at extreme temperatures, above 68 o C in Ca*+ absence), significant changes on fatty (L,, acid biosynthesis occur. Thus, decreases among the
Effect of Ca’ + on fatty acid composition Addition of 2.5 mM Ca*+ to cultures of B. stearothermophilus has a pronounced effect on fatty acid composition, specially at suboptimal temperatures.
TABLE
and Ca’+
II
General characteristics
of fatty acid distribution of B. stearothermophilus
Fatty acids
lipids
45OC
48OC
Ca*+
Ca*+
control
Ca*+
control
Ca2+
control
Ca*+
Total branched Total straight
77.4 20.6
74.7 22.7
82.3 15.2
71.4 26.3
69.2 28.5
63.9 32.8
64.3 29.1
50.0 36.2
Branched, odd (15 + 17) Branched, even (14 + 16)
62.5 14.9
61.1 13.2
73.2 9.1
62.7 8.2
60.4 8.2
57.9 6.0
57.8 6.5
44.2 5.1
Straight, odd (15+ 17) Straight, even (14+ 16)
1.2 17.9
1.7 19.9
1.6 12.1
2.0 23.1
2.0 25.0
2.4 28.1
2.1 23.6
1.9 30.0
6O“C
72OC
68OC
20 branched-chain species (i-C,,, i-C,, and a-C,,) are observed, with a consequent increase in the amounts of n-C,, (Table I). Furthermore, a very significant increase is observed in oleic acid, since its amount jumps from 2.7% of total, at 68°C to 11.5% at the extreme 72°C. This event is particularly interesting, since the presence of this species is extremely rare in bacteria (actually not previously described in B. stearothermophilus). The existence of oleic acid was systematically ascertained in a series of independent experiments. Its origin may come from either biosynthesis or uptake from the culture medium. Apparently, its occurrence and increase at extreme temperatures presumably contributes for a stabilizing effect induced by CaZf and is probably related to the membrane itself as a whole, favouring the adjustment of a variety of lipid species, rather than to the individual properties of this acid, since its cis double bond, in principle, induces disordering effects. Acknowledgements This work was supported Foundation and DUCT.
by
INIC,
Gulbenkian
References 1 Brock, T.D. (1967) Science 158, 1012-1019. 2 Daron, H.H. (1970) J. Bacterial. 101, 145-151. 3 Yao, M., Walker, H.W. and Lillard, D.A. (1970) J. Bacterial. 102, 877-878. 4 Ray, P.H., White, D.C. and Brock, T.D. (1971) J. Bacterial. 108, 227-235. 5 Weerkamp. A. and Heinen, W. (1972) J. Bacterial. 109, 443-446. 6 De Rosa, M., Gambacorta, A. and Bu’Lock. J.D. (1974) J. Bacterial. 117, 212-214. 7 Souza, K.A., Kostiw, L.L. and Tyson, B.J. (1974) Arch. Microbial. 97, 89-102. 8 Rilfors, L., Wieslander, A. and Stahl, S. (1978) J. Bacterial. 135, 1043-1052. 9 Hasegawa, Y., Kawada, N. and Nosoh, Y. (1980) Arch. Microbial. 126,103-108.
10 Sinensky, M. (1974) Proc. Natl. Acad. Sci. USA 71, 522-525. 11 Shen. P.Y., Coles, E., Foote, J.L. and Stenesh, J. (1970) J. Bacterial. 103, 479-481. 12 Chan, N., Himes, R.H. and Akagi, J.M. (1971) J. Bacterial. 106. 876-881. 13 14 15 16
Ljunger, C. (1970) Physiol. Plant 23, 351-364. Stahl, S. and Ljunger, C. (1976) FEBS Lett. 63, 1844187. Stahl, S. (1978) FEMS Microbial. Lett. 4, 77-81. Mosley, G.A., Card, G.L. and Koostra. W.L. (1976) Can. J. Microbial. 22, 468-474. 17 Jurado, M.A.S., Santana, A.C., Costa, M.S. and Madeira, V.M.C. (1987) J. Gen. Microbial. 133, 507-513. 18 Trauble, H. and Eibl, H. (1974) Proc. Natl. Acad. Sci. USA 71, 214-219.
19 Van Dijck, P.W.M., Ververgaert, P.H.J.Th., Verkleij. A.J., Van Deenen, L.L.M. and De Gier. J. (1975) Biochim. Biophys. Acta 406. 465-478. 20 Jacobson, K. and Papahadjopoulos, D. (1975) Biochemistry 14, 152-161. 21 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. 22 Bottcher, C.J.F., Van Gent, C.M. and Pries, C. (1961) Anal. Chim. Acta 24. 203-204. 23 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. 24 Kaneda, T. (1977) Bacterial. Rev. 41, 391-418. 25 Rand, R.P. and Sengupta, S. (1972) Biochim. Biophys. Acta 255, 4844492. 26 Cullis, P.R., Verkleij, A.J. and Ververgaert, P.H.J. (1978) B&him. Biophys. Acta 513, 11-20. 27 Vasilenko, I., De Kruijff, B. and Verkleij. A.J. (1982) Biochim. Biophys. Acta 684, 282-286. 28 De Kruijff, B. and Cullis, P.R. (1980) Biochim. Biophys. Acta 602, 477-490. 29 Cullis, P.R. and Verkleij, A.J. (1979) B&him. Biophys. Acta 552. 546-551. 30 Tilcock, C.P.S. and Cullis, P.R. (1981) Biochim. Biophys. Acta 641, 189-201. 31 Farren. S.B. and Cullis. P.R. (1980) Biochem. Biophys. Res. Commun. 97, 182-191. 32 Nayar, R.. Schmid, S.L., Hope, M.J. and Cullis, P.R. (1982) Biochim. Biophys. Acta 688. 169-176. 33 Wieslander, A., Christiansson, A., Rilfors, L. and Lindblom, G. (1980) Biochemistry 19, 3650-3655.
BBALIP
et Biophysics
17
Acta, 1045 (1990) 17-20
53426
Composition of polar lipid acyl chains of Bacillus stearothermophilus as affected by temperature and calcium Ligia 0. Martins,
Amhlia S. Jurado
and Vitor M.C. Madeira
Centro de Biologia Celular, Universidade de Coimhra, Coimbra (Portugal)
Key words:
Fatty acid; Growth
(Received
27 December
temperature;
Calcium;
1989)
Polar acyl chain;
(Thermophilic
bacteria)
Bacillus stearothemophilus was grown within the temperature range of 48 to 68°C in a complex medium and in the range of 45 to 72” C in the presence of 2.5 mM Ca *+. The main fatty acids of lipid extracts contain 15 to 17 carbon atoms, mostly branched-chain species. The most prevalent saturated straight-chain fatty acid is n-C16. The total amount of branched-chain species decreases with increasing temperature of growth from 48 to 68” C, whereas the straight-chain species increase. Thus, n-C,, almost doubles while i-C ,6, i-C,, and a-C,, decrease by 41.2, 28.9 and 41.9%, respectively. In the presence of Ca”, the lipid metabolism favours the biosynthesis of straight-chain fatty acids with depression of branched-chain species, especially at lower temperatures. At high temperatures, Ca” has a less pronounced effect in the lipid biosynthesis. However, above 68”C, a significant decrease is observed among the branched-chain fatty acids i-C,,, i-C,, and a-C,, with a consequent increase in n-C,,. Furthermore, a remarkable increase is observed in oleic acid (from 2.7% at 68°C to 11.5% at the extreme 72“C).
Introduction Attempting to explain the molecular basis of thermophily, Brock [l] suggested that the plasma membrane would be one of the most critical and vulnerable cellular components. It must possess an inherent stability, since it is the structure most directly exposed to the hot external environment. Thermophilic as well as mesophilic bacteria alter the fatty acid composition of membrane lipids upon changing the growth temperature [2-91. Such a change on fatty acid composition has been taken as a prerequesite for maintaining a stable fluidity of membrane lipids over the entire growth temperature range, a process termed ‘ homeoviscous adaptation’ [lo], with a consequent improvement of membrane thermostability. Most of research on the lipids of thermophilic bacteria have been concerned with fatty acid composition and it has been shown that iso and anteiso branched-chain fatty acids predominate in the thermophilic eubacteria as compared with the mesophilic counterparts [2,3,11,12]. Increasing the temperature of growth, the proportion of the relatively high melting point fatty acids (saturated straight-chain and iso acids)
Correspondent: V.M.C. Madeira, Centro de Biologia versidade de Coimbra, 3049 Coimbra Codex, Portugal.
OOOS-2760/90/$03.50
0 1990 Elsevier Science Publishers
Celular,
Uni-
B.V. (Biomedical
increases. Conversely, the proportion of the lower melting point fatty acids (unsaturated and anteiso acids) decreases [2-5,8,9,12]. Ions, specially divalent cations, also have a role in thermostability [13-161. Calcium extends the range of maximal temperature for growth in B. stearothermophilus, including higher specific growth rates, higher bacterial yields and shorter lag phases [17]. Mosley et al. [16] have suggested that calcium increases the cell stability by binding to acidic membrane phospholipids forming cationic bridges, rather than to cellular proteins as proposed by Ljunger [13]. Several studies have shown that different cations induce temperature shifts of the lipid phase transition to higher values, thus stabilizing the membrane against the perturbation of order induced by heat excess [18-201. This study describes the temperatureand calciumdependent changes on fatty acid composition of membrane polar lipids from the thermopmlic eubacteria B. stearothermophilus. Materials and Methods A description of the organism and conditions for its maintenance and culture have been reported previously [17]. Cultures were grown in one liter Erlenmeyer flasks containing 200 ml of medium, shaken at 110 t-pm in a New Brunswick Scientific water bath shaker; growth Division)
18 was measured by turbidimetry at 610 nm in a Bausch & Lomb Spectronic 21 instrument.
linear relationship within an homologous the logarithm of the relative retention number of carbon atoms.
series between times and the
Preparation of fatty acid methyl esters Growth was stopped at the beginning of the stationary phase and cells were immediately collected by centrifugation and washed once with 0.9% NaCl. The lipids of B. stearothermophilus were extracted by the method of Bligh and Dyer [21]. The amount of lipid was determined by phosphate quantification in the extracts, after solvent evaporation. The lipid hydrolysis was carried out by the procedure described by Botcher et al. [22] and phosphate was determined by the Bartlett method [23]. The fatty acid methyl esters were prepared by heating the lipid with 3 ml of BF,-methanol complex (14%, w/v) at 100°C for 2-3 min. After addition an equal volume of water, the methyl esters were extracted in hexane and the extract was dried over anhydrous Na,SO,. After evaporation of solvent, the methyl esters residues were dissolved in a small volume of CS,.
B. stearothermophilus exhibited a maximum growth temperature at 68 o C and no growth below 48 o C, under the present culture conditions. Calcium had stimulatory effect on growth, extending the Tmin and 7&, for growth to 45 and 72” C, respectively. In the presence of 2.5 mM Ca2+, cultures showed shorter lag phases, higher specific growth rates and higher amounts of growth (Fig. 1).
Identification of fatty acids The fatty acid methyl esters were analysed with a gas-liquid chromatograph Varian Aerograph (series 2700) equipped with a flame ionization detector. Nitrogen was used as the carrier gas at a flow rate of 16.7 ml per min. The injector and detector temperatures were set at 210 and 230 o C, respectively. Chromatography was routinely carried out on a polar column, 4 m x l/8”, packed with 10% SP-2340 (75% cyanopropylsilicone) on Chromosorb WAW 100/120). A non-polar open tubular glass column coated with methylsilicone (OV-1) 50 m x 0.75 mm was also used to confirm identifications made on the polar column. The analysis were carried out isothermally at oven temperatures of 160°C and 180°C. Fatty acid esters were identified by comparing their relative retention times (with n-C,, as reference) with those of standard fatty acid methyl esters and by the
Effect of temperature on fatty acid composition The fatty acid composition of B. stearothermophilus at different growth temperatures is shown in Table I. The dominating fatty acids representing between 84 to 92.5% of the total were 13-methyltetradecanoic (i-c,,), 12-methyltetradecanoic (a-C,,), 14-methylpentadecanoic (i-c,,), palmitic (n-C,,), 15-methylhexadecanoic (i-C,,) and 14-methylhexadecanoic acid (a-C,,). Branched-chain species were the predominating (Table II), with i-C,, and a-C,, in significant amounts, 38.7 to 49.2% of the total fatty acids. The most prevalent saturated straight-chain acid was palmitic (n-C,,). As the temperature of growth increases to 68 o C, the total of branched-chain fatty acids decreases by 18% (relative value) and the amount of linear fatty acids doubles (Table II). The major shifts are the decrease in the relative contents of a-C,, (41.9%), i-C,, (41.2%) and i-C,, (28.9%) and the increase in the amount of n-C,,.
1
Quantitative determination of fatty acids The relative amount (mol%) of the fatty acids present in the lipid extracts were calculated by three different methods: (1) triangulation; (2) product of the peak height by the relative retention time; (3) weighing the recorded peak after cutting it from the chart. The mean of the values was taken as representative. Results and Discussion
A
0.5 i
a01
1,
0
1
2 TI ME
3 I HOURS
L I
5
6
7
8
L5
50
55 GROWTH
60 TEMP
65
L
70
75
(~Cl
Fig. 1. Effect of Ca*+ addition on the growth of B. stearofhermophilur. (A) 6S” C in control medium (Q) and medium containing 2.5 mM Ca*+. (0); (B) over the entire range of growth temperatures in control medium, (k); and medium containing 2.5 mM Ca*+. (0).
19 TABLE I Fatty acid composition of B. stearothermophilw Fatty acids
i-C,,
n-Cl4 i-C,,
a-% n-C,, i-C,, a-C,, n-C,, i-C,, a-C,, n-C,7 n-G 18 : lcis a-% 19: ltrans
lipids as affected by temperature 60°C
72OC
45OC
48°C
Ca2+
Ca*+
control
Ca*+
control
Ca*+
control
Ca*+
0.9 1.9 19.7 7.3 0.6 13.0 1.0 16.0 14.0 21.5 0.6 1.5 1.4 _
1.2 2.8 20.7 7.7 1.2 12.0 _
0.6 1.4 23.0 8.1 0.8 8.5
0.8 3.6 25.2 6.7 1.4 7.4
0.7 3.3 22.9 6.5 1.3 7.5
1.5 2.9 22.4 8.9 1.6 5.0
_
_
0.7 3.6 21.6 7.2 1.6 5.3 _
0.5 3.2 17.7 6.0 1.6 4.6 _
17.1 13.8 18.9 0.5 1.1 2.3 0.4 0.5
10.7 15.9 26.2 0.8 1.5 2.7
19.5 14.9 15.9 0.6 1.2 1.8 0.5 0.4
21.7 15.2 15.8 0.7 1.5 1.7 0.6 0.3
24.5 12.5 16.6 0.8 2.3 2.7
20.7 11.3 15.2 0.5 3.4 5.7 _
26.8 9.2 11.3 0.3 4.3 11.5 0.7 1.8
_
68°C
which almost doubles, whereas the contents of branched C,, fatty acids remains relatively constant (Table I). In general, our findings are in agreement with data reported by several workers [2-5,7,11,12,24]. A predominance of branched-chain fatty acids at lower temperatures suggests the presence of lipid complexes with decreased phase transition temperatures. Conversely, the presence of n-C,, acid in relatively large amounts in cells grown at T,,, indicates lipids with higher phase transition temperatures. Therefore, these results are consistent with the idea that B. stearothermophilus controls the lipidic composition of the membrane, in order to compensate the destabilizing effect of excess heat, and, therefore, preserves a relatively constant order compatible with a functional biomembrane.
_ 0.4
1.1
At 48°C (Tmi, in Ca*+ absence), the proportion of linear fatty acids increases with a consequent decrease in the proportion of branched-chain species (Table II). Since linear fatty acids induce membrane order and, therefore, increased membrane stability, these results are, in principle, strange and puzzling. However, as it becomes clear from the work of several researchers [25-331, Ca*+ may exert destabilizing effects by inducing higher proportions of H,, phase in some lipid species. Cardiolipin is particularly prone to undergo cone shapes on interaction with Ca*+, thus inducing Hi, phases [25-271. Therefore, synthesis of linear fatty acids may oppose the destabilizing effect of Ca*+ by increasing the proportion of cylindrical versus conical shapes. Below 48 o C, where growth can only proceed in the presence of Ca*+, the changes observed may serve the purpose of counteracting the ordering effect of temperature upon the aliphatic chains of the membrane lipids. Additionally, at extreme temperatures, above 68 o C in Ca*+ absence), significant changes on fatty (L,, acid biosynthesis occur. Thus, decreases among the
Effect of Ca’ + on fatty acid composition Addition of 2.5 mM Ca*+ to cultures of B. stearothermophilus has a pronounced effect on fatty acid composition, specially at suboptimal temperatures.
TABLE
and Ca’+
II
General characteristics
of fatty acid distribution of B. stearothermophilus
Fatty acids
lipids
45OC
48OC
Ca*+
Ca*+
control
Ca*+
control
Ca2+
control
Ca*+
Total branched Total straight
77.4 20.6
74.7 22.7
82.3 15.2
71.4 26.3
69.2 28.5
63.9 32.8
64.3 29.1
50.0 36.2
Branched, odd (15 + 17) Branched, even (14 + 16)
62.5 14.9
61.1 13.2
73.2 9.1
62.7 8.2
60.4 8.2
57.9 6.0
57.8 6.5
44.2 5.1
Straight, odd (15+ 17) Straight, even (14+ 16)
1.2 17.9
1.7 19.9
1.6 12.1
2.0 23.1
2.0 25.0
2.4 28.1
2.1 23.6
1.9 30.0
6O“C
72OC
68OC
20 branched-chain species (i-C,,, i-C,, and a-C,,) are observed, with a consequent increase in the amounts of n-C,, (Table I). Furthermore, a very significant increase is observed in oleic acid, since its amount jumps from 2.7% of total, at 68°C to 11.5% at the extreme 72°C. This event is particularly interesting, since the presence of this species is extremely rare in bacteria (actually not previously described in B. stearothermophilus). The existence of oleic acid was systematically ascertained in a series of independent experiments. Its origin may come from either biosynthesis or uptake from the culture medium. Apparently, its occurrence and increase at extreme temperatures presumably contributes for a stabilizing effect induced by CaZf and is probably related to the membrane itself as a whole, favouring the adjustment of a variety of lipid species, rather than to the individual properties of this acid, since its cis double bond, in principle, induces disordering effects. Acknowledgements This work was supported Foundation and DUCT.
by
INIC,
Gulbenkian
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