APPLIED AND ENVIRONMENTAL MIcRoBIoLOGY, June 1976, p. 864-869 Copyright X 1976 American Society for Microbiology
Vol. 31, No. 6 Printed in U.SA.
Macromolecule Synthesis in Escherichia coli BB Under Various Growth Conditions TETSUJI CHOHJI, TATSURO SAWADA,'*
AND
SIGERU KUNO
Department of Chemical Engineering, Faculty of Technology, and Department of Biochemistry, School of
Medicine, Kanazawa University, Kanazawa, Japan Received for publication 31 December 1975
The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures was investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above a certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.
Various authors have reported that microorganisms are able to change cell growth characteristics according to environmental conditions. Since Monod (18, 19) presented the microbial growth model for predicting the growth rate of cells under various conditions, numerous other models (5, 22-24, 26) have been proposed. In most of these models, however, growth rates are generally expressed as the rate of changes in average dry cell mass. Recent studies have revealed that cell growth can mainly be attributed to an overall increase in the contents of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein, and that the synthesis of these substances is regulated by the environment. Therefore, for a model of microbial growth to be precise, it has to include parameters characterizing the rates of macromolecule synthesis (1, 17, 20, 21). The model should be expressed in terms of the contents of the macromolecules per cell instead of the contents of the macromolecules per average dry cell mass, because only the former varies according to the cell age. In the present study, we have investigated kinetically the growth of Escherichia coli BB during balanced growth in a variety of media under different temperatures. The results have indicated that average mass, DNA, RNA, and protein contents per cell can be described as
exponential functions of the specific growth
rate at any given temperature, and that in a
given medium the cell size and composition are almost independent of the growth temperature, as reported by Schaechter et al. (25). In the logarithmic plot between the cell mass, DNA content, and RNA content or protein content per cell and the specific growth rate at a given temperature, the extrapolated point to the zero specific growth rate is constant. Kinetic behavior of macromolecules synthesis is interpreted based on these results.
MATERIALS AND METHODS Strain and culture media. The wild-type strain of E. coli BB was used throughout this investigation. The media used and the growth rates determined are listed in Table 1. Glucose, glycerol, or sodium succinate was used as a carbon source, and in some media Casamino Acids (Difco) was also included. Casamino Acids, sugar, and a solution of MgSO4-7H20 were mixed after separate sterilization. All media were adjusted to pH 7. The change in pH during growth in the above media was maintained 7.0 + 0.1. Growth condition. Cells from overnight cultures in each medium at 37 C were inoculated in fresh medium (10 ml) and shaken with a Monod-type shaker at a given temperature. When the optical density at 660 nm of the culture reached 0.2, the culture was poured into 100 ml of the fresh medium and shaken with the Monod-type shaker at the same 'Present address: Department of Chemical Engineering, temperature. Samples (2 to 3 ml) withdrawn from Kansas Satate University, Manhattan, Kan. 66506. the cultures at intervals were used for measurement 864
VOL. 31, 1976
MACROMOLECULE SYNTHESIS IN E. COLI BB TABLE 1. Culture media Medium no.
Carbon source Glucose Glycerol Sodium succinate Salt solution Casamino Acids
Concn
(mgl
1 + -
2 + -
3
4
5
-
-
+
+
-
-
-
-
-
+
+
+
+
_a
+
+ -
+
-
+
+
1.0
ml) 3.0 3.0 3.0
Avg doubling 52 29 58 32 38 time at 37 C (min) a Salt solution (milligrams per milliliter): (NH4),HPO., 2.5; KH2PO4,i.5; NaCl, 5.0; sodium glutamate, 3.0;
865
the value of the cell mass concentration at time specific growth rate was calculated from the following equation: [(ln X1 - ln X2)/(t, t2)] = ,u, in which Xl and X2 are the values of cell mass concentration at times t1 and t2, respectively. Thus, the following correlation between ,t and the doubling time [6] is obtained: ,u = In2/0.) As reported by Schaechter et al. (25), the macromolecule content per cell could be expressed as an exponential function of the specific growth rate. At a given temperature, the slopes, a, of the curve for DNA content, protein content, and mass are approximately identical, whereas that for RNA content is 1.6 to 1.7 times t. The
MgSO4-7H20, 0.1.
2
of the optical density and cell number to determine the doubling time. When the optical density reached 0.1, the cell cultures were cooled quickly with crushed ice and used for measurement of DNA, RNA, and protein content. The culture temperatures were 21.5, 27.0, 32.0, and 37.0 C. Measurement of cell number and optical density. To assess the specific growth rate and cell mass, the optical density was measured by a photoelectric photometer (Spectronic 20, Shimadzu Bausch-Lomb) at 660 nm. The cell number was microscopically counted with a bacteria counting chamber (Petroff-Hausser and Helber counting chambers, C. A. Hausser & Son Ltd.). When necessary, samples were adequately diluted with saline. Chemical analysis. A 0.5-ml amount of 10 N perchloric acid was added to 10 ml of a cold sample, and the resulting mixture was agitated vigorously with a thermomixer. After keeping it in ice, the suspension was centrifuged at 10,000 rpm for 10 min at 0 C. The sediment was washed twice with cold 0.5 N perchloric acid. After decantation of the supernatant, the sediment was suspended in 0.4 ml of cold 0.5 N perchloric acid and heated at 90 C for 15 min. After cooling with running tap water, the suspension was centrifuged at 10,000 rpm for 5 min. The supernatant was used for measurement of DNA and RNA, whereas the sediment was dissolved in 2 ml of 1 N NaOH and used for measurement of protein content. The DNA, RNA, and protein contents were analyzed by the methods of Burton (3), Mejbaum (11), and Lowry et al. (7), respectively.
RESULTS Balanced growth was maintained in the different media listed in Table 1 at various tempertures, and samples were analyzed for mass (optical density), DNA content, RNA content, protein content, and number of cells. The results are presented in Fig. 1 in which logarithms of mass and DNA, RNA, and protein contents per cell (CM, CD), CR, and Cp) are plotted against the specific growth rate, ,L. (The specific growth rate [,u is defined by the following equation:) [1IX] [dXldt] = t, in which X is
S
(00 O 0
U N
S1
0
0.5
1.0
1.5
SpclfIc growth rote (hr') FIG. 1. Relationship between macromolecule contents per cell and the specific growth rate. (A) Optical density at 660 nm per cell. (B) RNA content per cell. (C) Protein (solid line) and DNA (broken line) contents per cell. Cells were logarithmically grown at 21.5 C (a), 27 C (b), 32 C (c), and 37 C (d), respectively.
866
CHOHJI, SAWADA, AND KUNO
APPL. ENVIRON. MICROBIOL.
as much as those for others. Note that the extrapolation of mass and macromolecule contents per cell to zero specific growth rate gives rise to a unique value. It follows that each component per cell can be expressed as function of the specific growth rate: (1) CM = 1.2 x 10- "' exp ( aM ,u) (2) Cl, = 4.1 x 10-32 exp (aD ,u) CH = 1.1 x 10-" exp (aR A) 'lJ) C = 3.2 x 10-" exp (ap,/) (4) where a,M, a^D, aR, and ap are the function of temperature. Figure 2 plots the specific growth rate against a reciprocal of temperature for different culture media. For a given medium, the relationship between , and 1T will satisfy Arrhenius' law, as reported by Ingraham (6). The slopes of the Arrhenius plots are essentially identical for all media, and the energy of activation calculated from the slopes is approximately 15.4 kcal. Similar plots between 1/a and the reciprocal of temperature, 1/T, are given in Fig. 3. These plots again obey Arrhenius' reaction, and the slopes of the plots for mass and all macromolecules are identical. The activation energy calculated from the slopes is again 15.4 kcal. Schaechter et al. (25) have reported that the size and chemical composition of the cell depends on the culture medium and are not influenced by the temperature of cultivation. This observation has been confirmed in the present work. Figure 4 shows that the mass and macro(*C)
Temp. 42 5.0
C
2.0
0
1.0
37
U
27
'C
A~~~~
0.5
0.2 0.I
17
0
U
1) n)
22
01-U'AS
t-
g
32
_
3.2
3.25
3.3
3.35
3.4
3.45
I/T 103 ( K'1) FIG. 2. Arrhenius plots of the specific growth rate. The media used were No. 1 (A), No. 2 (O), No. 3 (A), No. 4 (0), and No. 5 (*) in Table 1. x
Temp. 42 2.0 r
_
37
32
I
I
( C) 27
22
17
+8p A
0.5k
A.
O
A56
-1
0.2 1-
0.J
3.2
3.25
3.3
3.35
3.4
3.45
x 10
/T (OK-K) FIG. 3. Arrhenius plots of the values of aM (0), al) (O), at, (A), and a,p (A).
molecule contents per cell are only dependent culture medium and are not affected by the growth temperature. Therefore, it is reasonable to assume that the physiological condition is only dependent on the culture medium, and cells are capable of adapting without any lag to sudden change in growth temperature. Figure 5 illustrates the response of the cell growth to the temperature change as measured by the increase in optical density. It can be seen that the cells are capable of accommodating immediately to a new condition created by the upward or downward shift in temperature. on the
DISCUSSION The mass and macromolecule contents per cell are known to be exponential functions of the growth rate (25), each of which is expressed as
C = CO exp (a-ji) (5) where C is the mass or macromolecule content per cell. Since C0, is constant for each component and independent of temperature, its value at the zero growth rate corresponds probably to the minimal amount required to maintain viability without growth. We may assume that the content of DNA at the zero growth rate is equivalent to the mass or weight of a single genome. Indeed, a value of 2.46 x 109 daltons calculated from the extrapolated value (4.1 x 10-12 mg/cell) is in good agreement with the molecular weight (2.8 x 109) of DNA of E. coli reported by Cairns (4). The contents of RNA and protein at the zero growth rate are three to ten times that of DNA and are several thousand molecules per cell. Let us consider the relationship between ,u and 1T for each medium culture plotted Fig. 2.
VOL. 31, 1976
MACROMOLECULE SYNTHESIS IN E. COLI BB
2
Note that the slope of each plot in Fig. 3 is identical to that of each plot in Fig. 2. The biological significance of a in equation 7 is unknown. It is, however, conceivable that 1/a
(A) IXc 0
5
_
is the overall reaction rate of the macromolecule synthesis, because all the plots of 1/a versus 1IT obey the Arrhenius-type equation. Since the activation energies for all the macromolecule synthesis reactions are identical, a rate-limiting step probably exists, which is common to all these synthesis reactions and regulates the growth rate. From equations 6 and 7, we have
_
0
40(0
2 _
A
0
-lo) - #
~I
iC
I
I
(B)
aA.
10o -0 o-8--
1C
5
(C) 4)1
2
_
867
]zz*-
f
-
E
16
= K
(8)
which is independent of the growth temperature. Thus, we can conclude that the macromolecule content per cell as expressed by equation 5 is only dependent on the culture medium and not on the growth temperature. However, the biological significance of this fact is unclear at this stage, and it will be a subject for further
investigation. According to the definition of the specific growth rate, the rate of RNA synthesis can be expressed as a function of the cellular RNA content, CR, as
(9) dCRIdt = ACR Although there are three types of RNA, trans5 I fer, messenger, and ribosomal (rRNA), we have (D) measured only the total RNA content. It is well known that rRNA is a major part of the cellular 2 _ -_*i _**_ RNA. Based on the works by Miller et al. (12-o1 _ 16), in which the transcription mode of the 10 rRNA gene was electronmicroscopically visualized, the following hypotheses on the transcripM_a a a= tion of the rRNA gene may be proposed. (i) -L 20 25 30 35 40 The spatial distance between two adjacent RNA polymerases in an rRNA gene is constant. (OC) Temp. The distance can be expressed by the DNA FIG. 4. Dependency ofcell mass (optical density a t 660 nm [OD66o) and DNA, RNA, and protein con content between the adjacent RNA polymerase, CpoJv(D)) (ii). The rate of RNA synthesis per RNA tents per cell on temperature in various media. Thi polymerase, Vp,O,V(R), is also constant during all growth media used were No. 1 (A), No. 2 (-), No. cell cycles. (A), No. 4 (0), and No. 5 (A) in Table 1. (A) OD66 per cell. (B) RNA content per cell. (C) Protein contenlt Suppose that the DNA content of all rRNA per cell. (D) DNA content per cell. genes is CID. Then the ratio of CID and Cp,..,..()), [CGD/CP,IY(D)], is the number of RNA polymerIt obeys the following Arrhenius-type equatiorn ases attached to all rRNA genes, N, and the with the slope, b, and the frequency factor, z: rate of the RNA synthesis in a cell can be ,u = z exp (-blT) (6 O written as __
--
--
-
-
Similarly, the relationship between 1/a and 1T for the mass or macromolecule synthesis obeys the following Arrhenius-type equation, as shown in Fig. 3: 1 a
dCR/dt
=
N* VP,,,y(H)
Therefore, (11) It has been shown in the present study, that a N * VP,,IY(R) =,:CR
=
z' exp(-b/T)
(7)
(10)
868
CHOHJI, SAWADA, AND KUNO
0
2
Time
APPL. ENVIRON. MICROBIOL.
3
0
2
(hr)
Time
3
(hr)
FIG. 5. Effects of temperature shift on growth rate of E. coli. The solid and broken lines represent the growth curve with media No. 2 and No. 1 in Table 1, respectively. At the time indicated by the arrow, growth temperature shifted from 37 to 27 C (A) and from 27 to 37 C (B).
cell growing with a specific growth rate of 0.83/ h at 37 C contains 4.3 x 10-l' mg of RNA per cell. The rate of RNA synthesis was reported to be approximately 30 nucleotides by Zimmermann and Levinthal (28), Mangiarotti et al. (9), and Manor et al. (10). Therefore, the number of RNA polymerases attached to rRNA genes has been calculated as 660/cell. According to Miller et al. (14), the number of RNA polymerases attached to a single rRNA genome is 100 to 150. Thus, it is apparent that four to six rRNA genes are contained in a cell, or two or three genes are contained in a single genome of E. coli. These values are comparable to the values reported by Attardi et al. (2) and Yankofsky and Spiegelman (27). RNA and protein are copied, respectively, from DNA and RNA. Efficiencies of these macromolecules syntheses can be estimated from the rate of RNA synthesis per DNA molecule, VD)R, and that of protein synthesis per RNA
molecule, V,E. Thus, VI,^ = (1/CD,) (dCR,dt) VR,.
=
(1/CR)
(dCpldt)
=1
L m
a.
(12) (13)
It is apparent from equations 2, 3, 4, and 9 that
VD)R =,ACRH/CI) = (C,/ICD,4l exp (aR a[))iL (14) V,itt = /.LC,/CR = (CP0ICRd)p exp (ap aR),u (15) where C,,, and CR, are the value of DNA and RNA contents at zero growth rate. In Fig. 6, (CR/CI))I and (CX,/CJ,A are plotted against A. As expected, both (CR/IC[I) and (Cp/CRt4, increase with increasing ,u. However, (CR/CI,u4 increases almost exponentially with increasing ,u, and the rate of increase is higher at a lower temperature. On the other hand, (Cp/CR)pI increases more or less rather hyperbolically, and the rate of increase is higher at a higher temperature. Since a, is much greater than aD and
0
0.5
1.0
1.5
2.0
( hr-1) Specific growth rote FIG. 6. Efficiency of RNA and protein synthesis at various growth rates. (A) UR; (B) U,.. The growth temperatures were 21.5 C (A), 27 C (a), 32 C (A), and 37 C (0). The solid lines represent VI)R and VR.I calculated from equations (14) and (15) with various (a,-a,) and (a-aR) values, respectively. ap (Fig. 1), the above results are expected from equations 14 and 15. In the present investigation, we have only measured the total RNA content. Variations of the total RNA content to
VOL. 31, 1976
MACROMOLECULE SYNTHESIS IN E. COLI BB
that of messenger RNA content can probably be neglected. However, since the transfer RNA/ DNA ratio is constant and the rRNA/DNA ratio is a function of , (8), the proportion of transfer RNA in the total RNA can be neglected only in the cells growing at a high rate. Therefore, if the RNA content in equations 14 and 15 is replaced with rRNA content, the plots between (C,1JC&p and A may become much steeper, and the increase in V,{,, with increasing ,u may become slower. It has been widely believed that ribosomes work at a constant, near maximum rate for protein synthesis in microorganisms. However, the results together with the prediction from equation 15 suggest that the efficiency of rRNA (and consequently of ribosomes) in protein synthesis increases gradually with an increasing growth rate but decreases above a certain growth rate. LITERATURE CITEI) 1. Aiba, S., M. Nagatani, and H. Furuse. 1967. Some analysis of lag phase in the growth of microbial cell. J. Ferment. Technol. 45:475-480. 2. Attardi, G., P. C. Huang, and S. Kabat. 1965. Recogni-
3.
4.
5. 6.
7. 8.
9.
10. 11.
tion of ribosomal RNA sites in DNA. I. Analysis of the E. coli system. Proc. Natl. Accad. Sci. U.S.A. 53:1490-1498. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. .T 62:315-323. Cairns, J. 1963. The chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 28:43-45. Herbert, D)., R. Elsworth, and R. C. Telling. 1956. The continuous culture of bacteria; a theoretical and experimental study. J. Gen. Microbiol. 14:601-622. Ingraham, J. L. 1958. Growth of psychrophilic bacteria. J. Bacteriol. 76:75-80. Lowrv, 0. H., N. J. Rosebrough, A. L.Farr., and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. 3iol. Chem. 193:265-275. Maal*e, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis. W. A. Benjamin, Inc., New York. Mangiarotti, G,., l). Apirion, l). Schlessinger, and L. Silengo. 1968. Biosynthetic precursors of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry 7:456-472. Manor, H., l). Goodman, and G. S. Stent. 1969. RNA chain growth rates in Escherichia coli. J. Mol. Biol. 39:1-29. Mejbaum, W. 1939. uber die Bestimmung kleiner Pen-
869
tosemengen, insbesondere in Derivaten der AdenylZ. Physiol. Chem. 258:117-120. 12. Miller, 0. L., Jr., and A. H. Bakken. 1972. Morphologisaure.
cal studies of transcription. Acta Endocrinol. (Covenhagen) 168:155-177. 13. Miller, 0. L., Jr., and B. R. Beatty. 1969. Portrait of a gene. J. Cell. Physiol. 74(Suppl. 1):225-232. 14. Miller, 0. L., Jr., B. R. Beatty, B. A. Hamkalo, and C. A. Thomas, Jr. 1970. Electron microscopic visualization of transcription. Cold Spring Harbor Symp. Quant. Biol. 35:505-512. 15. Miller, 0. L., Jr., and B. A. Hamkalo. 1972. Visualization of RNA synthesis on chromosomes. Int. Rev. Cytol. 33:1-25. 16. Miller, 0. L., Jr., B. A. Hamkalo, and C. A. Thomas, Jr. 1970. Visualization of bacterial gene in action. Science 169:392-395. 17. Miura, Y., K. Tsuchiya, K. Nishikawa, T. Obata, and M. Okazaki. 1974. Behaviour of cell structural components in steady and transient states of growth of Bacillus subtilis. J. Ferment. Technol. 52:100-105. 18. Monod, J. 1942. Recherches sur la croissance des cultures bacteriennes. Hermann et Cie, Paris. 19. Monod, J. 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3:371-394. 20. Nagai, S., Y. Nishizawa, and S. Aiba. 1969. Kinetics of nucleic acid synthesis in the growth of Azotobacter vinelandii. J. Gen. Appl. Microbiol. 15:427-438. 21. Nagai, S., Y. Nishizawa. I. Endo, and S. Aiba. 1968. Response of a chemostatic culture of Azotobacter vinelandii to a delta type of pulse in glucose. J. Gen. Appl. Microbiol. 14:121-134. 22. Ramkrishna, D., A. G. Fredrickson, and H.M. Tsuchiya. 1967. Dynamics of microbial propagation: models considering inhibitors and variable cell composition. Biotechnol. Bioeng. 9:129-169. 23. Sawada, T., and Y. Kojima. 1974. Mathematical model of microbial reaction; effect of micromixing on continuous operation. J. Ferment. Technol. 52:848-854. 24. Sawada, T., Y. Kojima, and T. Takamatsu. 1974. Growth model and control of biochemical reaction. J. Chem. Eng. Jpn. 7:368-373. 25. Schaechter, M., 0. Maaloe, and N. 0. Kjeldgaard. 1958. Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 19:592-606. 26. Topiwala, H., and C. G. Sinclair. 1971. Temperature relationship in continuous culture. Biotechnol. Bioeng. 13:795-813. 27. Yankofsky, S. A., and S. Spiegelman. 1962. The identification of the ribosomal RNA cistron by sequence complementarity. II. Saturation of and competitive interaction at the RNA cistron. Proc. Natl. Acad. Sci. U. S. A. 48:1466-1472. 28. Zimmermann, R. A., and C. Levinthal. 1967. Messenger RNA and RNA transcription time. J. Mol. Biol. 30:349-370.
Vol. 31, No. 6 Printed in U.SA.
Macromolecule Synthesis in Escherichia coli BB Under Various Growth Conditions TETSUJI CHOHJI, TATSURO SAWADA,'*
AND
SIGERU KUNO
Department of Chemical Engineering, Faculty of Technology, and Department of Biochemistry, School of
Medicine, Kanazawa University, Kanazawa, Japan Received for publication 31 December 1975
The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures was investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above a certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.
Various authors have reported that microorganisms are able to change cell growth characteristics according to environmental conditions. Since Monod (18, 19) presented the microbial growth model for predicting the growth rate of cells under various conditions, numerous other models (5, 22-24, 26) have been proposed. In most of these models, however, growth rates are generally expressed as the rate of changes in average dry cell mass. Recent studies have revealed that cell growth can mainly be attributed to an overall increase in the contents of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein, and that the synthesis of these substances is regulated by the environment. Therefore, for a model of microbial growth to be precise, it has to include parameters characterizing the rates of macromolecule synthesis (1, 17, 20, 21). The model should be expressed in terms of the contents of the macromolecules per cell instead of the contents of the macromolecules per average dry cell mass, because only the former varies according to the cell age. In the present study, we have investigated kinetically the growth of Escherichia coli BB during balanced growth in a variety of media under different temperatures. The results have indicated that average mass, DNA, RNA, and protein contents per cell can be described as
exponential functions of the specific growth
rate at any given temperature, and that in a
given medium the cell size and composition are almost independent of the growth temperature, as reported by Schaechter et al. (25). In the logarithmic plot between the cell mass, DNA content, and RNA content or protein content per cell and the specific growth rate at a given temperature, the extrapolated point to the zero specific growth rate is constant. Kinetic behavior of macromolecules synthesis is interpreted based on these results.
MATERIALS AND METHODS Strain and culture media. The wild-type strain of E. coli BB was used throughout this investigation. The media used and the growth rates determined are listed in Table 1. Glucose, glycerol, or sodium succinate was used as a carbon source, and in some media Casamino Acids (Difco) was also included. Casamino Acids, sugar, and a solution of MgSO4-7H20 were mixed after separate sterilization. All media were adjusted to pH 7. The change in pH during growth in the above media was maintained 7.0 + 0.1. Growth condition. Cells from overnight cultures in each medium at 37 C were inoculated in fresh medium (10 ml) and shaken with a Monod-type shaker at a given temperature. When the optical density at 660 nm of the culture reached 0.2, the culture was poured into 100 ml of the fresh medium and shaken with the Monod-type shaker at the same 'Present address: Department of Chemical Engineering, temperature. Samples (2 to 3 ml) withdrawn from Kansas Satate University, Manhattan, Kan. 66506. the cultures at intervals were used for measurement 864
VOL. 31, 1976
MACROMOLECULE SYNTHESIS IN E. COLI BB TABLE 1. Culture media Medium no.
Carbon source Glucose Glycerol Sodium succinate Salt solution Casamino Acids
Concn
(mgl
1 + -
2 + -
3
4
5
-
-
+
+
-
-
-
-
-
+
+
+
+
_a
+
+ -
+
-
+
+
1.0
ml) 3.0 3.0 3.0
Avg doubling 52 29 58 32 38 time at 37 C (min) a Salt solution (milligrams per milliliter): (NH4),HPO., 2.5; KH2PO4,i.5; NaCl, 5.0; sodium glutamate, 3.0;
865
the value of the cell mass concentration at time specific growth rate was calculated from the following equation: [(ln X1 - ln X2)/(t, t2)] = ,u, in which Xl and X2 are the values of cell mass concentration at times t1 and t2, respectively. Thus, the following correlation between ,t and the doubling time [6] is obtained: ,u = In2/0.) As reported by Schaechter et al. (25), the macromolecule content per cell could be expressed as an exponential function of the specific growth rate. At a given temperature, the slopes, a, of the curve for DNA content, protein content, and mass are approximately identical, whereas that for RNA content is 1.6 to 1.7 times t. The
MgSO4-7H20, 0.1.
2
of the optical density and cell number to determine the doubling time. When the optical density reached 0.1, the cell cultures were cooled quickly with crushed ice and used for measurement of DNA, RNA, and protein content. The culture temperatures were 21.5, 27.0, 32.0, and 37.0 C. Measurement of cell number and optical density. To assess the specific growth rate and cell mass, the optical density was measured by a photoelectric photometer (Spectronic 20, Shimadzu Bausch-Lomb) at 660 nm. The cell number was microscopically counted with a bacteria counting chamber (Petroff-Hausser and Helber counting chambers, C. A. Hausser & Son Ltd.). When necessary, samples were adequately diluted with saline. Chemical analysis. A 0.5-ml amount of 10 N perchloric acid was added to 10 ml of a cold sample, and the resulting mixture was agitated vigorously with a thermomixer. After keeping it in ice, the suspension was centrifuged at 10,000 rpm for 10 min at 0 C. The sediment was washed twice with cold 0.5 N perchloric acid. After decantation of the supernatant, the sediment was suspended in 0.4 ml of cold 0.5 N perchloric acid and heated at 90 C for 15 min. After cooling with running tap water, the suspension was centrifuged at 10,000 rpm for 5 min. The supernatant was used for measurement of DNA and RNA, whereas the sediment was dissolved in 2 ml of 1 N NaOH and used for measurement of protein content. The DNA, RNA, and protein contents were analyzed by the methods of Burton (3), Mejbaum (11), and Lowry et al. (7), respectively.
RESULTS Balanced growth was maintained in the different media listed in Table 1 at various tempertures, and samples were analyzed for mass (optical density), DNA content, RNA content, protein content, and number of cells. The results are presented in Fig. 1 in which logarithms of mass and DNA, RNA, and protein contents per cell (CM, CD), CR, and Cp) are plotted against the specific growth rate, ,L. (The specific growth rate [,u is defined by the following equation:) [1IX] [dXldt] = t, in which X is
S
(00 O 0
U N
S1
0
0.5
1.0
1.5
SpclfIc growth rote (hr') FIG. 1. Relationship between macromolecule contents per cell and the specific growth rate. (A) Optical density at 660 nm per cell. (B) RNA content per cell. (C) Protein (solid line) and DNA (broken line) contents per cell. Cells were logarithmically grown at 21.5 C (a), 27 C (b), 32 C (c), and 37 C (d), respectively.
866
CHOHJI, SAWADA, AND KUNO
APPL. ENVIRON. MICROBIOL.
as much as those for others. Note that the extrapolation of mass and macromolecule contents per cell to zero specific growth rate gives rise to a unique value. It follows that each component per cell can be expressed as function of the specific growth rate: (1) CM = 1.2 x 10- "' exp ( aM ,u) (2) Cl, = 4.1 x 10-32 exp (aD ,u) CH = 1.1 x 10-" exp (aR A) 'lJ) C = 3.2 x 10-" exp (ap,/) (4) where a,M, a^D, aR, and ap are the function of temperature. Figure 2 plots the specific growth rate against a reciprocal of temperature for different culture media. For a given medium, the relationship between , and 1T will satisfy Arrhenius' law, as reported by Ingraham (6). The slopes of the Arrhenius plots are essentially identical for all media, and the energy of activation calculated from the slopes is approximately 15.4 kcal. Similar plots between 1/a and the reciprocal of temperature, 1/T, are given in Fig. 3. These plots again obey Arrhenius' reaction, and the slopes of the plots for mass and all macromolecules are identical. The activation energy calculated from the slopes is again 15.4 kcal. Schaechter et al. (25) have reported that the size and chemical composition of the cell depends on the culture medium and are not influenced by the temperature of cultivation. This observation has been confirmed in the present work. Figure 4 shows that the mass and macro(*C)
Temp. 42 5.0
C
2.0
0
1.0
37
U
27
'C
A~~~~
0.5
0.2 0.I
17
0
U
1) n)
22
01-U'AS
t-
g
32
_
3.2
3.25
3.3
3.35
3.4
3.45
I/T 103 ( K'1) FIG. 2. Arrhenius plots of the specific growth rate. The media used were No. 1 (A), No. 2 (O), No. 3 (A), No. 4 (0), and No. 5 (*) in Table 1. x
Temp. 42 2.0 r
_
37
32
I
I
( C) 27
22
17
+8p A
0.5k
A.
O
A56
-1
0.2 1-
0.J
3.2
3.25
3.3
3.35
3.4
3.45
x 10
/T (OK-K) FIG. 3. Arrhenius plots of the values of aM (0), al) (O), at, (A), and a,p (A).
molecule contents per cell are only dependent culture medium and are not affected by the growth temperature. Therefore, it is reasonable to assume that the physiological condition is only dependent on the culture medium, and cells are capable of adapting without any lag to sudden change in growth temperature. Figure 5 illustrates the response of the cell growth to the temperature change as measured by the increase in optical density. It can be seen that the cells are capable of accommodating immediately to a new condition created by the upward or downward shift in temperature. on the
DISCUSSION The mass and macromolecule contents per cell are known to be exponential functions of the growth rate (25), each of which is expressed as
C = CO exp (a-ji) (5) where C is the mass or macromolecule content per cell. Since C0, is constant for each component and independent of temperature, its value at the zero growth rate corresponds probably to the minimal amount required to maintain viability without growth. We may assume that the content of DNA at the zero growth rate is equivalent to the mass or weight of a single genome. Indeed, a value of 2.46 x 109 daltons calculated from the extrapolated value (4.1 x 10-12 mg/cell) is in good agreement with the molecular weight (2.8 x 109) of DNA of E. coli reported by Cairns (4). The contents of RNA and protein at the zero growth rate are three to ten times that of DNA and are several thousand molecules per cell. Let us consider the relationship between ,u and 1T for each medium culture plotted Fig. 2.
VOL. 31, 1976
MACROMOLECULE SYNTHESIS IN E. COLI BB
2
Note that the slope of each plot in Fig. 3 is identical to that of each plot in Fig. 2. The biological significance of a in equation 7 is unknown. It is, however, conceivable that 1/a
(A) IXc 0
5
_
is the overall reaction rate of the macromolecule synthesis, because all the plots of 1/a versus 1IT obey the Arrhenius-type equation. Since the activation energies for all the macromolecule synthesis reactions are identical, a rate-limiting step probably exists, which is common to all these synthesis reactions and regulates the growth rate. From equations 6 and 7, we have
_
0
40(0
2 _
A
0
-lo) - #
~I
iC
I
I
(B)
aA.
10o -0 o-8--
1C
5
(C) 4)1
2
_
867
]zz*-
f
-
E
16
= K
(8)
which is independent of the growth temperature. Thus, we can conclude that the macromolecule content per cell as expressed by equation 5 is only dependent on the culture medium and not on the growth temperature. However, the biological significance of this fact is unclear at this stage, and it will be a subject for further
investigation. According to the definition of the specific growth rate, the rate of RNA synthesis can be expressed as a function of the cellular RNA content, CR, as
(9) dCRIdt = ACR Although there are three types of RNA, trans5 I fer, messenger, and ribosomal (rRNA), we have (D) measured only the total RNA content. It is well known that rRNA is a major part of the cellular 2 _ -_*i _**_ RNA. Based on the works by Miller et al. (12-o1 _ 16), in which the transcription mode of the 10 rRNA gene was electronmicroscopically visualized, the following hypotheses on the transcripM_a a a= tion of the rRNA gene may be proposed. (i) -L 20 25 30 35 40 The spatial distance between two adjacent RNA polymerases in an rRNA gene is constant. (OC) Temp. The distance can be expressed by the DNA FIG. 4. Dependency ofcell mass (optical density a t 660 nm [OD66o) and DNA, RNA, and protein con content between the adjacent RNA polymerase, CpoJv(D)) (ii). The rate of RNA synthesis per RNA tents per cell on temperature in various media. Thi polymerase, Vp,O,V(R), is also constant during all growth media used were No. 1 (A), No. 2 (-), No. cell cycles. (A), No. 4 (0), and No. 5 (A) in Table 1. (A) OD66 per cell. (B) RNA content per cell. (C) Protein contenlt Suppose that the DNA content of all rRNA per cell. (D) DNA content per cell. genes is CID. Then the ratio of CID and Cp,..,..()), [CGD/CP,IY(D)], is the number of RNA polymerIt obeys the following Arrhenius-type equatiorn ases attached to all rRNA genes, N, and the with the slope, b, and the frequency factor, z: rate of the RNA synthesis in a cell can be ,u = z exp (-blT) (6 O written as __
--
--
-
-
Similarly, the relationship between 1/a and 1T for the mass or macromolecule synthesis obeys the following Arrhenius-type equation, as shown in Fig. 3: 1 a
dCR/dt
=
N* VP,,,y(H)
Therefore, (11) It has been shown in the present study, that a N * VP,,IY(R) =,:CR
=
z' exp(-b/T)
(7)
(10)
868
CHOHJI, SAWADA, AND KUNO
0
2
Time
APPL. ENVIRON. MICROBIOL.
3
0
2
(hr)
Time
3
(hr)
FIG. 5. Effects of temperature shift on growth rate of E. coli. The solid and broken lines represent the growth curve with media No. 2 and No. 1 in Table 1, respectively. At the time indicated by the arrow, growth temperature shifted from 37 to 27 C (A) and from 27 to 37 C (B).
cell growing with a specific growth rate of 0.83/ h at 37 C contains 4.3 x 10-l' mg of RNA per cell. The rate of RNA synthesis was reported to be approximately 30 nucleotides by Zimmermann and Levinthal (28), Mangiarotti et al. (9), and Manor et al. (10). Therefore, the number of RNA polymerases attached to rRNA genes has been calculated as 660/cell. According to Miller et al. (14), the number of RNA polymerases attached to a single rRNA genome is 100 to 150. Thus, it is apparent that four to six rRNA genes are contained in a cell, or two or three genes are contained in a single genome of E. coli. These values are comparable to the values reported by Attardi et al. (2) and Yankofsky and Spiegelman (27). RNA and protein are copied, respectively, from DNA and RNA. Efficiencies of these macromolecules syntheses can be estimated from the rate of RNA synthesis per DNA molecule, VD)R, and that of protein synthesis per RNA
molecule, V,E. Thus, VI,^ = (1/CD,) (dCR,dt) VR,.
=
(1/CR)
(dCpldt)
=1
L m
a.
(12) (13)
It is apparent from equations 2, 3, 4, and 9 that
VD)R =,ACRH/CI) = (C,/ICD,4l exp (aR a[))iL (14) V,itt = /.LC,/CR = (CP0ICRd)p exp (ap aR),u (15) where C,,, and CR, are the value of DNA and RNA contents at zero growth rate. In Fig. 6, (CR/CI))I and (CX,/CJ,A are plotted against A. As expected, both (CR/IC[I) and (Cp/CRt4, increase with increasing ,u. However, (CR/CI,u4 increases almost exponentially with increasing ,u, and the rate of increase is higher at a lower temperature. On the other hand, (Cp/CR)pI increases more or less rather hyperbolically, and the rate of increase is higher at a higher temperature. Since a, is much greater than aD and
0
0.5
1.0
1.5
2.0
( hr-1) Specific growth rote FIG. 6. Efficiency of RNA and protein synthesis at various growth rates. (A) UR; (B) U,.. The growth temperatures were 21.5 C (A), 27 C (a), 32 C (A), and 37 C (0). The solid lines represent VI)R and VR.I calculated from equations (14) and (15) with various (a,-a,) and (a-aR) values, respectively. ap (Fig. 1), the above results are expected from equations 14 and 15. In the present investigation, we have only measured the total RNA content. Variations of the total RNA content to
VOL. 31, 1976
MACROMOLECULE SYNTHESIS IN E. COLI BB
that of messenger RNA content can probably be neglected. However, since the transfer RNA/ DNA ratio is constant and the rRNA/DNA ratio is a function of , (8), the proportion of transfer RNA in the total RNA can be neglected only in the cells growing at a high rate. Therefore, if the RNA content in equations 14 and 15 is replaced with rRNA content, the plots between (C,1JC&p and A may become much steeper, and the increase in V,{,, with increasing ,u may become slower. It has been widely believed that ribosomes work at a constant, near maximum rate for protein synthesis in microorganisms. However, the results together with the prediction from equation 15 suggest that the efficiency of rRNA (and consequently of ribosomes) in protein synthesis increases gradually with an increasing growth rate but decreases above a certain growth rate. LITERATURE CITEI) 1. Aiba, S., M. Nagatani, and H. Furuse. 1967. Some analysis of lag phase in the growth of microbial cell. J. Ferment. Technol. 45:475-480. 2. Attardi, G., P. C. Huang, and S. Kabat. 1965. Recogni-
3.
4.
5. 6.
7. 8.
9.
10. 11.
tion of ribosomal RNA sites in DNA. I. Analysis of the E. coli system. Proc. Natl. Accad. Sci. U.S.A. 53:1490-1498. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. .T 62:315-323. Cairns, J. 1963. The chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 28:43-45. Herbert, D)., R. Elsworth, and R. C. Telling. 1956. The continuous culture of bacteria; a theoretical and experimental study. J. Gen. Microbiol. 14:601-622. Ingraham, J. L. 1958. Growth of psychrophilic bacteria. J. Bacteriol. 76:75-80. Lowrv, 0. H., N. J. Rosebrough, A. L.Farr., and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. 3iol. Chem. 193:265-275. Maal*e, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis. W. A. Benjamin, Inc., New York. Mangiarotti, G,., l). Apirion, l). Schlessinger, and L. Silengo. 1968. Biosynthetic precursors of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry 7:456-472. Manor, H., l). Goodman, and G. S. Stent. 1969. RNA chain growth rates in Escherichia coli. J. Mol. Biol. 39:1-29. Mejbaum, W. 1939. uber die Bestimmung kleiner Pen-
869
tosemengen, insbesondere in Derivaten der AdenylZ. Physiol. Chem. 258:117-120. 12. Miller, 0. L., Jr., and A. H. Bakken. 1972. Morphologisaure.
cal studies of transcription. Acta Endocrinol. (Covenhagen) 168:155-177. 13. Miller, 0. L., Jr., and B. R. Beatty. 1969. Portrait of a gene. J. Cell. Physiol. 74(Suppl. 1):225-232. 14. Miller, 0. L., Jr., B. R. Beatty, B. A. Hamkalo, and C. A. Thomas, Jr. 1970. Electron microscopic visualization of transcription. Cold Spring Harbor Symp. Quant. Biol. 35:505-512. 15. Miller, 0. L., Jr., and B. A. Hamkalo. 1972. Visualization of RNA synthesis on chromosomes. Int. Rev. Cytol. 33:1-25. 16. Miller, 0. L., Jr., B. A. Hamkalo, and C. A. Thomas, Jr. 1970. Visualization of bacterial gene in action. Science 169:392-395. 17. Miura, Y., K. Tsuchiya, K. Nishikawa, T. Obata, and M. Okazaki. 1974. Behaviour of cell structural components in steady and transient states of growth of Bacillus subtilis. J. Ferment. Technol. 52:100-105. 18. Monod, J. 1942. Recherches sur la croissance des cultures bacteriennes. Hermann et Cie, Paris. 19. Monod, J. 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3:371-394. 20. Nagai, S., Y. Nishizawa, and S. Aiba. 1969. Kinetics of nucleic acid synthesis in the growth of Azotobacter vinelandii. J. Gen. Appl. Microbiol. 15:427-438. 21. Nagai, S., Y. Nishizawa. I. Endo, and S. Aiba. 1968. Response of a chemostatic culture of Azotobacter vinelandii to a delta type of pulse in glucose. J. Gen. Appl. Microbiol. 14:121-134. 22. Ramkrishna, D., A. G. Fredrickson, and H.M. Tsuchiya. 1967. Dynamics of microbial propagation: models considering inhibitors and variable cell composition. Biotechnol. Bioeng. 9:129-169. 23. Sawada, T., and Y. Kojima. 1974. Mathematical model of microbial reaction; effect of micromixing on continuous operation. J. Ferment. Technol. 52:848-854. 24. Sawada, T., Y. Kojima, and T. Takamatsu. 1974. Growth model and control of biochemical reaction. J. Chem. Eng. Jpn. 7:368-373. 25. Schaechter, M., 0. Maaloe, and N. 0. Kjeldgaard. 1958. Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 19:592-606. 26. Topiwala, H., and C. G. Sinclair. 1971. Temperature relationship in continuous culture. Biotechnol. Bioeng. 13:795-813. 27. Yankofsky, S. A., and S. Spiegelman. 1962. The identification of the ribosomal RNA cistron by sequence complementarity. II. Saturation of and competitive interaction at the RNA cistron. Proc. Natl. Acad. Sci. U. S. A. 48:1466-1472. 28. Zimmermann, R. A., and C. Levinthal. 1967. Messenger RNA and RNA transcription time. J. Mol. Biol. 30:349-370.
