JouRNAL OF BACTERIOLOGY, July 1979, p. 185-194
`0021-9193/79/07-0185/10$02.00/0
`
`Vol. 139, No. 1
`
`Levels of Major Proteins of Escherichia coli During Growth
`at Different Temperatures
`SHERRIE L. HERENDEEN, RUTH A. VANBOGELEN, AND FREDERICK C. NEIDHARDT*
`Department ofMicrobiology, The University ofMichigan, Ann Arbor, Michigan 48109
`
`Received for publication 26 March 1979
`
`The adaptation of Escherichia coli B/r to temperature was studied by mea-
`suring the levels of 133 proteins (comprising 70% of the cell's protein mass) during
`balanced growth in rich medium at seven temperatures from 13.5 to 460C. The
`growth rate of this strain in either rich or minimal medium varies as a simple
`function of temperature with an Arrhenius constant of approximately 13,500 cal
`(ca. 56,500 J) per mol from 23 to 370C, the so-called normal range; above and
`below this range the growth rate decreases sharply. Analysis of the detailed
`results indicates that (i) metabolic coordination within the normal (Arrhenius)
`range is largely achieved by modulation of enzyme activity rather than amount;
`(ii) the restricted growth that occurs outside this range is accompanied by marked
`changes in the levels of most of these proteins; (iii) a few proteins are thermom-
`eter-like in varying simply with temperature over the whole temperature range
`irrespective of the influence of temperature on cell growth; and (iv) the temper-
`ature response of half of the proteins can be predicted from current information
`on their metabolic role or from their variation in level in different media at 370C.
`
`In general, individual bacteria can grow over
`a range of approximately 40 (Celsius) degrees
`(23). For Escherichia coli, a typical mesophile,
`balanced growth can be sustained from 10 to
`almost 490C (depending at the upper extreme
`on the nutrients present in the medium). In the
`middle of this temperature range-from approx-
`imately 20 to 37°C-the rate of growth of E. coli
`varies with temperature as though cellular
`growth, no matter what the medium, were a
`simple chemical process with a temperature
`characteristic (j) of 12,000 to 14,000 cal (ca.
`50,230 to 58,600 J) per mol (8). Increasing the
`temperature above 400C, or decreasing it below
`200C, leads to progressively slower growth until
`finally growth ceases altogether at 9 or 490C.
`It is not known how E. coli manages its met-
`abolic affairs so as to maximize growth rate
`between 20 and 370C, nor is it understood what
`prevents this optimization outside this range. A
`prevalent and reasonable view is that one or
`more reactions become rate limiting above 370C
`(16) and below 200C (2, 3, 8, 13) as a result of
`the inability of the cell to compensate for ther-
`mally (hot or cold) induced changes in confor-
`mation of proteins. The "nornal" or "Arrhen-
`ius" range in this view is therefore the range
`over which the cell is successful in adjusting
`reaction rates that get out of line: adjustment
`that might entail changing the amount of the
`proteins involved, their activity, or both.
`Experimental exploration of this view has not
`
`been extensive, largely because of the previous
`inadequacy of methods to measure the levels of
`many enzymes simultaneously. Work has mostly
`been directed at the sub- and supranormal tem-
`peratures to learn what proteins might be lim-
`iting growth. An initiation factor in protein syn-
`thesis has been implicated as the vulnerable
`element both at high (16) and low (2) tempera-
`ture. Other work has shown that growth at high
`temperature in minimal medium can be re-
`stricted by inactivation of a (methionine) bio-
`synthetic enzyme (20, 21). In some sense the
`isolation of mutants with temperature-sensitive
`enzymes has provided a ready source of possible
`analogs of the wild-type cell at high and at low
`temperature (e.g., ref. 7). No systematic study
`has been made of the levels of individual pro-
`teins as a function of temperature within or
`beyond the normal range, but Schaechter et al.
`(22) showed that cell volume, mass, RNA, DNA,
`and the number of nuclei per cell in Salmonella
`typhimurium were nearly constant for a given
`medium at 37 and 250C.
`With the advent of methods for resolving total
`cell protein (14, 15) and accurately measuring
`their levels (e.g., ref. 17), comprehensive studies
`are now possible. We recently reported unex-
`pected changes in transient rates of synthesis of
`individual proteins after quite modest shifts in
`growth temperature (9). Here we present the
`first picture of how the levels of the major pro-
`teins of E. coli vary during steady-state growth
`185
`
`GNE 2002
`Page 1
`
`

`
`186
`
`HERENDEEN, VANBOGELEN, AND NEIDHARDT
`
`J. BACTwERIOL.
`
`33.45'a
`
`.: 1.0
`
`4
`
`j
`
`25'~~~~~q
`
`3
`
`at different temperatures within and beyond the
`nornal, Arrhenius range.
`MATERIALS AND METHODS
`Bacterial strain. The E. coli B/r derivative NC3
`(11) was used in all experiments.
`Media. All media used were based on the defined
`MOPS medium (11) and were made by supplementing
`the MOPS medium with 0.4% glucose (wt/vol), amino
`acids (minus leucine; 0.12 mM valine and 0.08 mM
`isoleucine), five vitamins, and four bases in concentra-
`tions given previously (26). At 13.5, 15, 42, and 46°C
`twice the normal concentration of MOPS was neces-
`sary to obtain steady-state growth at the desired cell
`densities.
`Bacterial growth. Cells were grown aerobically on
`a rotary shaker at seven temperatures. The tempera-
`ture was controlled to within +0.10C with a thermistor
`probe. All cultures were started at an optical density
`at 420 nm of 0.01 and grown to an optical density of
`1.0, approximately 108 cells per ml. In all cases growth
`was monitored with flasks growing in paallel to the
`flask containing radioactive isotope.
`Radioactive labeling. Steady-state cultures of
`strain NC3 were grown in rich medium containing
`['4C]leucine (356 mCi/mmol; 40 pCi/ml) at each of
`seven chosen temperatures (13.5, 15, 23, 30, 37, 42, and
`46°C). A reference culture was prepared at 37°C in the
`same medium but with [3H]leucine (502 mCi/mmol;
`63 yCi/ml). CelLs were harvested, and portions of cells
`from the reference culture were mixed with cells of
`each of the seven experimental cultures.
`Determination of steady-state levels. Extracts
`were prepared in the manner described by Blumenthal
`et al. (1). Portions (20 pl) of extracts were processed
`by the O'Farrell equilibrium and nonequilibrium gel
`systems (14, 15). The amounts of 14C and 3H isotopes
`in the individual protein spots and in the unresolved
`extracts were measurd, after sample oxidation, in the
`manner described by Lemaux et al. (9). The '4C:3H
`value for each protein, divided by the '4C:3H ratio of
`the unresolved mixture, is the level of that protein in
`the experimental culture relative to the reference cul-
`ture.
`
`RESULTS
`The growth rate of E. coli NC3 (or its close
`derivative strain NC81) was measured in a series
`of steady-state cultures at different tempera-
`tures in rich and in minimal media. The log of
`the specific growth rate constant (k) is plotted
`as a function of the inverse of absolute temper-
`ature in Fig. 1. This function appears linear
`between 21 and 37°C, and has approximately
`the same ,u value (13,000 to 14,000 cal; ca. 54,400
`to 58,600 J) in rich and in minimal medium. On
`the basis of this information, three temperatures
`within the linear range were chosen for study:
`23, 30, and 37°C. Outside this range four tem-
`peratures were chosen to provide celLs with var-
`ious levels of restricted growth (defined as per-
`centage of the value predicted at each tempera-
`
`21l
`0.5
`3.023
`00.4
`0.3 19.Gn
`
`LI)
`
`0.2-
`
`1 5'
`
`0.11I
`3.1
`3.2
`
`3.4
`
`3.5
`
`3.3
`1000/T(OK)
`FIG. 1. Growth rate of E. coli Bir as a fuinction of
`temperature. Cultures were grown to steady state at
`each temperature, and the rate ofgrowth was meas-
`ured. The logarithm of the growth rate constant, k
`(h-I), isplotted on the ordinate against the inverse of
`the absolute temperature (-K) on the abscissa. Indi-
`vidual data points are marked with the correspond-
`ing degrees Celsius. (0) Strain NC3 in glucose-rich
`medium; (0) strain NC81 (identical to strain NC3
`except for lacI lacP37 lacP5 thi) in glucose minimal
`medium.
`
`ture by extrapolating the linear Arrhenius rela-
`tionship): 13.50C, 56% normal rate; 15°C, 63%
`normal rate; 420C, 72% normal rate; and 46°C,
`28% normal rate.
`The steady-state levels of individual proteins
`resolved by the O'Farrell technique were meas-
`ured at each of the seven temperatures relative
`to their levels in reference cells growing at 370C.
`The results are presented in Table 1, which also
`contains, where available, the identification of
`the individual proteins, their metabolic class,
`and their chemical abundance in the cell. We
`have prepared several additional displays of
`these data to aid in analysis.
`In Fig. 2 we have grouped the proteins accord-
`ing to the magnitude of variation in their level
`within the normal range of temperature, 23 to
`370C. This histogram reveals that the amounts
`of most of the 111 measured proteins change
`very little throughout the normal Arrhenius
`temperature range: only 2 change more than 2.5-
`fold, and they change only 4-fold, and 83 of the
`111 change less than 1.6-fold. No transcription
`or translation protein changed more than 1.4-
`fold, and most changed 1.2-fold or less.
`A similar histogram has been prepared for the
`133 proteins for which we have data over the
`entire range of 13.5 to 460C. Figure 3 displays
`
`GNE 2002
`Page 2
`
`

`
`VOL. 139, 1979
`
`PROTEIN LEVELS AT DIFFERENT TEMPERATURES
`
`187
`
`TABLE 1. Steady-state levels of individual proteins
`Weight fraction
`of total protein
`in glucose rich
`at 37°a
`aedium
`(ca/eu)
`a'
`
`Metabolic
`Regulation
`Groupc)
`
`13.5 C
`
`15°C
`
`Level at each temperature relative to level at 37 C
`
`23 C)
`
`300C
`
`42°C
`
`0.82
`0.77
`0.68
`1.02
`1.17
`1. 38
`1.26
`1.03
`0.96
`0.84
`1.65
`0.94
`0.97
`1.42
`1.30
`1.02
`1.00
`1.02
`1.19
`1.15
`0.79
`1.24
`1.02
`
`46°C
`
`0.50
`25.03
`0. 37
`1.49
`1.35
`2.16
`0.93
`1.05
`1.69
`0.62
`7.22
`0. 52
`0.46
`3.06
`5.65
`1.11
`0.55
`0.60
`0.81
`1.90
`0.65
`0.66
`0.29
`
`Ic
`
`Ic
`Ia4
`
`Ib
`IIbl
`lb
`
`Ic
`
`Ia4
`Ic
`
`Ic
`
`Ic
`
`Ic
`
`Ic
`
`Ic
`
`Ic
`
`Ic
`
`Ic
`
`Ib
`Ib
`IIal
`
`IIal
`
`-
`
`_
`
`Ic
`
`Ic
`
`Ic
`
`Ic
`Ic
`
`Ilbl
`IIal
`
`Ia3
`
`3.05
`
`9.96
`1. 68*
`1.44*
`7.17*
`0.85e
`5.27
`5.30
`9.66
`
`16.47
`
`3. 01*
`
`26.27
`
`14.09
`
`0.68
`2.04*
`
`2.12
`5.65
`2.39
`5.17
`
`3.12*
`
`4.01
`
`1. 68*
`
`2.61*
`
`4. 18*
`4.45*
`2.18*
`
`3.27*
`4. 29*
`1. 36
`1.24
`
`1.05
`2.71*
`2. 55*
`
`2.21
`0.91
`0.25
`3.40
`1.28
`0.95
`0.63
`0.89
`1.15
`1.19
`0.94
`1.16
`0.73
`0.66
`0.63
`0.58
`0.80
`0.84
`0.42
`1.48
`0.84
`0.44
`2.06
`
`-
`0.24
`
`-
`
`1.92
`0.66
`0.68
`0.62
`0.87
`1.26
`1.08
`0.85
`1.05
`0.78
`0.69
`-
`0.62
`0.55
`0.79
`1.01
`0.73
`0.84
`0.49
`1.25
`
`-
`0.79
`
`-
`
`1.60
`0.63
`0.69
`0.56
`0.99
`1.29
`1.31
`0.61
`1.14
`0.97
`0.63
`
`-
`
`0.89
`0.88
`0.79
`1.02
`0.68
`1.08
`0.73
`1.18
`
`(0.96)
`
`(1.10)
`
`(0.95)
`(0.54)
`(0.99)
`
`(1.17)
`(0.68)
`
`(1.16)
`(0.59)
`
`(0.82)
`(0.82)
`
`(1.07)
`
`(0.46)
`
`(0.89)
`(1.19)
`
`1.01
`0.89
`0.74
`1.30
`1.11
`0.87
`0.86
`1.19
`1.18
`1.28
`0.76
`1.07
`1.00
`0.78
`0.55
`0.93
`1.00
`1.02
`1.00
`0.85
`1.13
`0.88
`1.10
`
`1.02
`0.82
`0.51
`0.87
`0.85
`1.32
`0.97
`0.83
`1.05
`1.27
`1.02
`
`Protein
`Muxiura)
`
`Protein
`Identifi-
`cationb)
`
`L7
`
`L12
`
`a
`
`RNP,
`ATPase, a
`
`Al
`
`groE
`
`S1
`
`EF-Ts
`
`EF-Ts
`
`A13.0
`
`A165
`813.0
`B18.4
`B18. 7
`B20.9
`B35. 1
`840. 7
`B46. 7
`850.3
`B56. 5
`
`861
`B65
`B66
`C15. 3
`C22.7
`
`C30. 7
`
`C31.6
`
`C34. 3
`C40. 3f)
`C44. 2
`
`C44.6
`
`C56
`
`C58. 5
`C60. 7
`C62. 5
`C62. 7
`
`C70
`
`C78
`
`D31.5
`
`D32. 5
`
`D40.7
`D44.5
`D46
`
`D47
`
`1.59
`0.57
`0.49
`0.79
`0.73
`3.22
`0.63
`0.53
`5.60
`2.30
`0.89
`1.49
`1.63
`1.12
`0.88
`2.16
`1.02
`0.72
`0.75
`1.88
`0.73
`1.04
`0.50
`0.55
`1.08
`1.41
`0.89
`1.12
`
`0.73
`0.49
`0.36
`0.78
`0.64
`0.56
`1.28
`2.47
`1.43
`1.49
`0.73
`2.03
`1.27
`1.85
`2.81
`0.76
`
`1.32
`0.62
`0.59
`0.61
`0.59
`3.21
`0.63
`0.57
`4.03
`1.45
`0.75
`1.03
`0.97
`1.02
`0.85
`2.16
`1.08
`1.03
`0.89
`2.05
`1.03
`1.01
`0.51
`0.67
`0.87
`0.76
`0.77
`1.14
`
`0.84
`0.59
`0.49
`0.79
`0.98
`0.71
`
`-
`
`1.29
`1.15
`1.00
`0.59
`1.61
`1.15
`1.51
`1.52
`0.74
`
`1.11
`0.70
`0.47
`0.59
`0.64
`2.01
`0.75
`0.68
`1.53
`1.32
`0.65
`0.75
`0.99
`0.85
`0.99
`1.82
`1.10
`1.08
`0.89
`1.79
`1.29
`1.20
`0.74
`0.82
`0.95
`0.97
`0.85
`1.42
`
`0.93
`0.71
`0.52
`1.17
`1.68
`0.92
`
`-
`
`0.99
`1.01
`0.98
`0.65
`0.60
`1.41
`1.20
`1.22
`0.82
`
`(1.04)
`
`(1.09)
`
`(0.97)
`(1.35)
`(0.99)
`(1.24)
`
`(0.84)
`
`(0.96)
`(0.87)
`
`(1.09)
`
`(1.24)
`
`(1.17)
`
`1.12
`0.83
`1.94
`1.09
`1.11
`0.93
`1.27
`1.16
`1.34
`1.46
`1.21
`1.18
`1.08
`1.08
`0.90
`0.57
`0.94
`0.79
`1.08
`0.81
`0.90
`0.90
`0.82
`0.81
`1.24
`0.93
`1.05
`1.49
`
`0.96
`1.19
`1.16
`1.22
`0.85
`l.09
`0.78
`1. 37
`1.15
`1.07
`0.97
`2.04
`1.28
`1.08
`1.16
`1.01
`
`1.10
`0.55
`2.73
`0.63
`0.69
`0.56
`1.98
`1.06
`4.35
`2.06
`3.06
`1.02
`1. 50
`0.93
`0.49
`0.24
`0.49
`0.65
`0.58
`1.36
`0.60
`0.60
`0.50
`0. 51
`1.80
`1.06
`0.93
`2.63
`
`0.59
`0.82
`0.55
`0.85
`2.47
`
`0.66
`0.58
`2.42
`2.40
`1.01
`0.46
`9.03
`2.75
`1. 16
`1.46
`0.91
`
`0.93
`1.02
`0.97
`1.04
`1.45
`1.12
`1.09
`1.08
`1.41
`0.94
`1.09
`0.88
`0.87
`0.94
`1.17
`1.02
`1.32
`
`0.91
`0.77
`0.69
`1.05
`1.52
`0.91
`1.00
`1.11
`1.05
`1.11
`0.82
`0.82
`1.35
`1.30
`1.23
`0.92
`
`LysS
`
`EF-G
`
`PheS,
`
`LeuS
`
`FRP,0
`
`EF-Tu
`
`ArgS
`GlyS
`
`VailS
`
`Ic
`
`Ic
`
`Ic
`lb
`
`Ic
`
`Ia3
`
`Ic
`
`Ic
`
`hIal
`
`Ib
`
`Ib
`_
`Ic
`
`_
`
`Ic
`
`Ic
`
`IIb2
`
`Ic
`
`IIa2
`
`2.82*
`2. 38^
`1. 31*
`1.48
`4.99
`28.22
`2.45*
`2.26
`
`1.05
`2.05*
`
`7.43
`0.64*
`1. 08*
`1.25*
`0.63
`
`84.92
`2.69*
`
`1.37
`
`2.74
`
`4.69
`
`2.11
`
`0.86
`
`- 1.11
`
`Ia4
`
`Ib
`
`-
`
`-
`
`Ic
`
`Ia3
`IIa2
`
`112
`
`lb
`
`-
`
`0. 85*
`
`16.20
`
`1.20*
`1.41*
`4.02
`
`0.48
`
`2.06
`
`2.94
`
`2.74*
`
`6.40*
`
`D47.5
`D49.2
`
`D58. 5
`D74
`D84
`
`D87. 5
`D94
`
`D99
`
`D100
`
`D157
`E22.8
`
`023
`
`E24. 8
`E38.5
`
`E42
`
`E48. 7
`
`E58
`
`E77. 5
`
`E79
`
`E106
`
`E133
`E140
`F14.7
`
`F24.5
`
`F24.6
`
`F26
`
`F30.2
`
`F32. 3
`
`P32. 5
`
`F38
`F39
`F42 .2
`
`GNE 2002
`Page 3
`
`

`
`188
`
`HERENDEEN, VANBOGELEN, AND NEIDHARDT
`
`J. BACTERIOL.
`
`Level at each temperature relative to level at 37 C
`
`TABLE 1-Continued
`Weight fraction
`of total protein
`in glucose rich
`at 37°d
`medium
`' (pg/mg)
`
`13.5 C
`
`150C
`
`Protein
`Numbera)
`
`Protein
`Identifi-
`cationb)
`
`l4etabolic
`Regulation
`Groupc)
`
`GluS, 8
`
`Ic
`
`Ib
`
`Ic
`Ia3
`
`Ia2
`
`F43.8
`
`F43.9
`F48.1
`
`F50.3
`
`F56
`
`F56.2
`
`F56.5
`
`10.54*
`3.53
`
`1.51
`0.24
`
`2.00*
`5.03
`
`1.44
`
`1.74
`
`0.95
`
`2.64
`
`0.68
`
`0.63
`
`0.81
`
`1.51
`
`1.33
`
`0.70
`
`1.95
`1.14
`0.65
`
`0.61
`
`238Ce)
`
`1.43
`1.28
`0.81
`
`1.15
`0.98
`
`0.86
`
`0.98
`
`(0.87)
`
`(1.13)
`
`300C
`
`1.04
`
`1.43
`
`0.75
`
`0.87
`
`0.95
`
`0.94
`
`1.23
`
`1.22
`
`42°C
`
`1.11
`
`0.79
`1.15
`1.22
`1.27
`1.03
`
`1.39
`
`0.85
`
`46°C
`
`1.23
`
`0.58
`
`0.78
`
`2.84
`1.86
`0.63
`
`1.87
`
`0.46
`
`AspS
`
`F58.5
`
`F60.3
`
`F63.4
`
`F63.5
`
`Ic
`-
`Ic
`Ia3
`
`Ic
`-
`
`Ia4
`IIal
`
`Ia3
`IIa2
`
`1.69
`1.64l
`0.23
`
`0.56
`
`1.24
`
`1.78
`
`2.91
`
`0.19
`
`0.79
`
`1.48
`
`2.16
`
`0.66
`
`0.57
`
`1.29
`
`0.82
`
`5.35
`
`2.20
`
`1.15
`
`1.70
`
`0.99
`
`1.04
`
`0.74
`
`0.94
`
`4.06
`
`1.58
`
`1.25
`
`1.00
`
`0.78
`
`0.80
`
`0.83
`
`0.89
`
`2.15
`
`1.28
`
`0.93
`
`1.23
`
`0.77
`
`0.89
`
`0.97
`
`1.13
`
`1.25
`
`0.51
`
`1.45
`
`0.90
`
`0.88
`
`1.18
`
`0.89
`
`1.17
`
`1.01
`
`0.94
`
`2.76
`
`0.97
`
`0.97
`
`0.73
`0.70
`
`1.59
`1.84
`
`4.66
`
`(2.20)
`
`(2.41)
`
`F63.8
`F64.5
`F82.5
`F84
`F84.1
`
`F88
`
`IleS
`
`Ia4
`IIa3
`
`Ic
`Ic
`
`Ib
`
`Ib
`
`Ial
`
`-
`
`0.73
`
`1.13
`
`24.12
`
`2.79
`
`0.63
`
`2.01
`
`0.45*
`0.50*
`
`0.28
`
`2.79
`
`0.69
`
`1.08
`
`1.90
`
`1.14
`
`3.19
`
`23.90
`
`0.35
`
`5.71
`
`0.58
`
`1.23
`
`2.42
`
`0.80
`
`3.10
`
`1.77
`
`0.46
`
`4.18
`
`0.62
`
`1.20
`
`1.58
`
`0.78
`
`2.30
`
`1.20
`
`(1.34)
`
`(1.26)
`
`(2.31)
`
`1.02
`
`0.71
`
`1.08
`
`1.20
`
`1.04
`
`1.52
`
`0.94
`
`0.88
`
`0.98
`
`0.78
`
`-
`
`1.27
`
`0.91
`
`1.44
`
`0.57
`
`0.47
`
`0.83
`
`0.53
`
`1.54
`
`0.30
`
`0.84
`
`F99
`F107
`F178
`G25.3
`G27.2
`G29.6
`G32.8
`G36
`
`G41
`
`G41.2
`
`G41.3
`
`PheS, a
`
`-
`
`Ic
`
`-
`
`Ib
`
`Ic
`
`0.75
`
`1.12
`
`1.55a
`
`2.07
`
`5.49
`
`0.41
`
`1.01
`
`(0.58)
`
`(0.75)
`
`0.65
`
`1.16
`
`1.79
`
`5.95
`
`0.28
`
`2.30
`
`0.96
`
`1.41
`
`0.77
`
`0.49
`
`1.40
`
`0.39
`
`0.93
`
`1.88
`
`0.96
`
`0.78
`
`0.77
`
`1.16
`
`0.47
`
`1.53
`1.75
`
`0.66
`
`0.82
`
`1.08
`
`1.02
`
`0.70
`
`1.34
`
`1.43
`
`1.19
`
`0.38
`
`1.07
`
`0.95
`
`1.01
`
`0.93
`
`0.85
`
`1.52
`
`1.12
`
`0.56
`
`0.69
`
`2.92
`
`0.89
`
`0.25
`
`2.87
`
`1.08
`
`2.15
`
`IIa2
`IIa2
`IIa2
`Ia4
`
`Ib
`
`IIbl
`Ia4
`Ic
`
`Ic
`
`Ic
`
`ATPase, B
`
`GlnS
`
`1.99
`
`3.54*
`2. 69*
`6.49
`
`6.00
`
`3.62
`
`0.54
`
`1.49
`
`1.42
`
`0.55
`
`0.57
`
`0.94
`
`0.99
`
`1.37
`
`0.59
`
`0.93
`
`0.99
`
`0.42
`
`0.54
`
`0.72
`
`1.19
`
`0.89
`
`0.86
`
`0.82
`
`0.82
`
`1.28
`
`0.49
`
`0.72
`
`0.75
`
`1.27
`
`0.86
`
`1.12
`
`0.83
`
`0.72
`
`(0.95)
`
`0.73
`
`0.86
`
`1.09
`
`1.21
`
`0.95
`
`1.08
`
`0.85
`
`0.78
`
`1.00
`
`1.13
`
`1.18
`
`0.99
`
`1.02
`
`0.97
`
`0.94
`
`0.64
`
`0.91
`
`1.16
`
`1.20
`
`1.73
`
`0.91
`
`0.72
`
`0.83
`
`1.75
`1.54
`
`Ia4
`Ia2
`Ia3
`Ic
`Ia4
`Ia3
`
`Ic
`Ib
`
`.38
`2.34
`
`-
`
`0.88
`
`1.16
`
`0.60
`
`-
`
`-
`
`4.07
`
`1.68
`1.66
`
`1.83
`
`0.69
`
`0.78
`
`2.42
`
`1.79
`
`1.08
`
`0.31
`
`3.21
`
`1.77
`
`0.74
`0.80
`2.68
`
`1.65
`
`1.01
`
`0.41
`
`2.10
`
`(2.99)
`
`(0.89)
`
`(1.14)
`
`3.47
`
`1.17
`
`1.26
`
`0.91
`
`1.46
`
`1.34
`
`1.04
`
`0.62
`
`1.89
`
`(0.38)
`
`0.95
`
`1.24
`
`1.13
`
`1.00
`
`0.90
`
`1.09
`
`0.89
`
`0.98
`
`1.38
`
`0.77
`
`1.07
`
`1.04
`
`1.13
`
`1.12
`
`1.04
`
`0.99
`
`1.32
`
`1.08
`
`1.24
`
`0.97
`
`1.17
`
`1.41
`
`1.03
`
`0.99
`
`0.36
`
`1.52
`
`G41.4
`G43.2
`G43.8
`G43.9
`G44
`G50.5
`G51
`G54.7
`G57
`G61
`G62.8
`G74
`G76
`G78
`G93
`G97
`G117
`G127
`H52.7
`H54.6
`I14.2
`814.4
`I14.7
`116.4
`I17.2
`I19.5
`I21.1
`I21.3
`I21.4
`I23.0
`I26.0
`I26.3
`I33.5
`I35.2
`847.2
`I49.6
`I58.4
`I63.5
`
`Ribosomal Pro.
`Ribosomal Pro.
`Ribosomal Pro.
`
`-
`
`Ic
`
`Ic
`
`Ic
`
`-
`
`Ib
`
`Ic
`Ic
`-
`
`Ic
`
`-
`
`-
`
`-
`8.63*
`8.58*
`
`-
`
`5.60*
`1.24*
`2.34*
`1.71*
`
`1.41
`0.98
`
`0.66
`
`0.65
`
`0.76
`1.50
`
`1.15
`
`0.93
`0.70
`0.13
`5.82
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`0.99
`
`0.98
`
`1.01
`
`1.00
`
`0.95
`
`0.98
`
`1.12
`
`0.90
`
`1.64
`
`1.02
`
`0.97
`
`0.96
`
`1.04
`
`1.17
`
`1.08
`
`0.91
`
`0.05
`
`0.77
`
`0.49
`
`0.45
`
`0.48
`1.28
`1.25
`
`0.85
`
`0.41
`0.05
`0.42
`
`a Proteins are numbered as described in Pedersen et al. (17). Letter prefixes refer to the position of the
`protein in the axis parallel to the isoelectric focusing dimension (A-I indicates progression from acid to base);
`
`Ribosomal Pro.
`Ribosomal Pro.
`Ribosomal Pro.
`Ribosomal Pro.
`Ribosomal Pro.
`
`Ic
`Ia2
`Ic
`Ic
`Ic
`
`Ic
`
`Ic
`
`-
`
`-
`
`-
`
`0.53
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`3.81*
`
`1.08
`
`0.78
`
`0.75
`
`0.63
`
`0.69
`0.74
`
`2.06
`
`0.58
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`1.08
`
`1.05
`
`1.03
`
`0.96
`
`0.99
`
`1.33
`
`o.82
`1.06
`
`0.90
`
`0.99
`
`0.98
`
`0.93
`
`1.07
`
`1.00
`
`1.28
`
`0.97
`
`0.69
`
`0.75
`
`0.49
`
`0.55
`
`0.43
`
`0.54
`0.47
`
`1.02
`0.50
`
`0.64
`1.30
`
`GNE 2002
`Page 4
`
`

`
`VOL. 139, 1979
`
`PROTEIN LEVELS AT DIFFERENT TEMPERATURES
`
`189
`
`TABLE 1-Continued
`numbers refer to the apparent molecular weight of the protein as determined by the distance moved in the
`sodium dodecyl sulfate electrophoresis dimension (56.5 indicates molecular weight of 56,500).
`b Abbreviations used: S-aminoacyl-tRNA synthetase; L7, L12, Si-identified ribosomal proteins; Ribosomal
`Pro.-proteins known to be ribosomal proteins, but not identified further; RNP, a-RNA polymerase, a subunit;
`RNP, /?-RNA polymerase, ,B subunit; EF-Ts, EF-Tu, EF-G-protein synthesis elongation factors Ts, Tu, and
`G; A, groE-the A protein of Subramanian et al. (25), also identified as the product of the groE gene by Drahos
`and Hendrix (personal communication).
`'The proteins have been grouped according to the way in which their relative levels change in cells grown in
`different media. The numerical values are published in Pedersen et al. (17), and the behavior of each class is
`shown in Fig. 6.
`d Most of these values were measured by determining the total radioactivity in individual spots on a gel made
`from cells grown on uniformly labeled ['4C]glucose, and are taken from Table 1 of Pedersen et al. (17). The
`values with an asterisk were measured in the same manner, but with cells grown on ['4C]leucine; these values
`are influenced by any differences in the leucine content of the individual proteins relative to the average for E.
`coli protein, and therefore should be regarded as approximate.
`'The values in parentheses were measured in a separate experiment (strain NC81) in which both the
`experimental culture (230C) and the reference culture (37°C) were grown in glucose minimal medium.
`IProtein spot C40.3 has recently been recognized to contain, in most gels, two individual proteins. The data
`shown reflect the behavior of the dominant protein of this pair, but must be regarded as approximate.
`
`G29.6 (30-fold) and A165 (100-fold), are virtually
`constant throughout the normal temperature
`range, and are nearly invisible in autoradiograms
`of gels made from cells grown at these temper-
`atures. Their behavior is presented in Fig. 4.
`Three proteins, D74, G44, and G27.2, have
`levels that vary as linear functions of tempera-
`ture over the entire range of 13.5 to 460C (Fig.
`5), despite the multiphasic effect of temperature
`upon cell growth over this temperature span
`(Fig. 1). These three "thermometer" proteins,
`plus 10 (B40.7, D49.2, E24.8, E42, F42.2, F74.5,
`G61, G127, I35.2, and I47.2) that show less than
`a 1.5-fold change at any temperature, are the
`major exceptions to the general rule that pro-
`teins vary little in the normal range (23 to 370C)
`and vary significantly above and below this
`range.
`Many of the proteins measured in this study
`were included in our recent report on the
`amounts of individual proteins during balanced
`growth at 370C in five different media (17). In
`Fig. 6 we present all of the proteins for which
`both "metabolic regulation" and temperature
`response data are available. The left side of each
`panel shows the variation of proteins as a func-
`tion of growth rate in different media at 370C,
`and the right side of each panel shows the vari-
`ation of these same proteins as a function of
`temperature in rich medium. Of the 133 proteins
`in the current study, 13 were not measured in
`our studies of metabolic regulation, and 10 ex-
`hibited individual and unique patterns of behav-
`ior in the five media and are therefore not dis-
`played in Fig. 6. Of those remaining, 50 belong
`to three classes of metabolic regulation (Ia4, Ib,
`and Ic) that display a heterogeneous response to
`temperature; there is no way of predicting how
`a protein belonging to these three metabolic
`
`40
`
`All Proteins( 111) 23tC to 37c
`
`3
`
`45'
`
`10°
`
`Transcriptional and Translational Proteins(18)
`230Cto37*C
`
`,o0
`
`0nE z
`
`1
`
`6
`
`2
`3
`5
`4
`Maximum Level of Protein/Minimum Level of Protein
`FIG. 2. Distribution ofE. coliproteins with respect
`to magnitude of variation in level within the normal
`temperature range, 23 to 37°C. For each protein its
`maximum level between 23 and 37°C was divided by
`its minimum level in this range. Proteins were then
`grouped according to the magnitude of their varia-
`tion: 1.0-1.2, 1.2-1.4, 1.4-1.6. The upper panel shows
`all 111 proteins for which data are available; the
`lower panel shows 18 transcriptional and transla-
`tional proteins. All data were taken from Table 1.
`
`what is evident to the unaided eye from the
`autoradiograms of the O'Farrell gels; the relative
`abundance of individual proteins at 460C is eas-
`ily distinguished from that at 370C. Of 133 pro-
`teins, 83 vary more than 2-fold, 18 vary more
`than 5-fold, and 9 vary more than 10-fold. The
`two proteins with the greatest change in level,
`
`GNE 2002
`Page 5
`
`

`
`190
`
`HERENDEEN, VANBOGELEN, AND NEIDHARDT
`
`J. BACTERIOL.
`
`All Proteins(133) 13.50C to 46°C
`
`,,
`
`40-
`
`30-
`
`20-
`
`i
`
`.& 10-
`4--0
`0.
`
`1 I
`
`10
`
`0 0
`
`.0
`E
`z
`
`m M
`a
`FI/I
`Th
`I
`20
`25
`10
`15
`5
`1
`l
`
`a-
`'
`30
`
`f
`
`I
`100
`
`I
`
`a u
`-'MI
`10
`15
`--I
`
`Transcriptional and Translational Proteins(25) 1 3.5°C to 460C
`
`, .
`
`1
`a
`
`1
`
`5
`
`105
`
`100
`30
`25
`20
`15
`10
`Maximum Level of Protein/Minimum Level of Protein
`FIG. 3. Distribution ofE. coli proteins with respect to magnitude of variation in level over the temperature
`range 13.5 to 460C. For each protein its maximum level between 13.5 and 46C was divided by its minimum
`level in this range. Proteins were then grouped according to the magnitude of their variation: 1.0-1.5,1.5-2.0,
`2.0-2.5, etc. The upper panel shows all 133 proteins for which data are available; the lower panel shows 25
`transcriptional and translational proteins. AU data were taken from Table 1.
`Ribosomal protein L7 (protein A13.0) is an
`acetylated form of L12 (protein B13.0). The sum
`of these proteins varies coordinately with the
`other ribosomal proteins, both at different tem-
`peratures (Table 1) and in different media (17).
`The ratio of the acetylated form to the unace-
`tylated form, however, differs dramatically at
`low temperature from that at moderate and high
`temperature (Table 2).
`As might be expected, there is coordinate con-
`trol at all temperatures for the measured poly-
`peptide subunits of 30S and 50S ribosomes and
`of the three enzymes, ATPase, RNA polymer-
`ase, and phenylalanyl-tRNA synthetase (Table
`1).
`
`classes will vary in level in response to temper-
`ature. For the 60 remaining proteins, one can
`predict how they will respond to temperatures
`by knowing either that they belong to metabolic
`regulation classes Ia2, Ia3, fIal, IIa2, and IIbl,
`or that they are a transcriptional or translational
`protein of class Ic. Because this group of 60
`proteins includes only 11 of the ribosomal pro-
`teins and 10 ofthe aminoacyl-tRNA synthetases,
`a reasonable guess is that 115 proteins can now
`have their temperature response predicted by
`their metabolic response and function.
`Translational and transcriptional proteins are
`reduced in level at both high and low tempera-
`tures (Fig. 6). From Table 1, it can be calculated
`that these proteins (together with the as yet
`unmeasured remai g ribosomal proteins) con-
`stitute 38% of the cell's total protein mass at
`37°C, but only 22% at 460C. The summed de-
`crease in these proteins is in fact equal to the
`increase in just three proteins (B56.5, B66.0, and
`F24.5) that together constitute 4% of the cell's
`protein at 370C, but 20% at 460C. It is the
`changes in these three proteins, in the transcrip-
`tional and transational proteins, and in protein
`A165 (Fig. 4) that produce the different appear-
`ance of autoradiograms of 460C cells and 370C
`cells.
`
`DISCUSSION
`From the data displayed in Fig. 2, it appears
`that the evolution of protein catalysts in E. coli
`has led to the ability to grow efficiently between
`23 and 37°C with only a small number of pro-
`teins having to be adjusted in level. We do not
`know how close to a value of 14,000 cal (ca.
`58,600 J) per mol is the temperature character-
`istic of each reaction in the cell over this tem-
`perature range, but whatever their individual
`values, the reaction rates can remain coordi-
`nated simply by modulations of enzyme activity
`
`GNE 2002
`Page 6
`
`

`
`VOL. 139, 1979
`
`PROTEIN LEVELS AT DIFFERENT TEMPERATURES
`
`191
`
`fold at 13.50C. Exploring the function of these
`proteins may help answer the question of what
`restricts growth at these temperature extremes.
`A third protein in this group, B56.5, has already
`been shown to be related to macromolecule syn-
`thesis and to be growth essential; it is the prod-
`uct of the groE gene (6; David J. Drahos and
`Roger W. Hendrix, personal communication).
`The synthesis of this protein has been shown to
`be subject to transient, extreme changes in rate
`following temperature shifts; these transient
`rates permit rapid attainment (within 10 to 15
`min) of the new steady-state level of this poly-
`peptide.
`O'Farrell gels produced from cells grown at 23
`and 370C are indistinguishable to the untrained
`eye. In contrast, the pattern at 460C is quite
`distinct with respect to the relative abundance
`of different proteins. The rate of degradation of
`E. coli protein increases at temperatures above
`370C (24), so we have initiated a study to learn
`whether the pattern at 460C reflects changes in
`the rates of synthesis of individual proteins, or
`changes in their rates of degradation. The most
`important preliminary result is easily summa-
`rized: protein degradation is enhanced at 460C
`compared to 370C, but the levels of proteins at
`
`f
`
`I
`
`.
`
`I
`
`.
`
`II
`
`I
`
`3.0 -
`
`G 27.2
`
`2.0 H
`
`D 74.0
`
`0)
`-J
`0L)
`4r..'
`)
`
`* 0
`
`1.0 F---
`
`50
`
`0
`
`10
`
`40
`30
`20
`Temperature (0C)
`FIG. 5. Variation in level of three "thermometer"
`proteins of E. coli as a function of temperature. The
`level of each protein relative to its level at 37°C is
`plotted as a function of temperature. The data were
`taken from Table 1. (0) Protein G27.2; (0) protein
`G44.0; (A) protein D74.0.
`
`- t(
`
`U
`6(
`
`40
`
`45
`
`15
`
`20
`
`30
`25
`35
`Temperature(C)
`FIG. 4. Variation in level of two proteins ofE. coli
`as a function oftemperature. The level ofeachprotein
`(log scale) relative to itslevel at 37°C is plotted as a
`function of temperature. The data were taken from
`Table 1. (0) Protein G2p.6; (0) protein A165.
`
`(as by allosteric effectors), and changes in en-
`zyme levels are not necessary.
`Three proteins vary in level in a simple, linear
`fashion with temperature. This regularity over
`the range from 13.5 to 460C is not readily ex-
`plained on the basis of the effect of temperature
`on promoter activity, on repressor activity, or on
`metabolic pools, because the absolute rate of
`growth is quite restricted at the high and low
`temperatures. A reasonable view is that the lev-
`els of these proteins are actively regulated by
`the cell, and that they are required in amounts
`that vary simply with temperature. A more pre-
`cise guess is not now possible.
`Nine proteins (A165, B56.5, D40.7, F32.3,
`F82.5, F84.1, G29.6, G41.2, and I63.5) are mark-
`edly elevated in level (5- to 25-fold) during
`growth at either high or low temperature. The
`impression gained is that these proteins are
`greatly increased amounts, but
`needed in
`whether this is because the proteins are not fully
`active at these temperatures or because their
`products are in greater demand cannot be told.
`Two of these proteins have particularly interest-
`ing behavior: A165 is constant and barely de-
`tectable at temperatures from 13.5 to 420C, and
`then abruptly increases 25-fold at 460C, whereas
`G29.6 is low and nearly constant in level from
`460C down to 150C and then abruptly jumps 23-
`
`GNE 2002
`Page 7
`
`

`
`192
`
`HERENDEEN, VANBOGELEN, AND NEIDHARDT
`
`J. BACTERIOL.
`
`5 0 _
`
`Medium
`
`Group2- Ribosomal Proteins
`5.0
`_
`Terature
`Medium
`
`T mparature
`
`.0
`
`2.0
`
`10
`
`0.5
`
`02
`
`50
`
`2.0
`
`025
`
`------
`
`1.0 --
`
`20
`
`0.5
`
`02
`
`Group 1a3
`Msd.v
`
`AminoacylktRNA Svnthetases,
`EF-G, Ef-Tu, EF-Ts, RNA Polymerase
`\~~~~~~~~II.~ ~ ~ ~ ~~~wu
`
`/
`
`Medium
`
`A
`
`T
`
`nperatureZ C5k(r.0
`
`Tem pwerstrfC
`
`025
`
`1.0
`
`0
`
`405
`
`-,
`5
`
`ru-a-1
`30
`40
`20
`2.010
`1.5
`1.0
`k(hr-1)ATemperature(T)
`Temperaoure
`(hr
`
`so
`-
`
`~~~~~~0 0.5
`1.0
`_50_Memk(hr)
`
`1.5
`
`la4
`
`rou
`2.010
`B
`
`30
`20
`Temperature(C)
`
`40
`
`so
`
`A_d
`
`Group fIal
`
`Group Ib
`
`3,z
`
`T--p- 1
`
`50
`1.
`
`5_Medium
`
`Tempereature _50Medium
`
`Tefnperature
`
`0,2
`
`5,0
`
`Group 1-a2
`
`Group lb
`
`0.2
`
`10
`
`20~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
`
`10~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`
`of the same regulatory class in glucose-rich medium at the indicated temperaturesrelativeato
`50each~50otein
`k(r5.0D Tmeatr(c
`k(hr-ium
`Ternperature(C
`37°C2.0These data were taken from Table1.
`
`0 2-
`
`50~~~~~~~~~~~~~~~~~~~0
`Tmeauet
`D
`(r)
`Temperaturelt
`MkdhrnI
`ofgrwth rte indifGroupei
`minbma Groupm(ac(tatemntimalk=d038
`t 7Crltvet lcs
`
`ofeachproteinofthesame reglatory class i glucose-rich mdium at the inicated temperatresTrelativer t
`2rCThs0 aawr ae rmTbe1
`
`GNE 2002
`Page 8
`
`

`
`VOL. 139, 1979
`
`PROTEIN LEVELS AT DIFFERENT TEMPERATURES
`
`193
`
`TABLE 2. Effect of temperature on acetylation of
`ribosomalprotein L7/L12
`Acetylateda
`Temp
`(OC)
`(%)
`13.5 ..................
`59.3
`28.6
`30.0 ..................
`37.0 .................. 23.4
`42.0 .................. 26.5
`28.4
`46.0 ..................
`a The levels of L7 and L12 at 13.5, 30, 42, and 460C
`relative to their level at 37°C (from Table 1) were
`multiplied by their weight fraction of total protein at
`37°C (a' value from Table 1) to find their weight
`fraction (a') at each of these temperatures. Because
`L7 and L12 have very nearly identical molecular
`weight, the percent acetylation of L7/L12 at each
`temperature was calculated by dividing the a' value
`(x100) of L7 by the total a' of L7 plus L12.
`460C reflect changes in rates of synthesis, not
`degradation. A gel made from pulse-labeled cells
`at 460C resembles one from steady-state-labeled
`cells at 460C, and a gel made from pulse-labeled
`cells at 370C resembles one from steady-state-
`labeled cells at that temperature (VanBogelen
`and Neidhardt, manuscript in preparation).
`At both extremes of the temperature range of
`growth, E. coli has lowered levels of ribosomal
`proteins and other translational and transcrip-
`tional proteins (Fig. 6). In this respect, the re-
`stricted growth at low and high temperature
`resembles restricted growth in general, and no
`diagnostic feature helps us identify what might
`be limiting the growth rate at these temperature
`extremes. Minimal medium will not support the
`growth of E. coli at temperatures above 440C,
`indicating that inactivation of biosynthetic en-
`zymes determines the maximum temperature of
`growth under this condition. In the present
`study, a rich medium (glucose-MOPS supple-
`mented with amino acids, purines and pyrimi-
`dines, and vitamins) was deliberately chosen to
`avoid growth restriction brought about by bio-
`synthetic insufficiency and to require that inac-
`tivation of some generally irreplaceable enzyme
`be responsible for growth restriction. As a brief
`survey, however, 40 proteins were monitored in
`cells grown in glucose miniimal medium as 23
`and 370C. The results (Table 1) indicate that
`about 90% of these proteins behave similarly
`over this span of temperature whether the cells
`are grown in rich or miniimal medium. The re-
`maining 10% (proteins F84.1, F88, G97, and
`H54.6) are clearly of interest, and must be sus-
`pected of being involved in amphibolic or bio-
`synthetic pathways.
`The enhanced level of acetylation of ribo-
`somal protein L7/L12 at 13.50C is interesting.
`Our earlier data (17) indicated that at 370C the
`
`level of acetylation varies regularly with growth
`rate, from 23.4% in glucose-rich medium to 90.4%
`in acetate minimal medium