- Email: [email protected]
Thermal stability of ribosomes and RNA from Thermus aquaticus
512
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96449
THERMAL
STABILITY OF RIBOSOMES AND RNA FROM THERMUS
A Q UA TIC US
J. G. ZEIKUS, M. \V. TAYLOR AND T. D. BROCK
Department o/ Microbiology, Indiana University, Bloomington, Ind. 47402- (U.S.A.) (Received November ioth, 1969)
SUMMARY
This p a p e r presents d a t a on the p h y s i c a l a n d chemical p r o p e r t i e s of the ribosomes a n d R N A of Thermus aquaticus, an o r g a n i s m able to grow at t e m p e r a t u r e s u p to 79 °. U l t r a c e n t r i f u g a l analysis of purified ribosomes in o . o i M Mg 2+ d e m o n s t r a t e d c o m p o n e n t s s e d i m e n t i n g a t 50 S, 7 ° S a n d IOO S. Chemical analysis of ribosomes revealed an average c o n t e n t of 59 % protein a n d 4 1 % R N A . W h e n h e a t e d in s t a n d a r d buffer, Escherichia coli ribosomes b e g a n g r a d u a l dissociation at 59 ° with a Tm of 71°. I n contrast, T. aquaticus ribosomes were stable up to 79 ° a n d h a d a Tm of 86 °. T. aquaticus r i b o s o m a l R N A (rRNA) was found to be more t h e r m a l stable t h a n t h a t of E. coli. However, t h e r m a l d e n a t u r a t i o n profiles of f r a c t i o n a t e d I6-S a n d 23-S r R N A of E. coli a n d T. aquaticus d e m o n s t r a t e d t h a t o n l y the 23-S species of T. aquaticus r R N A was more stable t h a n t h a t of E. coli. T h e r m a l d e n a t u r a t i o n studies revealed t h a t T. aquaticus transfer R N A ( t R N A ) was stable up to 68 ° a n d h a d a Tm of 86°; whereas, E. coli t R N A was only stable up to 55 ° a n d h a d a Tm of 8o °. The r R N A a n d t R N A of T. aquaticus was higher in guanine a n d cytosine t h a n similar c o m p o n e n t s of E. coli. There was a striking correlation between the t e m p e r a t u r e at which d e n a t u r a t i o n of ribosomes of T. aquaticus initiates a n d the m a x i m u m g r o w t h t e m p e r a t u r e . These d a t a p r o v i d e further evidence for the h e a t s t a b i l i t y of t h e r m o philic maeromolecules.
INTRODUCTION N u m e r o u s investigations a t t e m p t i n g to e x p l a i n the a b i l i t y of b a c t e r i a to proliterate at high t e m p e r a t u r e s in t e r m s ot molecular m e c h a n i s m s have been carried out d u r i n g the p a s t 20 years. The l i t e r a t u r e in this field has r e c e n t l y been reviewed 1. The a b i l i t y of some b a c t e r i a to grow at t e m p e r a t u r e s above 9 °0 (ref. 2) cert a i n l y reflects a p r o n o u n c e d degree of t h e r m a l s t a b i l i t y of their macromolecules. Studies on t h e s t a b i l i t y of t h e r m o p h i l i c ribosomes 3-5 have d e m o n s t r a t e d an increased h e a t s t a b i l i t y of Bacillus stearothermophilus ribosomes as c o m p a r e d to those of E. coli. Likewise, studies on r i b o s o m a l R N A (rRNA) ~-6 have shown t h a t the r R N A of B. stearothermophilus is nlore t h e r m a l l y stable t h a n t h a t of E. coli. On the other h a n d , transfer R N A ( t R N A ) 3,4,~ a n d D N A from t h e r m o p h i l i c b a c t e r i a 4,8 e x h i b i t e d no unusual h e a t s t a b i l i t y . However, it should be p o i n t e d out t h a t these a n d other studies
Biochim. Biophys..4cta, 204 (I97 o) 512-52o
RIBOSOMES AND RNA FROM T. aquaticus
513
concerning molecular mechanisms of thermophily have been limited primarily to
B. stearothermophilus. Recently, a new thermophilic bacterium, T. aquaticus, has been isolated 9. This gram negative non-sporulating rod is capable of growing at temperatures as high as 79 ° which is i i ° higher than the maximum growth temperature reported for B.
stearothermophilus 3. The present work reports some of the physical and chemical characteristics of the purified ribosomes, of the rRNA, and of the tRNA of T. aquaticus. These constituents were obtained from strain YT-I of T. aquaticus isolated from Mushroom Spring in Yellowstone National Park. A comparison was made with properties exhibited by the same constituents from E. coli which has been studied in detail by others.
M A T E R I A L S AND METHODS
(a) Medium and growth conditions T. aquaticus was grown in the salts-tryptone-yeast extract medium described by BROCK AND FREEZE9. Cultures were grown at 70-73 ° in a 2oo-1 fermenter with intense stirring and aeration; cells were harvested in the log phase, cooled and collected by centrifugation, resuspended in "Standard buffer" (o.oi M Tris-HC1 buffer, pH 7.8, o.oi M MgC12, 0.06 M KC1, and 0.006 M 2-mercaptoethanol) and recentrituged. The washed cells were stored at --7 °o until used. E. coli B was grown in tryptone phosphate broth (Difco) at 37 ° and processed as above.
(b) Isolation and puri/ication o] ribosomes The frozen bacteria were disrupted by passage through the French pressure cell at 18oo lb/inch 2. This and all subsequent steps were performed at 5 °. The cell debris was removed by two I5-min eentrifugations at 20 ooo ×g. The supernatant fluid was decanted and recentrifuged for 15 rain at 30 ooo ×g. Ribosomes were sedimented by centrifugation of the 30 ooo ×g supernatant at 122 ooo ×g for 9° min. The ribosomal pellets were rinsed and resuspended in standard buffer. The ribosomal suspension was layered onto i.o M sucrose, o.oi M Tris-HC1 buffer, pH 7.8, and o.oi M MgC12 and centrifuged at 15o ooo x g for 9° rain. The ribosomal pellets were washed and re-suspended in standard buffer and stored at --7 °o . The protein content of the ribosomes was determined by the method of LOWRY et al. TM, RNA content by the method of CERIOTT111.
(c) Isolation and puri]ication o! R N A Frozen cells were broken as described above, and tRNA was isolated as described by ZEIKUS et al. TM. The tRNA was further purified by the use of benzolated DEAE-cellulose column chromatography. Benzolated DEAE-cellulose was prepared according to GILLAN et al. ~3. 5000 A260nm units of tRNA were applied to benzolated DEAE-cellulose columns, washed with 15o ml of a solution containing o. 4 M NaC1, o.oi M MgC12 and 0.05 M sodium acetate (pH 4-5), then eluted with 200 ml of a solution containing IO ~o ethanol in I.O M NaC1. Absorbance was measured at 256 nm in a Gilson recording fraction collector. Tubes containing t R N A were precipitated with 2 vol. of cold 95 % ethanol, dissolved in triple distilled water and stored at --7 o°. Biochim. Biophys. Acta, 2o 4 (197 o) 5 1 2 - 5 2 o
514
J.G. ZEIKUS et al.
Ribosomal RNA was isolated from the frozen ribosomal suspension described previously by extraction with o.oi M Tris-HC1 buffer (pH 7.o)-satd. phenol until no protein was sedimented at the phenol-buffer interface. The rRNA was precipitated three times by addition of 2 vol. of 95 % ethanol and resuspended in o.15 M NaC1 and stored at --7 °0 until used. The rRNA was separated into I6-S and 23-S fractions by use of methylated albumin kieselguhr column chromatography. The methylated albumin kieselguhr was prepared according to the method of MANDELL AND HERSHEY14. Colunms of 0.8 cm ×20 cm were washed with o.oi M Tris-HC1 buffer (pH 7.0) containing 0.3 M NaC1 until all excess protein had been eluted. The rRNA was applied to the column, washed with the above mentioned buffer and eluted with a linear gradient of 0.2 M to 1. 3 M NaC1 in o.oi M Tris-HC1 buffer (pH 7.0). A total of 15o ml of elution buffer was used and 3-ml samples were collected. Peak tubes of the I6-S and 23-S rRNA fractions were collected and dialyzed against O.Ol5 M trisodium citrate-o.I 5 M NaC1 (pH 7.o), and stored at --7 o°.
(d) Sedimentation velocity determination Sedimentation velocity studies were performed in a Spinco (model E) analytical ultracentrifuge at 20 °. All runs were made in a I2-mm standard cell fitted with a Kel-F centerpiece. The sedimenting boundaries of ribosomes and RNA were followed with the Schlieren optical system. The sedimentation coefficients were corrected to 20 ° and water and are expressed in Svedberg units.
(e) Melting temperature determination Profiles of thermal denaturation of solutions of RNA and of ribosomes were determined in a Beckman DU spectrophotometer fitted with a thermospacer connected to a Colora circulating bath. Glass stoppered cuvettes were used and all samples had initial absorbances at 260 nm of o.3o-o.6o.
(/) Base composition analysis o/ R N A The purified RNA was hydrolyzed in 0.05 M KOH at 37 ° for 18 h according to the method of OSAWA et al. 15. The resulting 2'-3' nucleotides were separated on a Dowex I-SX formate column with a resin bed of 0. 9 cm in diameter by 25 cm high. The method of elution described by HURLBERT el al. TM was employed. Absorbance of the eluate was monitored at 256 nm in a Gilson recording fraction collector. The absorbance of each fraction was determined in the Gilford (Model 240o ) spectrophotometer.
RESULTS
Ribosomes Analytical ultracentrifugal analysis of purified T. aquaticus ribosomes in standard buffer revealed three peaks sedimenting at 50 S, 7° S and IOO S. Dialysis of the ribosomal preparation from standard butfer into o.oo1 M MgC12resulted in partial degradation into 36-S, 5o-S, and 7o-S components. Further dialysis against triple distilled water resulted in 36-S and 5o-S peaks. Biochim. Biophys. Acta, 204 (197 o) 512-52o
RIBOSOMES AND R N A FROM
T. aquaticus
515
The chemical analysis of purified preparation of T. aquaticus ribosomes tor protein and RNA showed an average content of 59 % protein and 41 ~o RNA. The thermal stability of T. aquaticus ribosomes was compared to E. coli ribosomes by following the increase in absorbance at 260 nm upon raising the temperature. Fig. I illustrates the effect of increasing the temperature on the stability of intact ribosomes which were suspended in standard buffer. The E. coli ribosomes were only stable up to 59 ° and displayed a Tm (the temperature at which 50 % of the hyperchromic effect is observed) of 7 l°. On the other hand, the ribosomes oI T. aquaticus did not show any rise in absorbance until 79 ° and they demonstrated a Tm of 86 °. B. stearothermophilus ribosomes denatured under similar conditions began melting at 72o and had a Tm of 77.9 ° (ref. 4).
1.6
i 1.5
~o~. 0 '--e
1.4 m
1.3 < m > <
1.2
/
I.I
/
I.C
I
20
I 30
I 40
~0
I 60
I 70
I 80
I 90
I 100
TEMPERATURE
Fig. i. Thermal denaturation profiles of purified ribosomes in standard buffer. O - O , E. coli; 0 - 0 , T. aquaticus.
Ribosomal R N A RNA was isolated from purified ribosomal suspensions of E. coli and T. aquaticus to compare the thermal stability of their ribosomes to the thermal stability of their rRNA. In Fig. 2 it can be seen that the Tm of E. coli rRNA in o.15 M NaC1 is 58°; whereas, that of T. aquaticus is 64 °. Although T. aquaticus rRNA is more thermal stable than that of E. coli, both rRNA's are significantly less stable than their corresponding ribosomes. The nucleotide composition of T. aquaticus and E. coli rRNA is shown in Table I. T. aquaticus rRNA is slightly higher than E. coli in both guanine and cytosine. For comparison with E. coli rRNA, data obtained by other investigators 17-19 are also reported. It is evident that the values obtained for the base composition of E. coli rRNA in this research are comparable with those reported by other investigators. Biochim. Biophys. Acta, 204 (197o) 51z-52o
516
j.G.
ZEIKUS et a[.
'~ 1.2 o
z 0
i/!//,,o
I.I
m
. . . . .
--
I
20
I
30
I
40
I
50
I
60
?0
~0
~0
I
100
tEMPERATURE
Fig. 2. T h e r m a l d e n a t u r a t i o n profiles of t o t a l r R N A in o.15 M NaCI. O - Q ,
E. coli; (2)-(2), T.
aquaticus. TABLE I NUCLEOTIDE COMPOSITION OF rR~'A
Nucleotide
A3cfP GMP UMP CMP
Composition (mole %) T. aquaticus
B. stearothermophilus (re/. 3)
2o.o 38.5 16.2 25.2
25.9 34.5 17. 5 22.0
E. coli B Authors
Re/. I7
Re[. z8
Re[. z 9
24.8 33.1 20.6 21.5
25.o 31.5 21. 4 22. I
26. 3 28.9 22.o 22.8
23.9 32.5 20. 7 22.9
To further investigate the nature of the increased thermal stability of T. aquaticus ribosomal RNA, rRNA was fractionated into I6-S and 23-S species by methylated albumin kieselguhr column chromatography and then employed in thermal denaturation studies. E. coli I6-S rRNA had a Tm of 58°, while the Tm of T. aquaticus I6-S rRNA was 57 ° (Fig. 3). In contrast to the similar thermal denaturation profiles for I6-S rRNA, the melting profiles for 23-S rRNA revealed differences as illustrated in Fig. 4. I t can be seen that 23-S rRNA from T. aquaticus (Tin 64 °) is somewhat more thermal stable than 23-S rRNA from E. coli (Tm) 58°), but not as stable as whole ribosomes.
Trans/er R N A Preparations of t R N A isolated from T. aquaticus showed one component in the analytical ultracentrifuge with a sedimentation coefficient of 4 S. The thermal stability of T. aquaticus t R N A was compared to E. coli t R N A in standard buffer and is shown in Fig. 5. E. coli t R N A began denaturing at 55 ° and had Biochim. Biophys. Acta, 204 (197 o) 512-52o
RIBOSOMES
AND
RNA
FROM
517
T. aquaticus
Fig. 3. Thermal denaturation profiles of 16-s rRNA trate. O-0, E. coli; 0-0, T. aquaticus.
in 0.15 M NaCl and 0.015 M trisodium
ci-
Fig. 4. Thermal denaturation profiles of 23-S rRNA trate. O-0, E. co&; m-0, T. aquaticus
in 0.15 M NaCl and 0.015 M trisodium
ci-
a T, of 80”; whereas, T. aquaticus tRNA began melting at 68” and had a Tm of 86”. Under similar conditions, other investigators 4,7have shown that thermal denaturation profiles of tRNA from B. stearothermophilus and E. coli are virtually identical. Fig. 6 illustrates the melting profile of tRNA in triple distilled water. Under these conditions, E. coli tRNA began melting at 46” and demonstrated a T,,, of 70~ while T. aqsaticus began denaturing at 56” and had a Tm of 81”. Biochim.
Biophys.
Acta,
204
(1970)
512-520
J.G. ZEIKUS et al.
518
L25
e/e-e "~
~#
1.2
./ /
o/°
,0
// /z /z
O ,D v
/°
z
=* =
e"
t.I
0 m
......... :i¢/°
< I.C
I
30
I 40
I
I
I
|
I
50
60
70
80
90
I tOO
TEMPERATURE
Fig.
5. T h e r m a !
denaturation
profiles
of purified
tRNA
in Standard
buffer.
0-0,
E. toll; G - O ,
T. aquaticus.
E ©
O 1.21
U Z m
H5
1,10
/
•
m >
o
1.05i
° ~o~°
so~O t0
,=c ~
!
B.c
,,..o~ .__o~O--,~'~o ~°~ 2'0
3'0
4'0
~o
~o
~o
Jo
~o
,~o
TEMPERATURE
Fig. 6. Thermal denaturation profiles of purified tRNA in triple distilled water. 0 - 0 , G - Q , 7". aquaticus.
E. coli;
Table I I illustrates t h e nucleotide composition of T. aquaticus t R N A a n d includes for c o m p a r i s o n the base composition of B. stearothermophilus a n d E. coli B. t R N A as r e p o r t e d b y MANGIANTINI et al. 3. F r o m this comparison it a p p e a r s t h a t the t R N A of T. aquaticus is higher in g u a n i n e plus cytosine t h a n b o t h E. coli B. a n d B. stearothermophilus.
Biochim. Biophys. Acta, 204 (197o) 512-52o
RIBOSOMES AND RNA FROM T. aquaticus
519
TABLE II NUCLEOTIDE COMPOSITION OF t R N A
Nucleotide
Composition (mole %) T. aquaticus
AMP GMP CM P U M P a n d p s e u d o u r i d y l i c acid
16. 5 38.4 25.1 20.2
(re/. 3)
E. coli B (re/. x9)
20. 5 31.5 26.5 21.i
20. 5 3I.o 28.5 20.0
B. stearothermophilus
DISCUSSION
From these data it is clear that the ribosomes and t R N A of the extrenle thermophile T. aquaticus are more heat stable than those of either the mesophile E. coli or the moderate thermophile B. slearothermophilus. The Tm of T. aquaticus ribosomes (86 °) is considerably higher than E. coli libosomes (71°) and of that reported for B. stearothermophilus (77.90) 4. However, the ribosomes of T. aquaticus sedimented into components typical of E. coli and B. stearothermophilus ribosomes under similar conditions 4,2°. At present the basis for the heat stability of thermophile ribosomes remains to be elucidated. Other investigators 4,5 have shown that total rRNA, I6-S RNA and 23-S RNA of B. stearothermophilus are more thermal stable than the corresponding species of RNA from E. coli. We have demonstrated that total rRNA from T. aqua~icus is more thermal stable than E. coli rRNA; however, this increased thermal stability resides only in the 23-S species and not in the I6-S species. The slightly higher guanine plus cytosine content of T. aquaticus rRNA m a y account for the increased thermal stability of the 23-S RNA. However, it seems likely that the rRNA alone does not account for the high degree of thermal stability of intact T. aquaticus ribosomes. The chemical composition of T. aquaticus ribosomes (59 % protein and 4 1 % RNA) differs from the chemical composition reported for E. coli ~° and B. stearothermophilus 7 both of which consist of approximately 59 % RNA and 4 1 % protein. The increased protein to RNA ratio m a y possibly be related to the elevated thermal stability of T. aquaticus ribosomes. Another possible mechanism related to ribosomal thermal stability could be the activity of a ribosome-bound ribonuclease. Other investigaters 3,21 have shown that rRNA in B. stearothermophilus ribosomes was more resistant to action by ribosomal ribonuclease than in mesophile ribosomes. The heat stability ot ribosomes m a y play an important role in governing the maximal growth temperature of bacteria. A study by PACE AND CAMPBELL22 demonstrates a correlation between the Tm of bacterial ribosomes and the maximal growth temperature from which they were derived. Our data with T. aquaticus are consistent with PACE AND CAMPBELL12 and extend their study to somewhat higher temperatures. Another interesting result of our studies was the finding of thermally stable tRNA. Other investigations 3,7 have shown that the thermal denaturation profiles of B. stearothermophilus t R N A and E. coli t R N A are virtually identical under a wide variety of ionic conditions. Furthermore, these investigators suggested that maintenance of the proper secondary structure of t R N A in the intact thermophile could be achieved by polycation stabilization rather than by alterations in the primary JBiochim. Biophys. Acta, 204 (197 o) 512-52o
520
j.G. ZEIKUS et al.
structure. It is apparent from our data that T. aquaticus t R N A is more thermal stable than E. coli t R N A with or without the presence of Mg 2+. The reason for this increased thermal stability in the t R N A m a y be a reflection of the increased guanine phts cytosine content in T. aquaticus as compared to lower percentages in both E. coli and B. stearothermophilus. Our data would therefore suggest that although Mg 2+ is extremely important in maintaining thermal stability of tRNA, inherent structural features of the tRNA may also be responsible for the increased thermal stability. It will be interesting to see how t R N A from organisms growing above 9 °° (ref. 2) compare to T. aquaticus in terms of thermal denaturation profile and base composition. In any event, the data presented here provide turther evidence for the heat stability of thermophile ribosomes and ribonucleic acids. Upon comparing the thermostability of ribosomes from T. aquaticus to the temperature at which it grows, there is a striking correlation between the temperature at which denaturation of ribosomes initiates and the maximum growth temperature, both of which are 79 ° . The high degree of thermal stability which the protein synthesizing machinery of T. aquaticus possess must play a most important part in allowing bacterium to proliferate at such high temperatures. Even though a thermal stable protein synthesizing apparatus enables the bacterium to synthesize macromolecules at high temperatures, it may not be the limiting factor in thermal death. It has been suggested 23, that the integrity of the membrane might be the limiting factor in thermal death. ACKNOWLEDGMENTS
Supported by research grants from the U.S. Public Health Service 5TI-6H-5o3 (to J.G.Z.) and CA-Io4I 7 (to M.W.T.) and the National Science Foundation GB78I 5 (to T.D.B.). REFERENCES I S. FREIDMAN, Bacteriol. Rev., 32 (1968) 27. 2 T. BOTT AND T. BROCK, Science, 164 (I969) 1411. 3 ]_V[. MANGIANTINI, G. TECCE, G. TOSCHI AND A. TRENTALENCE, Biochim. Biophys. deta, lO 3 (1965) 252. 4 0 . SAUNDERS AND L. CAMPBELL, J. Bacteriol., 91 (1966) 332. 5 S. FREIDMAN, R. AXEL AND I. WEINSTEIN, J . Bacteriol., 93 (1967) 1521. 6 J. STENESH AND A. HOLAZO, Biochim. Biophys. Acta., lO 3 (1967) 252. 7 S. FREIDMAN AND I. WEINSTEIN, Biochim. Biophys. Acta, 114 (1966) 593. 8 J. MARMUR, Biochim. Biophys. Acta, 38 (196o) 342. 9 T. BROCK AND H. FREEZE, J. Bacteriol., 99 (1969) 289. IO O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951 ) 265. I i G. CERIOTTI, J. Biol. Chem., 214 (1955) 59. 12 J. ZEIKUS, M. TAYLOR AND C. BUCK, J. Exptl. Cell Res., 57 (1969) 74. 13 I. GILLAN, S. MILLWARD, D. BLEW, M. VON TIGERSTROM, G. WIMMER AND G. TENER, Biochemistry, 6 (I967) 3043 . 14 J. MANDELL AND A. HERSHEY, Anal. Biochem., I (196o) 66. 15 S. OSAWA, K. TAKATA AND Y. HOTA, Biochim. Biophys. Acta, 28 (1958) 271. 16 R. HURLBERT, H. SCHMITZ, A. BRUMM AND V. POTTER, J . Biol. Chem., 209 {I954) 23. 17 P. SPAHR AND A. TlSSlERES, J. Mol. Biol., 2 (196o) IO. 18 U. LITTAUER AND H. EISENBERG, Biochim. Biophys. Acta, 32 (1959) 320. 19 K. MIURA, Biochim. Biophys. dcta, 55 (1962) 62. 20 A. TlSSlERBS, J. WATSON, D. SCHLESSlNGER AND B. HOLLINGWORTH, J . Mol. Biol., I (I959) 221. 21 J. STENESH AND C. YANG, J. Bacteriol., 93 (1967) 93 °. 22 B. PACE AND L. CAMPBELL, Bacteriol. Proc., 99 (1966) 98. 23 T. BROCK, Science, 158 (1967) lO12.
Biochim. Biophys. dcta, 2o 4 (197 o) 512-52o
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96449
THERMAL
STABILITY OF RIBOSOMES AND RNA FROM THERMUS
A Q UA TIC US
J. G. ZEIKUS, M. \V. TAYLOR AND T. D. BROCK
Department o/ Microbiology, Indiana University, Bloomington, Ind. 47402- (U.S.A.) (Received November ioth, 1969)
SUMMARY
This p a p e r presents d a t a on the p h y s i c a l a n d chemical p r o p e r t i e s of the ribosomes a n d R N A of Thermus aquaticus, an o r g a n i s m able to grow at t e m p e r a t u r e s u p to 79 °. U l t r a c e n t r i f u g a l analysis of purified ribosomes in o . o i M Mg 2+ d e m o n s t r a t e d c o m p o n e n t s s e d i m e n t i n g a t 50 S, 7 ° S a n d IOO S. Chemical analysis of ribosomes revealed an average c o n t e n t of 59 % protein a n d 4 1 % R N A . W h e n h e a t e d in s t a n d a r d buffer, Escherichia coli ribosomes b e g a n g r a d u a l dissociation at 59 ° with a Tm of 71°. I n contrast, T. aquaticus ribosomes were stable up to 79 ° a n d h a d a Tm of 86 °. T. aquaticus r i b o s o m a l R N A (rRNA) was found to be more t h e r m a l stable t h a n t h a t of E. coli. However, t h e r m a l d e n a t u r a t i o n profiles of f r a c t i o n a t e d I6-S a n d 23-S r R N A of E. coli a n d T. aquaticus d e m o n s t r a t e d t h a t o n l y the 23-S species of T. aquaticus r R N A was more stable t h a n t h a t of E. coli. T h e r m a l d e n a t u r a t i o n studies revealed t h a t T. aquaticus transfer R N A ( t R N A ) was stable up to 68 ° a n d h a d a Tm of 86°; whereas, E. coli t R N A was only stable up to 55 ° a n d h a d a Tm of 8o °. The r R N A a n d t R N A of T. aquaticus was higher in guanine a n d cytosine t h a n similar c o m p o n e n t s of E. coli. There was a striking correlation between the t e m p e r a t u r e at which d e n a t u r a t i o n of ribosomes of T. aquaticus initiates a n d the m a x i m u m g r o w t h t e m p e r a t u r e . These d a t a p r o v i d e further evidence for the h e a t s t a b i l i t y of t h e r m o philic maeromolecules.
INTRODUCTION N u m e r o u s investigations a t t e m p t i n g to e x p l a i n the a b i l i t y of b a c t e r i a to proliterate at high t e m p e r a t u r e s in t e r m s ot molecular m e c h a n i s m s have been carried out d u r i n g the p a s t 20 years. The l i t e r a t u r e in this field has r e c e n t l y been reviewed 1. The a b i l i t y of some b a c t e r i a to grow at t e m p e r a t u r e s above 9 °0 (ref. 2) cert a i n l y reflects a p r o n o u n c e d degree of t h e r m a l s t a b i l i t y of their macromolecules. Studies on t h e s t a b i l i t y of t h e r m o p h i l i c ribosomes 3-5 have d e m o n s t r a t e d an increased h e a t s t a b i l i t y of Bacillus stearothermophilus ribosomes as c o m p a r e d to those of E. coli. Likewise, studies on r i b o s o m a l R N A (rRNA) ~-6 have shown t h a t the r R N A of B. stearothermophilus is nlore t h e r m a l l y stable t h a n t h a t of E. coli. On the other h a n d , transfer R N A ( t R N A ) 3,4,~ a n d D N A from t h e r m o p h i l i c b a c t e r i a 4,8 e x h i b i t e d no unusual h e a t s t a b i l i t y . However, it should be p o i n t e d out t h a t these a n d other studies
Biochim. Biophys..4cta, 204 (I97 o) 512-52o
RIBOSOMES AND RNA FROM T. aquaticus
513
concerning molecular mechanisms of thermophily have been limited primarily to
B. stearothermophilus. Recently, a new thermophilic bacterium, T. aquaticus, has been isolated 9. This gram negative non-sporulating rod is capable of growing at temperatures as high as 79 ° which is i i ° higher than the maximum growth temperature reported for B.
stearothermophilus 3. The present work reports some of the physical and chemical characteristics of the purified ribosomes, of the rRNA, and of the tRNA of T. aquaticus. These constituents were obtained from strain YT-I of T. aquaticus isolated from Mushroom Spring in Yellowstone National Park. A comparison was made with properties exhibited by the same constituents from E. coli which has been studied in detail by others.
M A T E R I A L S AND METHODS
(a) Medium and growth conditions T. aquaticus was grown in the salts-tryptone-yeast extract medium described by BROCK AND FREEZE9. Cultures were grown at 70-73 ° in a 2oo-1 fermenter with intense stirring and aeration; cells were harvested in the log phase, cooled and collected by centrifugation, resuspended in "Standard buffer" (o.oi M Tris-HC1 buffer, pH 7.8, o.oi M MgC12, 0.06 M KC1, and 0.006 M 2-mercaptoethanol) and recentrituged. The washed cells were stored at --7 °o until used. E. coli B was grown in tryptone phosphate broth (Difco) at 37 ° and processed as above.
(b) Isolation and puri/ication o] ribosomes The frozen bacteria were disrupted by passage through the French pressure cell at 18oo lb/inch 2. This and all subsequent steps were performed at 5 °. The cell debris was removed by two I5-min eentrifugations at 20 ooo ×g. The supernatant fluid was decanted and recentrifuged for 15 rain at 30 ooo ×g. Ribosomes were sedimented by centrifugation of the 30 ooo ×g supernatant at 122 ooo ×g for 9° min. The ribosomal pellets were rinsed and resuspended in standard buffer. The ribosomal suspension was layered onto i.o M sucrose, o.oi M Tris-HC1 buffer, pH 7.8, and o.oi M MgC12 and centrifuged at 15o ooo x g for 9° rain. The ribosomal pellets were washed and re-suspended in standard buffer and stored at --7 °o . The protein content of the ribosomes was determined by the method of LOWRY et al. TM, RNA content by the method of CERIOTT111.
(c) Isolation and puri]ication o! R N A Frozen cells were broken as described above, and tRNA was isolated as described by ZEIKUS et al. TM. The tRNA was further purified by the use of benzolated DEAE-cellulose column chromatography. Benzolated DEAE-cellulose was prepared according to GILLAN et al. ~3. 5000 A260nm units of tRNA were applied to benzolated DEAE-cellulose columns, washed with 15o ml of a solution containing o. 4 M NaC1, o.oi M MgC12 and 0.05 M sodium acetate (pH 4-5), then eluted with 200 ml of a solution containing IO ~o ethanol in I.O M NaC1. Absorbance was measured at 256 nm in a Gilson recording fraction collector. Tubes containing t R N A were precipitated with 2 vol. of cold 95 % ethanol, dissolved in triple distilled water and stored at --7 o°. Biochim. Biophys. Acta, 2o 4 (197 o) 5 1 2 - 5 2 o
514
J.G. ZEIKUS et al.
Ribosomal RNA was isolated from the frozen ribosomal suspension described previously by extraction with o.oi M Tris-HC1 buffer (pH 7.o)-satd. phenol until no protein was sedimented at the phenol-buffer interface. The rRNA was precipitated three times by addition of 2 vol. of 95 % ethanol and resuspended in o.15 M NaC1 and stored at --7 °0 until used. The rRNA was separated into I6-S and 23-S fractions by use of methylated albumin kieselguhr column chromatography. The methylated albumin kieselguhr was prepared according to the method of MANDELL AND HERSHEY14. Colunms of 0.8 cm ×20 cm were washed with o.oi M Tris-HC1 buffer (pH 7.0) containing 0.3 M NaC1 until all excess protein had been eluted. The rRNA was applied to the column, washed with the above mentioned buffer and eluted with a linear gradient of 0.2 M to 1. 3 M NaC1 in o.oi M Tris-HC1 buffer (pH 7.0). A total of 15o ml of elution buffer was used and 3-ml samples were collected. Peak tubes of the I6-S and 23-S rRNA fractions were collected and dialyzed against O.Ol5 M trisodium citrate-o.I 5 M NaC1 (pH 7.o), and stored at --7 o°.
(d) Sedimentation velocity determination Sedimentation velocity studies were performed in a Spinco (model E) analytical ultracentrifuge at 20 °. All runs were made in a I2-mm standard cell fitted with a Kel-F centerpiece. The sedimenting boundaries of ribosomes and RNA were followed with the Schlieren optical system. The sedimentation coefficients were corrected to 20 ° and water and are expressed in Svedberg units.
(e) Melting temperature determination Profiles of thermal denaturation of solutions of RNA and of ribosomes were determined in a Beckman DU spectrophotometer fitted with a thermospacer connected to a Colora circulating bath. Glass stoppered cuvettes were used and all samples had initial absorbances at 260 nm of o.3o-o.6o.
(/) Base composition analysis o/ R N A The purified RNA was hydrolyzed in 0.05 M KOH at 37 ° for 18 h according to the method of OSAWA et al. 15. The resulting 2'-3' nucleotides were separated on a Dowex I-SX formate column with a resin bed of 0. 9 cm in diameter by 25 cm high. The method of elution described by HURLBERT el al. TM was employed. Absorbance of the eluate was monitored at 256 nm in a Gilson recording fraction collector. The absorbance of each fraction was determined in the Gilford (Model 240o ) spectrophotometer.
RESULTS
Ribosomes Analytical ultracentrifugal analysis of purified T. aquaticus ribosomes in standard buffer revealed three peaks sedimenting at 50 S, 7° S and IOO S. Dialysis of the ribosomal preparation from standard butfer into o.oo1 M MgC12resulted in partial degradation into 36-S, 5o-S, and 7o-S components. Further dialysis against triple distilled water resulted in 36-S and 5o-S peaks. Biochim. Biophys. Acta, 204 (197 o) 512-52o
RIBOSOMES AND R N A FROM
T. aquaticus
515
The chemical analysis of purified preparation of T. aquaticus ribosomes tor protein and RNA showed an average content of 59 % protein and 41 ~o RNA. The thermal stability of T. aquaticus ribosomes was compared to E. coli ribosomes by following the increase in absorbance at 260 nm upon raising the temperature. Fig. I illustrates the effect of increasing the temperature on the stability of intact ribosomes which were suspended in standard buffer. The E. coli ribosomes were only stable up to 59 ° and displayed a Tm (the temperature at which 50 % of the hyperchromic effect is observed) of 7 l°. On the other hand, the ribosomes oI T. aquaticus did not show any rise in absorbance until 79 ° and they demonstrated a Tm of 86 °. B. stearothermophilus ribosomes denatured under similar conditions began melting at 72o and had a Tm of 77.9 ° (ref. 4).
1.6
i 1.5
~o~. 0 '--e
1.4 m
1.3 < m > <
1.2
/
I.I
/
I.C
I
20
I 30
I 40
~0
I 60
I 70
I 80
I 90
I 100
TEMPERATURE
Fig. i. Thermal denaturation profiles of purified ribosomes in standard buffer. O - O , E. coli; 0 - 0 , T. aquaticus.
Ribosomal R N A RNA was isolated from purified ribosomal suspensions of E. coli and T. aquaticus to compare the thermal stability of their ribosomes to the thermal stability of their rRNA. In Fig. 2 it can be seen that the Tm of E. coli rRNA in o.15 M NaC1 is 58°; whereas, that of T. aquaticus is 64 °. Although T. aquaticus rRNA is more thermal stable than that of E. coli, both rRNA's are significantly less stable than their corresponding ribosomes. The nucleotide composition of T. aquaticus and E. coli rRNA is shown in Table I. T. aquaticus rRNA is slightly higher than E. coli in both guanine and cytosine. For comparison with E. coli rRNA, data obtained by other investigators 17-19 are also reported. It is evident that the values obtained for the base composition of E. coli rRNA in this research are comparable with those reported by other investigators. Biochim. Biophys. Acta, 204 (197o) 51z-52o
516
j.G.
ZEIKUS et a[.
'~ 1.2 o
z 0
i/!//,,o
I.I
m
. . . . .
--
I
20
I
30
I
40
I
50
I
60
?0
~0
~0
I
100
tEMPERATURE
Fig. 2. T h e r m a l d e n a t u r a t i o n profiles of t o t a l r R N A in o.15 M NaCI. O - Q ,
E. coli; (2)-(2), T.
aquaticus. TABLE I NUCLEOTIDE COMPOSITION OF rR~'A
Nucleotide
A3cfP GMP UMP CMP
Composition (mole %) T. aquaticus
B. stearothermophilus (re/. 3)
2o.o 38.5 16.2 25.2
25.9 34.5 17. 5 22.0
E. coli B Authors
Re/. I7
Re[. z8
Re[. z 9
24.8 33.1 20.6 21.5
25.o 31.5 21. 4 22. I
26. 3 28.9 22.o 22.8
23.9 32.5 20. 7 22.9
To further investigate the nature of the increased thermal stability of T. aquaticus ribosomal RNA, rRNA was fractionated into I6-S and 23-S species by methylated albumin kieselguhr column chromatography and then employed in thermal denaturation studies. E. coli I6-S rRNA had a Tm of 58°, while the Tm of T. aquaticus I6-S rRNA was 57 ° (Fig. 3). In contrast to the similar thermal denaturation profiles for I6-S rRNA, the melting profiles for 23-S rRNA revealed differences as illustrated in Fig. 4. I t can be seen that 23-S rRNA from T. aquaticus (Tin 64 °) is somewhat more thermal stable than 23-S rRNA from E. coli (Tm) 58°), but not as stable as whole ribosomes.
Trans/er R N A Preparations of t R N A isolated from T. aquaticus showed one component in the analytical ultracentrifuge with a sedimentation coefficient of 4 S. The thermal stability of T. aquaticus t R N A was compared to E. coli t R N A in standard buffer and is shown in Fig. 5. E. coli t R N A began denaturing at 55 ° and had Biochim. Biophys. Acta, 204 (197 o) 512-52o
RIBOSOMES
AND
RNA
FROM
517
T. aquaticus
Fig. 3. Thermal denaturation profiles of 16-s rRNA trate. O-0, E. coli; 0-0, T. aquaticus.
in 0.15 M NaCl and 0.015 M trisodium
ci-
Fig. 4. Thermal denaturation profiles of 23-S rRNA trate. O-0, E. co&; m-0, T. aquaticus
in 0.15 M NaCl and 0.015 M trisodium
ci-
a T, of 80”; whereas, T. aquaticus tRNA began melting at 68” and had a Tm of 86”. Under similar conditions, other investigators 4,7have shown that thermal denaturation profiles of tRNA from B. stearothermophilus and E. coli are virtually identical. Fig. 6 illustrates the melting profile of tRNA in triple distilled water. Under these conditions, E. coli tRNA began melting at 46” and demonstrated a T,,, of 70~ while T. aqsaticus began denaturing at 56” and had a Tm of 81”. Biochim.
Biophys.
Acta,
204
(1970)
512-520
J.G. ZEIKUS et al.
518
L25
e/e-e "~
~#
1.2
./ /
o/°
,0
// /z /z
O ,D v
/°
z
=* =
e"
t.I
0 m
......... :i¢/°
< I.C
I
30
I 40
I
I
I
|
I
50
60
70
80
90
I tOO
TEMPERATURE
Fig.
5. T h e r m a !
denaturation
profiles
of purified
tRNA
in Standard
buffer.
0-0,
E. toll; G - O ,
T. aquaticus.
E ©
O 1.21
U Z m
H5
1,10
/
•
m >
o
1.05i
° ~o~°
so~O t0
,=c ~
!
B.c
,,..o~ .__o~O--,~'~o ~°~ 2'0
3'0
4'0
~o
~o
~o
Jo
~o
,~o
TEMPERATURE
Fig. 6. Thermal denaturation profiles of purified tRNA in triple distilled water. 0 - 0 , G - Q , 7". aquaticus.
E. coli;
Table I I illustrates t h e nucleotide composition of T. aquaticus t R N A a n d includes for c o m p a r i s o n the base composition of B. stearothermophilus a n d E. coli B. t R N A as r e p o r t e d b y MANGIANTINI et al. 3. F r o m this comparison it a p p e a r s t h a t the t R N A of T. aquaticus is higher in g u a n i n e plus cytosine t h a n b o t h E. coli B. a n d B. stearothermophilus.
Biochim. Biophys. Acta, 204 (197o) 512-52o
RIBOSOMES AND RNA FROM T. aquaticus
519
TABLE II NUCLEOTIDE COMPOSITION OF t R N A
Nucleotide
Composition (mole %) T. aquaticus
AMP GMP CM P U M P a n d p s e u d o u r i d y l i c acid
16. 5 38.4 25.1 20.2
(re/. 3)
E. coli B (re/. x9)
20. 5 31.5 26.5 21.i
20. 5 3I.o 28.5 20.0
B. stearothermophilus
DISCUSSION
From these data it is clear that the ribosomes and t R N A of the extrenle thermophile T. aquaticus are more heat stable than those of either the mesophile E. coli or the moderate thermophile B. slearothermophilus. The Tm of T. aquaticus ribosomes (86 °) is considerably higher than E. coli libosomes (71°) and of that reported for B. stearothermophilus (77.90) 4. However, the ribosomes of T. aquaticus sedimented into components typical of E. coli and B. stearothermophilus ribosomes under similar conditions 4,2°. At present the basis for the heat stability of thermophile ribosomes remains to be elucidated. Other investigators 4,5 have shown that total rRNA, I6-S RNA and 23-S RNA of B. stearothermophilus are more thermal stable than the corresponding species of RNA from E. coli. We have demonstrated that total rRNA from T. aqua~icus is more thermal stable than E. coli rRNA; however, this increased thermal stability resides only in the 23-S species and not in the I6-S species. The slightly higher guanine plus cytosine content of T. aquaticus rRNA m a y account for the increased thermal stability of the 23-S RNA. However, it seems likely that the rRNA alone does not account for the high degree of thermal stability of intact T. aquaticus ribosomes. The chemical composition of T. aquaticus ribosomes (59 % protein and 4 1 % RNA) differs from the chemical composition reported for E. coli ~° and B. stearothermophilus 7 both of which consist of approximately 59 % RNA and 4 1 % protein. The increased protein to RNA ratio m a y possibly be related to the elevated thermal stability of T. aquaticus ribosomes. Another possible mechanism related to ribosomal thermal stability could be the activity of a ribosome-bound ribonuclease. Other investigaters 3,21 have shown that rRNA in B. stearothermophilus ribosomes was more resistant to action by ribosomal ribonuclease than in mesophile ribosomes. The heat stability ot ribosomes m a y play an important role in governing the maximal growth temperature of bacteria. A study by PACE AND CAMPBELL22 demonstrates a correlation between the Tm of bacterial ribosomes and the maximal growth temperature from which they were derived. Our data with T. aquaticus are consistent with PACE AND CAMPBELL12 and extend their study to somewhat higher temperatures. Another interesting result of our studies was the finding of thermally stable tRNA. Other investigations 3,7 have shown that the thermal denaturation profiles of B. stearothermophilus t R N A and E. coli t R N A are virtually identical under a wide variety of ionic conditions. Furthermore, these investigators suggested that maintenance of the proper secondary structure of t R N A in the intact thermophile could be achieved by polycation stabilization rather than by alterations in the primary JBiochim. Biophys. Acta, 204 (197 o) 512-52o
520
j.G. ZEIKUS et al.
structure. It is apparent from our data that T. aquaticus t R N A is more thermal stable than E. coli t R N A with or without the presence of Mg 2+. The reason for this increased thermal stability in the t R N A m a y be a reflection of the increased guanine phts cytosine content in T. aquaticus as compared to lower percentages in both E. coli and B. stearothermophilus. Our data would therefore suggest that although Mg 2+ is extremely important in maintaining thermal stability of tRNA, inherent structural features of the tRNA may also be responsible for the increased thermal stability. It will be interesting to see how t R N A from organisms growing above 9 °° (ref. 2) compare to T. aquaticus in terms of thermal denaturation profile and base composition. In any event, the data presented here provide turther evidence for the heat stability of thermophile ribosomes and ribonucleic acids. Upon comparing the thermostability of ribosomes from T. aquaticus to the temperature at which it grows, there is a striking correlation between the temperature at which denaturation of ribosomes initiates and the maximum growth temperature, both of which are 79 ° . The high degree of thermal stability which the protein synthesizing machinery of T. aquaticus possess must play a most important part in allowing bacterium to proliferate at such high temperatures. Even though a thermal stable protein synthesizing apparatus enables the bacterium to synthesize macromolecules at high temperatures, it may not be the limiting factor in thermal death. It has been suggested 23, that the integrity of the membrane might be the limiting factor in thermal death. ACKNOWLEDGMENTS
Supported by research grants from the U.S. Public Health Service 5TI-6H-5o3 (to J.G.Z.) and CA-Io4I 7 (to M.W.T.) and the National Science Foundation GB78I 5 (to T.D.B.). REFERENCES I S. FREIDMAN, Bacteriol. Rev., 32 (1968) 27. 2 T. BOTT AND T. BROCK, Science, 164 (I969) 1411. 3 ]_V[. MANGIANTINI, G. TECCE, G. TOSCHI AND A. TRENTALENCE, Biochim. Biophys. deta, lO 3 (1965) 252. 4 0 . SAUNDERS AND L. CAMPBELL, J. Bacteriol., 91 (1966) 332. 5 S. FREIDMAN, R. AXEL AND I. WEINSTEIN, J . Bacteriol., 93 (1967) 1521. 6 J. STENESH AND A. HOLAZO, Biochim. Biophys. Acta., lO 3 (1967) 252. 7 S. FREIDMAN AND I. WEINSTEIN, Biochim. Biophys. Acta, 114 (1966) 593. 8 J. MARMUR, Biochim. Biophys. Acta, 38 (196o) 342. 9 T. BROCK AND H. FREEZE, J. Bacteriol., 99 (1969) 289. IO O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951 ) 265. I i G. CERIOTTI, J. Biol. Chem., 214 (1955) 59. 12 J. ZEIKUS, M. TAYLOR AND C. BUCK, J. Exptl. Cell Res., 57 (1969) 74. 13 I. GILLAN, S. MILLWARD, D. BLEW, M. VON TIGERSTROM, G. WIMMER AND G. TENER, Biochemistry, 6 (I967) 3043 . 14 J. MANDELL AND A. HERSHEY, Anal. Biochem., I (196o) 66. 15 S. OSAWA, K. TAKATA AND Y. HOTA, Biochim. Biophys. Acta, 28 (1958) 271. 16 R. HURLBERT, H. SCHMITZ, A. BRUMM AND V. POTTER, J . Biol. Chem., 209 {I954) 23. 17 P. SPAHR AND A. TlSSlERES, J. Mol. Biol., 2 (196o) IO. 18 U. LITTAUER AND H. EISENBERG, Biochim. Biophys. Acta, 32 (1959) 320. 19 K. MIURA, Biochim. Biophys. dcta, 55 (1962) 62. 20 A. TlSSlERBS, J. WATSON, D. SCHLESSlNGER AND B. HOLLINGWORTH, J . Mol. Biol., I (I959) 221. 21 J. STENESH AND C. YANG, J. Bacteriol., 93 (1967) 93 °. 22 B. PACE AND L. CAMPBELL, Bacteriol. Proc., 99 (1966) 98. 23 T. BROCK, Science, 158 (1967) lO12.
Biochim. Biophys. dcta, 2o 4 (197 o) 512-52o