Thermal analysis of bacteria by differential scanning calorimetry: Relationship of protein denaturation in situ to maximum growth temperature

19

Biochimica et Biophysica Acta, 1055 (1990) 19-26 Elsevier

BBAMCR 12783

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature James R. Lepock

1,2,

Harold E. Frey 1 and William E. Inniss 2

I Guelph-Waterloo Program for Graduate Work in Physics, Waterloo Campus and 2 Department of Biology, University of Waterloo, Waterloo (Canada)

(Received 26 March 1990) (Revised manuscript received 6 June 1990)

Key words: Heat shock; Protein denaturation; Hyperthermia; DSC; (Bacillus)

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmx) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33°C below T ~ . The transition temperature (Tm) of the main melting of DNA varies from 88 to 92°C, 23-32"C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tt), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56°C, respectively. For cells grown between 4 and 200C below Tmx, TI is 2-40C lower than Tma~, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tt or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megatedum and B. psychrophilus, except that Tmax is 48 and 32.5°C (Tt--45-460C and 30°C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Introduction All organisms have a specific range of temperature over which they can function and grow. In general, bacteria can exist at any temperature at which water remains liquid [1]; however, each bacterial species is viable over a relatively narrow temperature range of 30-40°C with limits defined by the minimum (Tmin) and maximum (Tm~x) temperatures for growth and with an optimum temperature of maximum growth (Topt) near the upper end of the range [2]. Bacteria are generAbbreviations: DSC, differential scanning calorimetry. Correspondence: J.R. Lepock, Department of Physics, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.

ally grouped in three categories depending on their temperature range for growth: (1) psychrophiles and psychrotrophs with Topt generally from 5 to 25°C, (2) mesophiles with Topt from 25 to 45°C and (3) thermophiles with Topt at high temperatures, in some cases exceeding 100°C [1]. Since growth is normally defined by division, inhibition of growth is a consequence of inhibition of division. Above Toot organisms suffer irreversible heat damage that is dependent on both time and temperature of exposure [3]. The temperature relationship of the rate of killing is well described by the Arrhenius equation with, in general, a high temperature dependence for killing [3,4]. For example, most lines of mammalian cells [5] and yeasts [6] have activation energies for cell killing in excess of 100 kcal/mol. This is also true for the irreversible inactivation of most pro-

0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

20 teins [7], whereas most metabolic processes have far lower activation energies [3]. The mechanism of inhibition of growth and cell killing by supra-optimal temperatures is not known; however, nearly all cellular components have been suggested as targets. The general approach has been to identify changes between mesophilic and thermophilic organisms that appear to lead to a more thermally stable cell. Alterations to cellular constituents that correlate with growth at high temperatures include changes in membrane lipids yielding membranes that are less fluid and melt at a higher temperature [8], changes in the G C / A T ratio of nucleic acids also resulting in a higher melting point [9], and changes in extracted proteins, primarily amino acid sequence, yielding more thermostable proteins with a higher denaturation temperature [10-12]. Each of these components - lipids, nucleic acids and proteins - undergo a temperature-induced or thermotropic transition resulting in a less ordered conformation at high temperatures. Most isolated enzymes from thermophiles are stable in vitro; denaturation does not occur until several degrees above Tmax, implying that they are not the specific target for inhibition of growth and cell killing [10]. Other isolated enzymes denature well below Tmax, suggesting the presence of stabilizing factors within the cell. What is needed is to be able to determine the stability of cellular components in vivo and to identify components undergoing a thermotropic transition near Tmax. These thermolabile components must be considered as potential critical targets for heat damage. Differential scanning calorimetry (DSC) is a technique that has been widely used in detecting transitions in isolated proteins [13] and cellular organelles [14]. All thermotropic transitions directly induced by increasing temperature are endothermic and potentially detectable by DSC if of sufficient enthalpy. DSC and other techniques capable of measuring lipid transitions and lipid dynamics (generally referred to as fluidity) have been applied to the study of thermoadaptive mechanisms of membranes from a number of bacterial species [15,16]. Recently DSC has been applied to living mammalian cells [17,18], in which case the complex profile obtained is the sum of all thermotropic transitions in all cellular components separated on the basis of thermostability. We describe the application of DSC to the study of thermotropic transitions in the mesophile Bacillus megaterium, two strains of the thermophile Bacillus stearothermophilus and the psychrotroph Bacillus psychrophilus. Protein, lipid and DNA transitions are identified in situ and compared to the maximum temperatures of growth. Materials and Methods

Bacteria and Growth Conditions The bacteria used were chosen as representative of

procaryotic microorganisms capable of growing in one of the three major microbial temperature ranges. In addition, only members of the same genus, namely Bacillus, were used to eliminate any generic differences. The thermophile B. stearothermophilus (ATCC 12016), the mesophile B. rnegaterium (ATCC 13632) and the psychrotroph B. psychrophilus (ATTC 23304) were employed. In addition, another thermophilic strain of B. stearothermophilus (designated as B. stearothermophilus WAT), which had been adapted for growth at a lower temperature, although still in the thermophilic temperature range, was studied in order to compare two very similar bacteria with different optimum growth temperatures within the same major temperature range of growth. Cells were maintained in trypticase soy broth at the appropriate incubation temperatures. For use experimentally, cells were inoculated into two 250-ml Erlenmeyer flasks each containing 100 ml of trypticase soy broth to give an initial optical density of 0.05 at 650 nm. The flasks were then incubated at the various experimental temperatures on rotary shakers until the bacterial growth attained an A650 of 1.0, at which time they were used for DSC experimentation. Bacterial growth rates were spectrophotometrically determined using identical conditions.

Differential Scanning Calorimetry Cells to be used for DSC were quickly cooled to 4°C and harvested by centrifugation, washed twice in phosphate buffer at 4°C (10 mM, pH = 7.0) containing 100 /~M chloramphenicol (to prevent any further protein synthesis) and resuspended in the same solution. The cell suspension was degassed under mild vacuum at 4°C for 5 min immediately before addition to the calorimeter cells. DSC scans were obtained with a Microcal-2 calorimeter (1.21 ml sample cells) interfaced to a DEC Pro 380 computer as previously described [17]. The scan was started (1 °C/rain scan rate) when the sample and reference (identical solution lacking bacteria) reached equilibrium at 0°C (approx. 40 rain after addition of cells). The scan was run to 100-102°C, the sample cooled back to 0°C, and a second scan (the rescan) run. A baseline (reference solution in both the sample and reference cells) was usually obtained but these varied only slightly from day to day. Results

The uncorrected DSC profile (Cp vs. temperature) of the thermophile Bacillus stearothermophilus (ATCC 12016) grown at 65°C is shown in Fig. 1A. Curve a is the initial scan from 5 to 100°C. Positive deflections of Co represent heat absorbed due to endothermic processes and negative deflections represent heat re-

21 leased due to e x o t h e r m i c processes. A n o b v i o u s e n d o t h e r m i c region f r o m 6 5 - 1 0 0 ° C a n d a n e x o t h e r m at 1 5 - 2 0 ° C are r e a d i l y visible. T h e rescan (curve b), obt a i n e d i m m e d i a t e l y after c o o l i n g to 0°C, has two reversible e n d o t h e r m i c p e a k s at a p p r o x . 30 a n d 92°C. A baseline, o b t a i n e d with p h o s p h a t e b u f f e r b u t n o b a c t e r i a in b o t h the s a m p l e a n d reference cells, is also shown in Fig. 1 ( p a n e l A, curve c). It has a convex s h a p e similar to that of the rescan b u t w i t h o u t the two peaks. T h e scan a n d rescan were c o r r e c t e d for the curved b a s e l i n e b y s u b t r a c t i n g a baseline c o n s t r u c t e d b y rem o v i n g the two p e a k s from the rescan a n d fitting the r e m a i n i n g curve with a f o u r t h o r d e r p o l y n o m i a l . This yields a s m o o t h curve that follows the rescan except for the p e a k s at 30 a n d 92°C. T h e c o r r e c t e d scan a n d rescan are shown in Fig. 1B (curves a a n d b, respectively). T h e specific features are m o r e clearly illustrated b y these p l o t s which have h a d the intrinsic c u r v a t u r e o f the b a s e l i n e r e m o v e d . T h e m a j o r e x o t h e r m at 6 5 - 1 0 0 ° C consists o f at least four p e a k s labelled t r a n s i t i o n s A - D . T h e t r a n s i t i o n t e m p e r a t u r e s (Tin) are given in T a b l e I. T h e t e r m t r a n s i t i o n will b e used to refer to the m o l e c u lar events r e s p o n s i b l e for each p e a k ; however, these p e a k s p r o b a b l y d o n o t r e p r e s e n t a distinct t r a n s i t i o n in a single c o m p o n e n t b u t in general m u s t b e d u e to m u l t i p l e t r a n s i t i o n s at the s a m e o r closely space t e m p e r atures. T r a n s i t i o n D is at least p a r t i a l l y reversible, is p r e s e n t in the rescan a n d occurs at a b o u t the t e m p e r a ture (88°C) for m e l t i n g o r u n f o l d i n g o f D N A i s o l a t e d f r o m B. stearothermophilus [19]. T h e o t h e r t r a n s i t i o n s are irreversible a n d p r e s u m a b l y r e p r e s e n t p r i m a r i l y the d e n a t u r a t i o n of cellular p r o t e i n . Thus, this e x o t h e r m i c region m u s t b e c o m p o s e d of a large n u m b e r o f transitions, each r e p r e s e n t i n g the d e n a t u r a t i o n of a n i n d i v i d ual p r o t e i n o r p r o t e i n d o m a i n . T h e o b s e r v a b l e p e a k s are

d u e to t r a n s i t i o n s o f m a j o r c o m p o n e n t s , c o o p e r a t i v e t r a n s i t i o n s o f s t r o n g l y i n t e r a c t i n g c o m p o n e n t s , o r to the f o r t u i t o u s s u p e r p o s i t i o n o f several t r a n s i t i o n s [18]. T h e r e is usually an i n c r e a s e in specific heat, r e f e r r e d to as ACp, u p o n going f r o m the n a t i v e to the d e n a t u r e d state o f a protein. This p o s i t i v e shift in the b a s e l i n e can be r e m o v e d to give a true p r o f i l e o f d e n a t u r a t i o n if the value of Cp is k n o w n for the d e n a t u r e d state [20]. This c o r r e c t i o n was n o t p e r f o r m e d for a n y o f the b a c t e r i a l scans o f B. stearothermophilus or B. megaterium, since it a p p e a r s that d e n a t u r a t i o n m a y n o t b e c o m p l e t e even at 100°C. H o w e v e r , the c o r r e c t i o n for ACp was perf o r m e d for B. psychrophilus (Fig. 5) in which case it is clear that d e n a t u r a t i o n d o e s n o t exceed 100°C. Thus, the high t e m p e r a t u r e region o f the c o r r e c t e d scans m a y be artificially elevated, b u t this has n o effect o n a n y of the Tm valves r e p o r t e d . T h e o n s e t of d e n a t u r a t i o n is best c h a r a c t e r i z e d b y T l, which is d e f i n e d as the t e m p e r a t u r e at w h i c h the linear, low t e m p e r a t u r e side o f t r a n s i t i o n A intersects a horiz o n t a l line d r a w n t h r o u g h the lowest p a r t of the curve (see Fig. 1B). In a d d i t i o n , the t e m p e r a t u r e ( T l * ) o f first d e t e c t a b l e u p w a r d d e v i a t i o n f r o m the b a s e l i n e was d e t e r m i n e d for each curve ( T a b l e I). T h i s t e m p e r a t u r e is difficult to m e a s u r e a c c u r a t e l y a n d is v e r y subjective, b u t it is r e l a t e d to the m i n i m u m t e m p e r a t u r e o f den a t u r a t i o n . Tt (64.5°C) is a p p r o x i m a t e l y equal to the g r o w t h t e m p e r a t u r e ( 6 5 ° C ) for B. stearothermophilus ( A T C C ) used in Fig. 1. Thus, s o m e d e n a t u r a t i o n occurs when this g r o w t h t e m p e r a t u r e is e x c e e d e d b y even a small a m o u n t . In the rescan, there is a w e l l - d e f i n e d e n d o t h e r m i c t r a n s i t i o n (labelled L in Fig. 1B) c e n t e r e d at a b o u t 3 0 ° C with a h i g h - t e m p e r a t u r e limit at a b o u t 36°C. T h e low t e m p e r a t u r e limit is difficult to d e t e r m i n e a c c u r a t e l y

TABLE I Transition temperaturesfor the four main high-temperature transitions (TA -TD), transition L (determined from the rescan), and the onset of denaturation (Tl) determined from DSC scans of B. stearothermophilus (,4 TCC and WA T strains), B. megaterium (.4 TCC) and B. psychrophilus (A TCC) The column labels represent: Tfgrowth temperature, Ti-incubation temperature, ti-time of incubation, Tl*-minimum temperature of deviation from the baseline of transition A, Trextrapolation of low temperature side of transition A with baseline (onset of denaturation), TL-transition temperature of the L transition on rescan, and TL (high) high temperature end of L transition. Errors are S.E.M. of at least three measurements. Cells

T$

Ti

ti

Transition temperatures (°C)

(°C) (°C) (min) Tl.

B. stearothermophilus (ATCC) 65 B. stearothermophilus (ATCC) 56

-

-

B. stearothermophilus (WAT) B. stearothermophilus (WAT)

56 56

50

60

B. megaterium (ATCC) B. megaterium (ATCC) B. megaterium (ATCC) B. megaterium (ATCC)

44 37 37 30

30 -

60 -

B. psychrophilus (ATCC)

20

-

-

62.5 + 1 = 64

rl

TA

TB

64.5 + 1 65.5

= 73 = 72

81.0 + 0.7 ---85 81.4 = 87

92.2 + 0.6 29.5 + 1 93.5 30

68.0+0.7 67.2

--73 -- 71

92.1:t:0.5 25.3+-0.5 31+-1 92 26.4 32

59.5+-0.5 68.8+-1.0 60.6+0.7 69.4+0.7 ~ 56 70.7 -- 54 70.4

-

54.8+-0.2 56.1+-0.6 ---61 49.8 51.9 ~ 64 44.5+0.5 ---44 39 38 27

46.4+0.2 46.7+1 44.7 40.2 29.7

34.5

54.2

Tc

TD

88.2+0.2 88.1+0.7 88.5 89.5

71.2 88.2

TL

22.0+-1 21.9+-0.4 21.5 20.3 -

TL(high)

36 + 1 37

26+1 26+-0.5 24 25 -

22 B

B

A

C

A

D

>o "6

O 11)

I

v

O

O

. / L \

I' 0

I0

20

:50

40

50

60

70

80

I L I A ] ~ I ~

IO0

90

0

l0

20

30

T (°C)

40

50 T

60

70

80

90

100

(°C)

Fig. 1. DSC profile of Bacillus stearothermophilus (ATCC 12016) grown at 65°C. The original scans are given in panel A and the baseline corrected scans in panel B. The labelled curves are a: initial scan, b: rescan following the initial scan, and c: buffer-buffer baseline. The main endothermic transitions above the growth temperature are labelled A - D , T l is defined as the temperature of onset of denaturation, and L is the reversible low-temperature transition. The two arrows in panel B label the exotherms.

and may be below 0°C. This transition is not as well-defined in the original scan (Fig. 1B, curve a), partially because of the presence of a superimposed exotherm at about 18°C. This exotherm was present in about half of the scans and varied somewhat in temperature and magnitude. Transition L is more clearly shown in scans where the exotherm is not present (see Fig. 2). Because of its position and revisibility, transition L is identified as representing a membrane lipid transition. An endothermic transition in isolated membranes of B. stearothermophilus has been reported, although at a somewhat higher temperature [16]. Another exotherm is visible in the region of 50-65°C. This exotherm is eliminated by 10 mM K C N and is much stronger when the cells are scanned in medium containing glucose; thus, it must represent the heat of metabolism released during the scan. KCN has no effect on the remainder of the profile, including the exotherm superimposed on the lipid transition. Thus, the low temperature exotherm is not due to metabolism

and may represent an event triggered by the melting of the membrane lipid bilayer in intact cells. Growth curves of B. stearothermophilus (ATCC) were obtained from 65-71°C and the doubling times (td) are shown in Table II. Those give a maximum growth temperature (Tmax) of approx. 69°C, similar to the values of 7 0 - 7 5 ° C previously reported for B. stearothermophilus [21]. Tmax is approx. 4°C higher than T 1 (64.5°C - the onset temperature of denaturation). The denaturation occurring between 65 and 69°C appears to be sufficient to depress but not block growth, suggesting that a low level of denaturation of some proteins can be tolerated. The shape of the DSC profile of B. stearothermophilus (ATCC) is not altered by growth at 56°C (results not shown). The transition temperatures (Table I) are not significantly different than those of cells grown at 65°C. The second strain of B. stearothermophilus, referred to as the WAT strain, was derived from the ATCC strain but has been grown at 56°C for several years. The

D T A B L E II

Doubling times (td) used to determine the maximum growth temperatures

"6 /

Q)

T (°C)

t d (h)

65 67 69 71

1.0 1.3 1.2 25

B. stearothermophilus (WAT) B. stearothermophilus (WAT)

56 58

0.85 30

B. B. B. B.

44 46 48 50

0.49 0.60 0.59 7.2

__

S. S. S. S.

v

Q

,

0

I

10

,

I

20

~

I

30

~

I

40

,

I

50

,

I

60

,

I

70

i

I

80

,

l

90

,

100

T(*C)

Fig. 2. DSC profile of Bacillus stearothermophilus (WAT) grown at 56°C. The broken line is the first scan to 41°C which was followed by a full scan (solid line).

stearothermophilus stearothermophilus stearothermophilus stearothermophilus

megaterium megaterium megaterium megaterium

(ATCC) (ATCC) (ATCC) (ATCC)

(ATCC) (ATCC) (ATCC) (ATCC)

23

¢:x

//.J/ / / /

J/ ,

A . _~- -

45

Z~-

- - - I ~ , ,~"

50

,

i

I

55

60

65

T (°C) Fig. 3. Corrected DSC profiles in the region 45-65°C of Bacillus at 56°C after 0 ( ), 5 ( - - - - ) and 30 ( - --) min at 50°C. The D S C profile of cells incubated in 500 #M chloramphenicol at 50°C for 15 min is also shown ( - - -).

stearothermophilus (WAT) grown

DSC profile of the WAT strain is shown in Fig. 2. An initial scan to 41°C demonstrates the reversibility of the L transition. No low-temperature exotherm occurred during the L transition for this sample, but other scans of the WAT strain had an exotherm at about 20°C as shown in Fig. 1 for the ATCC strain. A metabolic exotherm centered at about 48°C can be seen in Fig. 2. Although the general shape of the DSC profile is the same, there are some significant changes in the transition temperatures of the WAT strain compared to the ATCC strain. The Tm valves of transitions A - C are about 12°C lower in the WAT strain (Table I), but transition D is unaltered. T~ occurs at 56°C, the growth temperature, which is 9°C lower than T~ for the ATCC strain. Thus, denaturation commences at the growth temperature for both strains. The Tm and the high-temperature end of the L transition are about 4-5°C lower, which is less than the shift in transitions A - C and T:. The maximum growth temperature for B. stearothermophilus (WAT) is 56°C (Table II). Tl is also 56°C

(Table I). Thus, denaturation commences at temperatures above Tg, as for the ATCC strain, but this correlates with an immediate cessation of growth, not at a temperature 4°C higher as for the ATCC strain. This suggests that some protein denaturation might be occurring during growth at 56°C. This possibility was investigated by growing the cells at 56°C, incubating them at 50°C for 5 to 60 min, and then obtaining DSC scans. The expanded region around Tj of the corrected profiles after incubation for 5 and 30 rain at 50°C is shown in Fig. 3. The onset of denaturation is shifted to lower temperatures as indicated by the reduction in Tl and Tl* to 52 and 50°C, respectively. There are no significant changes in any other transition temperatures (Table I). The reduction in T~ and T:* is blocked by the protein synthesis inhibitor chloramphenicol (Fig. 3, curve d), demonstrating the requirement of protein synthesis for the reduction in Tj and suggesting that thermolabile proteins denaturing between 52 and 56°C are synthesized during incubation at 50°C. DSC profiles of the mesophile Bacillus megaterium (ATCC 13632) grown at different temperatures are shown in Fig. 4. Growth temperatures of 44, 37 and 30°C were used and an incubation at 30°C after growth at 37°C was performed. The general shape of the profile of the endothermic transitions A - D is somewhat different than for B. stearothermophilus. Rather than a main peak B flanked by two minor peaks A and C separated somewhat from a high temperature peak D, only two peaks, well-resolved and labelled A and B, are present consistently in addition to peak D. Peaks A and B are very broad and probably consist of a number of unresolved transitions. The Tm of transition D is 88°C, about 4°C lower than that for both strains of B. stearothermophilus. In addition, transition D is usually but not always reversible (Fig. 4B), suggesting that under some conditions renaturation of the DNA is hindered. The cooling rate was approx. 10°C/rain at

B A

D

.=,,

A

o

CI)

QJ

0

0.

/f,--

i

O

I

I0

J

I

20

L

I

30

i

I

40

A

I

50

i

I

60

,

I

70

,

I

80

,

I

90

I0

,

K:)O

20

30

40

50 60 T (°C)

70

80

90

I00

Fig. 4. DSC profiles of Bacillus megaterium (ATCC 13632) grown under the following conditions: Ts = 44°C (curve a, - - ) , Ts ffi 37°C followed by incubation at 30°C for 60 rain (curve b, - . . . . . ), and Tg ffi 30°C (curve c, - - - - - ) . Panel A contains the original scans and panel B the rescans.

24 88°C and decreased to 2 - 3 ° C / r a i n at 40°C, considerably faster than the cooling rates of 1 / 2 - 2 / 3 ° C per min usually employed in studies of the renaturation of D N A [22], which may explain why renaturation was not always observed. The metabolic exotherm is much stronger for B. megaterium; thus, KCN (10 mM) was added during all scans. However, there is still some instability in the region of the L transition due to metabolism (see Fig. 4A). The Tm for the L transition, determined from the rescans, is 22°C, about 3 and 8°C lower than that for the WAT and ATCC strains of B. stearothermophilus, respectively (Table I). The onset of denaturation ( T 1) for the mesophilic B. megaterium is lower than TI for B. stearothermophilus and highly dependent on growth temperature. For Tg = 44°C, T I is 46°C, slightly above the growth temperature, and the Tm of transition A is 60°C (Fig. 4A, curve a). Doubling times for growth measured at 44-50°C (Table II) give a maximum growth temperature of 48°C, slightly greater than Tv This observation is consistent with the correlation between TI and Tmax found for B. stearothermophilus. Dropping T~ to 37°C does not affect T 1 (Table I), but the magnitude of transition A increases relative to transition B (results not shown). This indicates the presence of a greater proportion of more thermolabile proteins during growth at 37°C. Major changes in the DSC profile occur after growth or even short periods of incubation at 30°C (Fig. 4A, curves b and c). The Tm valves for transitions B and D are unaltered, but transition A broadens and shifts from 60°C to 54-56°C (Table I). This results in a decrease in T I of 6°C following growth at 30°C . Thus, there is a large increase in the fraction of thermolabile proteins denaturing in the region of 40 to 46°C. Rescans of the curves shown in Fig. 4A are given in 4B. There are no significant changes in the general shape of transition L or its transition temperature and, when present in the rescan, transition D still has a Tm of 88°C (Table I). Thus, there are major changes in protein stability during growth at 30°C but no detectable changes in the lipid or D N A melting temperatures. A corrected DSC scan of the psychrophile Bacillus psychrophilus (ATCC 23304) grown at 20°C is shown in Fig. 5. The general shape of the profile is similar to that of B. stearothermophilus; however, T I is at approx. 30°C and transitions A - C are spread over a wider temperature range (Table I). Transitions A, B and C in B. psychrophilus are 39, 27 and 14°C lower than their counterparts in B. stearothermophilus (ATCC). Whether or not the protein components of each transition are identical between B. psychrophilus and B. stearothermophilus is unknown. N o lipid transition is visible above 0°C, which is consistent with growth of B. psychrophilus at 0°C. The D N A transition (D) is at 88°C, the same temperature as for B. megaterium, but

B

tD

.>

O A

0

I0

20

30

C

40

,50

60

70

D

OO

9(3

I(30

T (*C) Fig. 5. DSC profile of Bacillus psychrophilus (ATCC 23304) grown at 20°C. The solid line is the scan and the broken line the rescan.

4°C lower than for B. stearothermophilus. The onset of denaturation ( T l = 30°C) is about 2°C lower than Tmax, which has previously been shown to occur at 32.5°C [23]. Thus, inhibition of growth of B. psychrophilus, which is due to inability to form septa [23], correlates with the denaturation of the cellular proteins comprising transition A. Discussion Studies of numerous organisms, both multi- and uni-cellular, have shown that, for most, a high activation energy in the range of 100-200 k c a l / m o l is associated with killing due to supra-optimal temperatures [3,4,24]. These activation energies are much greater than those for metabolic processes which are in the range of 10-20 kcal/mol. Most proteins denature with a high activation energy (indicating a strong temperature dependence), and this correspondence suggests that the rate-limiting step of cell killing may be protein denaturation. Other molecular transitions such as lipid and nucleic acid melting also have a strong temperature dependence because of the narrow temperature range over which they occur. Thus they miast also be considered as potential rate-limiting steps for cell killing. The involvement of a thermotropic transition, whether or not it is protein denaturation, in heat damage leads to a simple model for such damage - the existence of a rate-limiting, critical target [24,17,18]. Each process at the cellular level, e.g. growth and division or the maintenance of viability, is dependent on numerous cellular components and structures. These vary in thermostability and undergo transitions at different temperatures. Some critical component for each process is the most thermolabile and is irreversibly damaged first during heating. The rate at which this irreversible damage occurs in the critical target determines the rate of inactivation of the cellular process. Thus, it is rate-limiting and the target damaged is the critical target. The irreversibility of this transition implies that damage is

25 dependent on time and temperature of exposure, and the Tm of the critical target can be predicted from the rate and temperature-dependence of cell killing at hyperthermic temperatures [17,18]. DSC is ideal for the detection of thermotropic transitions in biological macromolecules, cellular organelles and intact cells. All transitions present in whole cells will contribute to the final profile in proportion to their calorimetric enthalpy. We have used DSC to study protein transitions in the mammalian Chinese hamster lung V79 line [17]. Protein denaturation commences slightly below the onset temperature for killing (40.5°C) and is highly irreversible even after short periods of heating at 43-45°C. The onset temperature for denaturation is increased in cells made thermotolerant by a prior heat exposure [18]. In addition, studies on isolated membranes and mitochondria from V79 cells using protein specific spin labels and intrinsic fluorescence has demonstrated protein denaturation commencing at about 40°C [25,26]. Protein denaturation in the erythrocyte membrane, beginning at 40-45°C, has been well characterized [27]. The concept of a critical molecular target determining the rate of a cellular process has been used to show that the lysis of human erythrocrytes exposed to temperatures in excess of 48°C correlates with an irreversible transition at 60°C, probably the denaturation of a membrane protein [20]. Several transitions are detectable by DSC in intact B. stearothermophilus, B. megaterium, and B. psychrophilus. A low temperature transition, apparently due to the melting of at least a portion of the membrane lipid, occurs above 0°C for the first two species and is centered 22-39°C lower than Tmax. Thus, Toot and Tmax are far above the temperature at which all lipids are in the liquid crystalline state, clearly showing that Tmax is not determined by the upper end of a lipid transition. This conclusion is consistent with previous studies of Acholeplasrna laidlawaii [28] and B. stearothermophilus [16] which also showed that Tm~x is much higher than any lipid transition and that membrane lipid fluidity is relatively unimportant for proper membrane function as long as the lipids are in the liquid crystalline state [29]. Although there is no correlation between the lipid transition and Tma~, the minimum temperature of growth [Tmin] might be related to the formation of gel lipid. The upper end of the lipid transition (36 + I°C) correlates very well with the Tminof 37°C determined by Babel et al. [30] for B. stearothermophilus (ATCC 12980). A number of endothermic transitions, labelled A - D , occur above Tmax. Transition D, centered at 92°C for B. stearothermophilus and 88°C for B. megaterium and B. psychrophilus, is the melting of DNA. The difference of 4°C in the Tm of transition D between B. stearothermophilus and B. megaterium is almost identical to the 3 ° difference previously measured for isolated D N A from these species in 0.15 M NaC1 [19]. This difference

can be accounted for by the increased G C content of B.

stearothermophilus and indicates that the greater Tm of transition D in intact B. stearothermophilus is not due to the presence of general stabilizers of macromolecular structure or specific stabilizers of D N A (e.g., polyamines). The main endothermic transitions labelled A - C must be due primarily to protein denaturation. However, other endothermic and exothermic processes should also contribute but to a lesser extent. These could include the unfolding of RNA, macromolecular polymerization or depolymerization, aggregation of denatured proteins and alterations in ligand binding [17]. In addition, chemical reactions induced by high temperature might contribute to the high temperature end of the profile. The onset temperature of denaturation, characterized by the parameter T~, correlates well with Tmax for both strains of B. stearothermophilus (grown at 50-65°C) and for B. psychrophilus grown at 20°C. Tmax is 2 - 3 ° C higher than Tt, indicating that a small amount of denaturation can be tolerated before growth and division are inhibited. A similar correlation holds for Tmax (48°C) and T 1 for B. megaterium grown above 37°C. Transitions A and B are distinctly resolved for B. psychrophi/us because of the wide range over which denaturation occurs (30-100°C), and it is clear that only the components of transition A can contribute to inhibition of growth at Tmax (32.5°C). Identification of these components should be easier in B. psychrophilus than in the other species. A close correspondence between T I and Tmax also exists for mammalian Chinese hamster lung V79 cells [17]. The synthesis of heat shock proteins (HSP's) is induced in all organisms, including Bacilli, by exposure to temperatures 5-15°C in excess of Toot [31]. In E. coli, the transcription activating factor has been identified as the RNA polymerase subunit 032; however, the initial signal for induction is unknown. Abnormal protein, specifically denatured protein, has been suggested to be the inducing agent [32,33]. Temperatures required for HSP synthesis in B. megaterium [34], B. stearothermophilus and B. psychrophilus [35] are similar to the values of T I (i.e., temperature required for protein denaturation) as determined by DSC. Thus, the induction of the heat shock response and the denaturation of cellular protein in these Bacilli correlate over the temperature range of 30 to 70°C. This study also illustrates the applicability of DSC to the investigation of heat-induced damage in cells. DSC separates, albeit at low resolution, all thermotropic transitions in cells, analogous to the separation of proteins on the basis of molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Thus, a profile of denaturation vs. temperature (i.e. thermostability) is generated for all cellular macromolecules under conditions present in the intact cell, which necessarily

26 accounts for the effect of all cellular stabilizers on Tm. Potentially this approach can be used to identify the specific proteins denaturing in the temperature range of Tmax, which must contain the critical targets limiting growth.

Acknowledgements This investigation was supported by PHS grant number CA40251 awarded by the National Cancer Institute, DHHS, to J.R.L. and by a grant awarded by the Natural Sciences and Engineering Research Council of Canada to W.E.I.

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