JouRNAL OF BACTERIOLOGY, Sept. 1978, p. 754-759

Vol. 135, No. 3

WX21-9193/78/0135-0754$02.00/0 Copyright C 1978 American Society for Microbiology

Printed in U.S.A.

Effect of Growth Temperature on Membrane Fatty Acid Composition and Susceptibility to Cold Shock of Bacillus amyloliquefaciens C. PATON, E. J. McMURCHIE, B. K. MAY,* AND W. H. ELLIOTT Depifnmerit of Biochemistry, The University of Adelaide, Adelaide, South Australia 5001 J.

Received for publication 18 April 1978

We investigated the fatty acid composition of the membrane of Bacillus amnyloliquefaciens grown at different temperatures. A decrease in growth temperature was accompanied by an increase in the ratio of branched- to straightchain fatty acids and a marked increase in the level of unsaturation of branchedchain fatty acids. When cells of this organism grown at 300C were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween 80 to cells caused the critical temperature zone for cold shocking to be lowered significantly. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock.

Permeability damage to membranes caused by the rapid chilling of cells (that is, cold shocking) has been shown in a number of gram-negative organisms (3, 4, 7, 10, 13) and was first observed in the gram-positive organism Bacillus amyloliquefaciens in this laboratory (12). It was shown by Smeaton and Elliott that for this organism a critical temperature zone existed through which cells must be rapidly chilled to induce the effect. The permeability of the cell was altered, allowing a fluorescent dye to penetrate the membrane and intracellular components, such as the intracellular ribonuclease inhibitor (a protein with a molecular weight of 12,000), to leak out. In the present work, this phenomenon was investigated in cells of B. amyloliquefaciens grown from spore inoculum at different temperatures. We found that the critical temperature zone for cold shock is markedly altered by changes in growth temperature.

of Casamino Acids (Difco) was reduced to 0.025% (wt/vol). Thick suspensions of cells maintained at their growth temperature were rapidly cooled by squirting 0.5-ml samples into 19.5-ml lots of stirred WCSM maintained at different temperatures. (This addition increased the temperature of the medium by not more than 0.5°C.) Alternatively, cells were slowly cooled by shaking flasks of cells in an ice water bath (the rate of temperature decrease being approximately 4°C/m) until the desired temperature was reached. Assay for the occurrence of cold shock. (i) Protease secretion. Samples (20 ml) of cells that had been cooled were returned to an orbital shaking water bath maintained at 300C (Paton model OW1412) and allowed to equilibrate for 5 min. Incubation was continued for 30 min, and samples were withdrawn at 10min intervals. These samples were centrifuged (4,000 x g for 5 min), and the amount of protease present in the supernatant fraction was assayed by the method of Rinderknecht et al. (9). (ii) Viability. Washed cells were sheared in a Sorvall Omnimixer at 18,000 rpm for 3 min, which resulted in the breakage of the normal B. amyloliquefaciens chains of cells into single cells. Cells were either rapidly or slowly cooled as described above. Cells were serially diluted in growth medium at 300C, and 0.1-ml samples of the appropriate dilutions were spread on prewarmed plates containing growth medium and 1.5% (wt/vol) agar (Difco). Triplicate plates for each temperature sample were scored for colonies after overnight incubation at 370C. Lipid extraction. Harvested cells were suspended in 50 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.3), and 1 volume of cell suspension was extracted with 1 volume of chloroform and 2.17 volumes of methanol by shaking at 20C for 2 h. Undissolved material was pelleted by centrifugation and extracted two more times. Extracts were pooled, and 0.2 volume of 10% (wt/vol) NaCl was added to form

MATERIALS AND MEUHODS Growth of organism. A mutant strain of B. amyloliquefaciens that does not secrete surfactin (10) was grown from a spore inoculum in a medium described previously by Both et al. (2). Flasks were incubated at 300C or other temperatures in an orbital shaking incubator (Paton model 461) oscillating at 300 rpm. The celis were harvested at the late exponential growth phase (absorbance at 600 nm, 3.6). Procedure for cooling cells. Cells were washed and suspended in washed cell suspension medium (WCSM), which was the same as the growth medium except that FeCl3 and yeast extract were omitted, thereby preventing cell growth, and the concentration 754

FATTY ACID COMPOSITION AND COLD SHOCK

VOL. 135, 1978

two phases. The chloroform phase was removed, and

the aqueous phase was extracted with an equal volume of chloroform. The chloroform extracts were pooled, dried over anhydrous sodium sulfate, concentrated in a rotary evaporator, and reduced under a stream of N2. Lipid extracts were dissolved and stored under N2 at -20°C. Poly-,-hydroxybutyrate (included as an antioxidant) was removed from the lipid extract by the method of Bishop et al. (1). Preparation and analysis of fatty acids. After removal of the solvent, lipids were saponified by refluxing for 3 h in 1 N NaOH in 50% (wt/vol) methanol under N2. Non-saponifiable material was removed by extraction with hexane. The aqueous layer was acidified with 20 N H2SO4, and the fatty acids were extracted with ether. The extract was dried over anhydrous sodium sulfate and reduced under a stream of N2. Fatty acids were converted to their methyl esters by heating at 750C for 1 h in the presence of 14% (wt/vol) BF3 in methanol. The sample was cooled on ice, 3 ml of water was added, and the methyl esters were extracted with petroleum ether (40 to 60°C fraction). Extracts were dried under N2 and taken up in a small volume of CS2. Fatty acid methyl esters were analyzed on a PerkinElmer model 880 gas chromatograph equipped with a flame ionization detector and connected to a Rikadenki millivolt recorder. The flame ionization detector was operated at flow rates of 30 and 450 ml/min for H2 and air, respectively. Four separate 6-foot (ca. 1.83m) columns were used for the identification of fatty acid methyl esters; these contained 3 and 5% Apiezon L, 10% EGSS-X (ethylene succinate-methyl silicone polymer, low silicone), and 25% DEGS (diethylene glycol-succinate coated onto 100- to 120-mesh, acidwashed, ilanized Chromosorb P; purchased from Applied Science Laboratories). The 3 and 5% Apiezon L columns were operated at 1900C with a carrier gas (H2) flow rate of 50 ml/min, the temperatures of the injector block and the detector being 240 and 2000C, respectively. DEGS and EGSS-X columns were operated at 1650C with a carrier gas flow rate of 40 ml/min, the injector block and detector temperatures being 215 and 1750C, respectively. Fatty acid methyl esters were identified by running unknowns and methyl ester standards (purchased from Applied Science Laboratories) on both polar and nonpolar columns, determining their Rf values relative to methyl palmitate, and plotting the logarithm of the Rf value against the carbon number. Unsaturated fatty acids were identified by their different behavior on the two types of columns together with their disappearance after hydrogenation when palladium on charcoal was used as a catalyst. Quantitative analysis was performed only on samples run on the nonpolar Apiezon L columns because these columns gave greater resolution between the branched-chain fatty acid methyl esters and their straight-chain homologs. Peak areas were determined by triangulation. RESULTS

Fatty acid analysis of cells grown at different temperatures. Cells were grown from

755

at different temperatures, and the fatty acids of the cells were extracted and analyzed as described above. When the growth temperature was lowered from 37 to 25°C, the proportion of normal saturated fatty acids decreasd markedly from 17.7 to 5.1%, whereas the proportion of saturated branched-chain fatty acids increased from 81.5 to 92.9% (Table 1). No unsaturated branched-chain fatty acids were detected in cells grown at 370C, but the mono-unsaturated normal C18:i acid was present in small amounts (0.4%). However, when the growth temperature was lowered to 25°C, this C18 acid could no longer be detected, but the mono-unsaturated branched-chain fatty acids iso-Cs and iso- and anteisO-C17:1 appeared, comprising 1.9% of total cellular fatty acids. Further reduction in the growth temperature to 20°C resulted in a further decrease of normal saturated fatty acids from 5.1 to 1.2% and a marked increase in the monounsaturated branched-chain fatty acids (1.9 to 9.4%). The proportion of saturated branchedchain fatty acids decreased slightly from 92.9 to 89.4%, presumably as a result of desaturation of the branched-chain acids contning 16 and 17 carbon atoms. Determination of the temperature zone for cold shocking cells grown at 300C. In previous studies of the cold shock phenomenon in this organism, release of intracellular ribonuclease inhibitor was employed as an assay for the occurrence of cold shock (12). A more convenient alternative procedure was used in the

spores

TABLE 1. Fatty acid comrposition of cells grown at different tenperatures Fatty acid % of total fatty acids at a growth temp (IC) methyl es ters

i-C14: n-C14O i+

a-C,50b

n-C150

i-C,6O n-C160

a-Ci7ob n-Cm7o n-Ci8:1 n-C,8o i+

i-CIe:I

i + a-C71b

of

37

30

25

20

4.4 1.0 39.6 0.7 14.1 13.3 23.8 0.5 0.4 2.2 0 0

7.3 0.9 43.5

8.3 0.1

8.7 0.2 58.0 0.1 11.2 0.9 11.5

1.1 12.8

12.0 20.4 1.2 0.2 0.4 0.1 0.1

48.5 0.3 15.9 4.5 19.8

0.7 0 0 1.1 0.8

0 0 0 4.3 5.1

aFatty acid methyl esters were analyzed as described in the text. n, i and a denote normal, iso, and anteiso fatty acids, respectively. Fatty acid methyl esters are denoted C.,, where x is the number of carbon atoms and y is the number of double bonds. b Some iso and anteiso compounds were not sufficiently separated to allow individual quantitation.

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PATON ET AL.

present study; loss of ability of cells to secrete extracellular protease after rapid cooling was examined. Cultures of cells grown at 300C were washed, suspended in fresh medium, and cooled (rapidly or slowly) to various temperatures. The cell suspensions were shaken at 300C, and, after a 5min equilibration time, the rate of protease secretion over the next 30 min was measured as described above. Protease production by washed cells was linear for 30 min, and the rates of protease secretion (expressed as a percentage of that of celLs that were not cooled) were plotted as a function of the temperature to which the celLs were cooled

,

9a

(Fig. 1). 0 15 2 5 0 20 10 ( c) TEMP. When cells were rapidly cooled from 30 to 10C, they completely lost the ability to syntheFIG. 2. Effect of cooling rate on the viability of size protease. The requirements for this cold cells cookd to various temperatures. Cells of B. amshock effect were rather precise. The critical yloliquefaciens grown at 30'C were washed and sustemperature zone through which the cells must pended in WCSM and then sheared in a Sorvall be cooled to obtain the effect was about 16 to Omnimixer for 3 min at 18,000 rpm. Samples were then rapidly (0) or slowly (0) cooled, and their 110C, and, as previously observed (12), rapid viability determined as described in the text. The chilling was essential for this phenomenon. viability was is expressed as a percentage of the viability When cell suspensions were slowly cooled from of ceUs that were not cooled. 30 to as low as 10C, subsequent protease secretion after return to 300C was normal (Fig. 1). from 30 to 10C completely lost viability, the When cell viability was measured as an assay critical temperature range measured in this way for the occurrence of cold shock, results imilar being 17 to 130C. When cells were slowly cooled to those found for protease secretion were ob- through this temperature range, the viability tained (Fig. 2). Cells that were rapidly cooled loss was less than 25% (Fig. 2). The cold shock effect was not dependent on 0 0 the magnitude of the temperature decrease or loo '0 100 on the starting temperature (provided it was above the critical zone). Cells slowly cooled to 19 from 300C and then rapidly cooled to tem60 peratures below 130C were completely inactive (data not shown). The critical temperature range determined I0 here was somewhat broader than the 16 to 140C range reported by Smeaton and Elliott (12) for U0 this organism when they measured the selective release of ribonuclease inhibitor. However, measurement of cold shock by release of ribonuclease inhibitor in the present work showed that the release occurred over a range of 17 to 130C (data not shown). Effect of growth temperature on the critCI) TIMP. FIG. 1. Effect of cooling rate on subsequent pro- ical temperature zone for cold shock. Cells tease secretion by cells cooled to various temperawere grown from a spore inoculum at 37, 30, 25, tures. Cells of B. amyloliquefaciens grown at 30(C and 200C and harvested when the absorbance of were washed and suspended in WCSM. They were the culture at 600 nm was 3.6 in each case, which then rapidly (0) or slowly (0) cooled to the indicated corresponded to the end of the exponential temperatures as described in the text. Samples of ceUs growth phase. Cell cultures grown at these tem(20 ml) were returned to a 30fC shaking water bath, peratures took 12, 18, 34, and 65 h, respectively, and after a 5-min equilibration the rate of protease secretion was measured over the next 30 min as to reach the required cell density. The dry described in the text. The rate ofprotease secretion is weights of the cells grown at the different temexpressed as a percentage of that of cells that were peratures were the same when the celLs were not cooled. harvested at the same absorbance value.

FAT1Y ACID COMPOSITION AND COLD SHOCK

VOL. 135, 1978

The effect of rapid cooling on viability or the ability to subsequently secrete protease at 300C was measured in cells grown at these temperatures as described above. (When suspended in WSCM, cells grown at these different temperatures synthesized protease linearly for at least 30 min and at comparable rates, such that the final level of protease after 30 min was about 50 U/ml.) From Fig. 3 it is clear that growing cells at temperatures lower than 300C had a dramatic effect on the critical temperature zone for the cold shock phenomenon, as measured by protease secretion. Cells grown at 20°C could be rapidly chilled to temperatures as low as 70C with no ill effect on the ability of the celLs to secrete, whereas chilling to 10C still resulted in a subsequent 50% rate of protease secretion. With cells grown at 25°C, somewhat intermediate results were obtained for protease secretion, whereas with those grown at 37°C there was only a slight shift in the critical zone to higher temperatures (Fig. 3). When cells grown at 200C were rapidly cooled to temperatures as low as 1°C, there was no significant loss of viability (Fig. 4). However, there was almost total loss of viability when cells grown at 30°C were similarly cooled to 1°C (Fig. 4). Effect of Tween 80 on the critical temperature zone for cold shock. Cells grown at 30 or 200C were washed and suspended in WCSM with or without the nonionic detergent Tween 80 (polyoxyethylene sorbitan monooleate; 1% [vol/vol]) and rapidly chilled to various temperatures in WCSM with or without 1% (vol/vol) Tween 80. The occurrence of cold shock was assayed by the ability of the cells to secrete protease after a return to 300C. The presence of the detergent caused no inhibition of protease secretion in control cells. Figure 5 shows that for

757

cells grown at 30°C, the presence of Tween 80 in the cooling medium caused a depression of the critical temperature for cold shock of about 5°C. However, for cells grown at 20°C, no such depression was observed. The observed effect of Tween 80 was not due to the presence of contaminating free oleate. Separate experiments, in which oleate was added to the WCSM during the washing and incubation of cells, established that cellular protein synthesis and protease production were significantly inhibited by concentrations of oleate as low as 0.9 ytg/ml. Concentrations of oleate

S. S

(*C)

TEMP.

FIG. 4. Effect of growth temperature on the loss of viability after rapid cooling. Cells of B. amyloliquefaciens grown at 30 (0) or 200C (0) were washed, suspended in WCSM, and sheared in a Sorvall Omnimixer (18,000 rpm for 3 min). They were then rapidly chilled to the indicated temperature, and their viability was determined as described in the text (expressed as a percentage of the viability of cells that were not cooled).

0oo z 0 l

F

so

60

6

uO 4c *- 40

.-,20 0

0

5

15

10

TEMP.

25

20

30

37

(C)

FIG. 3. Effect ofgrowth temperature on the loss of ability to secrete protease after rapid cooling to various temperatures. Cells of B. amyloliquefaciens grown at 37 (0), 30 (0), 25 (U), or 20°C (0) were washed, suspended in WCSM, and then rapidly cooled to the indicated temperature. Their ability to secrete protease (expressed as a percentage of that of cells that were not cooled) was determined as described in the text.

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PATON ET AL.

J. BACTERIOL.

first grown at low temperatures. There is an apparent correlation between the critical temperature zone required for cold shock and the fatty acid composition of cells grown at different temperatures. Those grown at 20°C have a marked increase both in the ratio of branchedto straight-chain fatty acids and in the level of unsaturation of the branched-chain fatty acids. Both of these effects result in a membrane with increased lipid fluidity. Correspondingly, the temperature zones for cold shock are significantly reduced. When celLs grown at 200C are compared with those grown at 30°C, the zone is TEMP. (0c) shifted from 16-11oC to 5-less than 10C. In FIG. 5. Effect of Tween 80 on the critical temperpractice this means that, although cells grown at ature zone for cold shock as determined by a decrease 300C and instantly chilled to 3°C totally lose in the rate ofprotease secretion. Cells grown at 30"C viability and ability to secrete protease, those were washed, suspended, and rapidly cooled to the indicated tenperature in WCSM (@) or WCSM + 1% grown at 200C can be chilled to this temperature (vol/vol) Tween 80 (0). Cells grown at 200C were with almost no effect on these parameters. Cells similarly treated in WCSM (U) or WCSM + 1% grown at 250C exhibit an intermediate fatty acid (vol/vol) Tween 80 (0). The subsequent rate of pro- composition and an intermediate critical temtease secretion (expressed as a percentage of that of perature zone for cold shock. cells that were not cooled) was determined after a Attempts were made to further alter the fatty return to 30"C as described in the text. acid composition by growth at temperatures lower than 200C. Although cells grew at 150C, below this figure did not affect the critical tem- the fatty acid composition was identical to those perature for cold shock. grown at 200C, and the critical temperature zone for cold shock was unaltered. Apparently there DISCUSSION is a limit to which the fatty acid composition The fatty acid composition of membranes (and hence the critical cold shock temperature) from B. amyloliquefaciens has not been re- can be altered by lowering growth temperature. ported previously, but is not significantly differThat the membrane lipid fluidity is important ent from other bacilli (6), being characterized by for determining the cold shock effect was further a low percentage of unsaturated fatty acids and substantiated by the finding that Tween 80, a high percentage of branched-chain fatty acids. which presumably perturbates the lipid bilayer However, the response of B. amyloliquefaciens and thereby promotes disorder, lowered the critto lowered growth temperature is different from ical temperature zone for cold shock by 50C. that reported for other bacilli. Growth of B. Because the Tween 80 effect occurs rapidly and amyloliquefaciens at low temperatures results does not involve cell growth in the presence of not only in a significant increase in the ratio of the detergent, it seems likely that its effect is on branched- to straight-chain fatty acids, but also the membrane itself. The combination of growth in a 20-fold increase in the degree of unsatura- at low temperature and Tween 80 treatment tion of branched-chain fatty acids. Kaneda (5) failed to reduce the critical temperature for cold found for B. cereus that lowered growth temper- shock beyond the reduction already achieved by ature resulted in no change in the ratio of growth at low temperatures alone. straight- to branched-chain fatty acids, but did Haest et al. (4) and Leder (7) proposed that in result in a threefold increase in the level of Escherichia coli the temperature for cold shock unsaturation of both normal and branched-chain was that at which the hydrocarbon core solidifatty acids. McElhaney and Souza (8) found fied. They suggested that upon rapid chilling the that, when the growth temperature of B. stear- solidification gave rise to discontinuities in the othermophilus was lowered, there was a signifi- membrane packing, resulting in the release of cant increase in the ratio of branched- to small molecules. straight-chain fatty acids and an increase in The results of the work presented in this paper straight-chain unsaturated fatty acids, but no are compatible with this suggestion. unsaturated branched-chain fatty acids were deI-.

40

20

0

10

15

20

25

20

tected. The most important finding in the present work is that the cold shock phenomenon in B. amyloliquefaciens can be avoided if cells are

LITERATURE CITMD L. Rutbrg, and B. amuelaon. 1967.

1. Bishop, D. G.,

The chemical composition of the cytoplasmic membrane of Bacillus subtilis. Eur. J. Biochem. 2:448 453.

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2. Both, G. W., J. L Mes, J. E. Hanlon, B. K. May, and W. H. Elliott. 1972. Evidence for an accumulation of mesenger RNA specific for extracellular proteae and its relevance to the mechanism of enzyme secetion in bacteria. J. MoL BioL 67:199-217. 3. Gorrill, R. H, and E. L McNeiL 1960. The effect of cold diluent on the viable count of Pseudomonas pyocyanea. J. Gen. Microbiol. 22:437-442. 4. Haest, C. W. M, J. de Gier, G. A. Es, A. J. Verkleij, and L L KL van Deene 1972. Fragility of the permeability barrier of Escherichia coli. Biochim. Biophysa Acta 288:43-53. 5. Kaneda, T. 1972. Positional preference of fatty acids in phoepholipids of BaciUus cereus and its relation to growth temperature. Biochim. Biophys. Acta 280:297-305. 6. Kaneda, T. 1977. Fatty acids of the genus BaciUuw an example of branched-chain preference. Bacteriol. Rev. 41:391418. 7. Leder, L G. 1972. Interrelated effects of cold shock and osmotic prere on the permeability ofthe Escherichia coli membrane to permease accumulated substrates. J. Bacteriol. 111:211-219. van

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8. McElhaney, R. N., and K. A. Souza. 1976. The relationship between environmental temperature, cell growth and the fluidity and physical sate of the membrane lipids in BaciUus stearothermophils. Biochim. Bio. phys. Acta 443:34839. 9. Rinderknecht, EL, KL C. Geokas, P. Silverman, and B. J. Kaverback. 1968. A new ultrasensitive method for the determination ofproteolytic activity. Clin. Chim Acta 21:197-203. 10. Sanders, R. L., and B. K. May. 1975. Evidence for extrusion of unfolded extracellular enzyme polypeptide chains through membranes of Bacilus amyloliquefaciens. J. Bacteriol. 23:806-814. 11. Sato, M., and He Taahambi 1968. Cold shock of bacteria I. General features of cold shock in Escherichia coli J. Gen. Appl. Microbiol. 14:417-428. 12. Smeaton, J. R., and W. Elliott. 1967. Selective releam of ribonucleaminhibitor from Bacilus subtilis cells by cold shock treatment. Biochem. Biophys. Res. Commun. 26:75-81. 13. Strange, R. E., and F. A. Dark. 1962. Effect of chilling onAerobacter aerogenes in aqueous suspension. J. Gen. Microbiol. 29:719-730.

Effect of growth temperature on membrane fatty acid composition and susceptibility to cold shock of Bacillus amyloliquefaciens.

JouRNAL OF BACTERIOLOGY, Sept. 1978, p. 754-759 Vol. 135, No. 3 WX21-9193/78/0135-0754$02.00/0 Copyright C 1978 American Society for Microbiology P...
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