~) INSTITUT PASTEUR/ELSEVIER Paris 1992
Res. Microbiol. 1992, 143, 173-181
Catabolic pools in Escherichia coli S.R. Pai(*) and H.E. Kubitschek Biological, Environmental and Medical Research Division, Argonne National Laboratory, Argonne, IL 60439-4833 (USA) SUMMARY
Methods are described for measuring soluble pool magnitudes in steady-state or exponentially growing cultures, and for distinguishing between anabolic, catabolic and total metabolic pools within cells. These methods were applied to the measurement of pool magnitudes for several amino acids and other precursors in Escherichia coli THU. Our results support the independence of the magnitudes of total metabolic pools and growth rate in steady-state cultures. Our results also show that the total metabolic pool size is much larger than previously published estimates, which failed to include the contribution of catabolic pools. The average value of the total soluble material in exponentialphase cells is estimated to be 8 to 9 % of the cell dry mass; pool values could be almost twice as large during midcycle because of the known increase in the magnitudes of protein and RNA precursor pool during the cell cycle.
Key-words: ~acterial growth, Escherichia coil; Metabolism, Nutrition, Anabolic and catabolic pools, Incorporation.
INTRODUCTION Cell dry mass in Escherichia coil consists mainly of macromolecular compounds that cannot be extracted with trichloroacetic acid, along with a much smaller fraction of soluble material located in metabolic pools (Roberts et aL, 1955; Umbarger, 1977; Ingraham et al., 1983; Neidhardt, 1987). In addition to ions, cofactors and vitamins, these metabolic pools consist of two major components, anabolic pools of chemical precursors newly transported into the cell or resident as soluble intermediates of synthesis, and catabolic pools, p~oducts arising from degradation of macromolecular compounds.
Earlier measurements of pool magnitudes provided estimates only of their anabolic components, as few, if any, studies determined the magnitudes of catabolic pools in E. coll. This was, perhaps, to be expected because, although several different kinds of experimental approaches were used, all were designed to examine precursor pool formation in the ab~c~ce of macromolecular degradation (Roberts et aL, 1955; Dennis and Bremer, 1974; Umbarger, 1977; Summerton, 1976; Ingraham et al., 1983 ; Neidhardt, 1987). The occurrence of catabolic pool~ in E. coli is amply supported by numerous studies of pro-
Submitted May 28, 1991, accepted August 2, 1991. (*) Correspondingauthor and presentaddress: Departmentof Pathobiology,Auburn University,Auburn, AL 36849-5519(USA).
174
S.R. P A l AND H.E. KUB1TSCHEK
teolytic degradation. Observed rates of degradation of individual proteins are initially low and diminish further with time. When a culture is pulse-treated with a radioactive amino acid, about 5 % of the radioactivity is released as free amino acid dering the first minute (Pine, 1970). This initial release may not constitute normal proteolysis, but a part of protdn synthesis or the failure to complete protein synthesis (A. L. Koch, 1979).
A~ = A / ( I - e - t / z ) , where t is the time after adding the label and x is the time for the cell population to increase by the factor e (Britten and McCarthy, 1962). In terms of the doubling time T, A ~t = A / ( 1 - 2 - t / T ) ) . The term in parentheses describes the relative accumulation of labelled material in the presence of total incorporated precursor, both labelled and unlabelled.
Degradation rates may ~hen become reduced to values as low as 0.5 to !.0 % per hour after several hours in glucose cultures (Pine, 1970; Willets, 1967) although individual proteins have widely different half-lives for degradation (Mosteller et el., 1980) and some degrade even more slowly. Nevertheless, total protein breakdown can still be significant; in chemostat-grown cultures labelled with valine, alr,:~ost 60 % of the label was released as acid-soluble components during a period of 3 days (Willets, 1967). This kind of evidence cle~.rly indicates the presence of catabolic pools, whose magnitude would also depend upon the efficiency of reincorporation of breakdown products into macromolecules.
Anabolic pools
We describe here experimental approaches that distinguish between anabolic and catabolic pools by using cultures labelled in the steadystate or experimental growth phase. In order to maintain steady growth after labelling~ the growth medium must first contain the unlabeiled precursor because de novo addition of even a very small amount of labelled precursor can qualitatively alter growth or transport kinetics (Kubitschek, ~968). Pool magnitudes are determined as the difference between paired culture samples: the first is washed with unlabelled growth medium to determine the total cellular rad--:.(.~activity, A, and the second is extracted with ice-cold trichloracetic acid before washing to determine the radioactivity of the pool is A - B and the fraction of the labelled precursor is (A - B)/A. Of greater interest, however, is the magnitude of the pool in terms of the total amount of precursor used by the cell after an indefinitely long labelling period. To good approximation, this maximal activity is given by
After addition of label, the radioactivity of the anabolic pool increases within a few minutes to a constant plateau value per cell (see for example Britten and McClure, 1962; fig. 3). As this time, the relative magnitude of the pool in terms of the total amount of precursor used by the cell is given by Pa = _A .-_.~B (1 --2-t/T). A
Catabolic pools
When the radioactivity of the anabolic pool reaches its maximum value, the rate of incorporation of precursor label into macromolecules also becomes maximum. The subsequent breakdown of labelled macromolecules then leads to an increase in the size of the labelled catabolic pool, which, in turn, might require several generations to reach its saturation value, Pc. Measurement of the metabolic pool at that time provides the sum Pt of the magnitudes of thc anabolic and catabolic pools, Pt = Pa + Pc, when these pools are formed independently. The magnitude of the catabolic pool can then be determined by the difference between the values of the total metabolic pool and the anabolic pool. Alternatively, the magnitude of the catabolic pool can be determined directly by labelling a culture for several generations and then washing the culture free of the extracellular label. The residual anabolic pool is rapidly used, after which the measured values then correspond to those for the catabolic pool. The catabolic pool then diminishes only very slowly, at a rate of the order of 1% per ho~r. Furthermore, this slow decrease in the magnitude of the catabolic pool is matched by the decrease in the labelled macromolecular fraction. Thus, the magnitude of the catabolic pool is given to good approximation by Pc = (A - B)/A. The criterion that Pa and Pt have reached their saturation values is provided by the constancy of these values during the experiment.
CATABOLIC POOLS IN ESCHERICHIA COLI In this paper, we report the use of these approaches to determine the magnitudes of some total metabolic and catabolic pools in E. coli. la a few ca~e.% magnitudes of anabolic pools were determh~ed for comparison.
MATERIALS AND METHODS Culture conditions E. coli 15 THU (thy his ura), CGSC strain 4908, was used because earlier studies with this strain had already provided accurate measurements of anabolie pool sizes for several amino acids. Cultures (30 ml) were grown to exponential phase in Erlenmyer flasks under conditions described earlier (Kubitschek and Pal, 1988), except that sucrose usually was omitted from the growth medium. In addition, growth was always carried out in the presence of an unlabelled precursor added to a concentration of 5 to 20 ~tg/ml. The average doubling time was 40 min. For some measurements of phosphorous and uracil pools, cultures were grown in nutrient both (Difco) or Casamino acids were added to 0 . 1 % to provide rapidly growing cultures. Growth rates also were sometimes increased, less markedly, by addition of methionine to a concentration of 40 ~g/ml. Occasionally, an inefficiently utilized carbon source such as glycerol was used to provide a very slowly growing culture. Growth was monitored as the increase in turbidity observed with a "Klett-Summersoa" colorimeter with a "Kodak 66" filter, or occasionally, as the increase in cell count as measured with a "Coulter" counter. Experiments were carried out with exponential-phase cultures inoculated the previous day.
Radioactive materials
H ,~32po4, 207 Ci/mg P; H232SO4, 43 Ci/mg S; L-(U-~'C)-glutamine, 200 mCi/mmol, were all from ICN Radiochemicals, Irvine, CA. (2-~4C)-uracil, 50 mCi/mmol; L-(U-TC)-histidine, 336 mCi/mmol, were from Amersham, Arlington Hts., IL. L-(U-laC)-valine, 250 mCi/mmol, was from Becton-Dickinson, Oxnard, CA. D-(U-14C)-glucose, 14.4 mCi/mmol; (U-t4C)glycine, 113 mCi/mmol; L-(35S)-methionine, 800 Ci/mmol; L-(U-t4C)-proline, 1 mCi/mg, were from New England Nuclear, Boston, MA.
175
Labelling and ~o_moi|ng Cultures were always labelled durin~ exponential phase growth. Labelled precursors were added to concentrations of 0.01 to 0.I ~tCi/mL Labelling and sampling protocols depended upon the kind of pool under study. For anabolic pools, radioactivity was added when cultures reached 1.2 x l0 s cells/ml (10 Klett units). After about 10 min, 0.1-mi samples were removed at 5-min intervals for a period of 30 min to determine total and macromolecular cellular radioactivity. Earlier e×l~eriments had shown that anabolic pools in our cultures became saturated within approximately 10 min (Kubitschek and Pai, 1988). To produce more fully labelled metabolic pools, cultures were labelled at lov,er call concentrations (1-2 × 107/ml) for periods of about 3 to 4 generations, providing cell activities that were 90 % or more of their maximum values. Samples were removed at intervals of 15 to 30 rain. The continued presence of the label assured that the anabolic pool remained fully labelled. Cells with labelled catabolic pools and unlabetled anabolic pools were prepared by washing and resuspending cultures in unlabelled growth medium following determination of the total metabolic pools through the method just described. Cultures were filtered over a nitrocellulose membrane filter, immediately washed with unlabelled medium as described earlier (Kubitschek and Pai, 1988) and suspended to volume.
Cell and pool radioactivity Culture samples (0.1 ml) were always taken in pairs. As described earlier in detail (Kubitschek and Pai, 1988), samples were washed in unlabelled growth medium to determine total cell radioactivity, or first extracted with ice-cold 10 % trichloroacetic acid and then washed to determine radioactivity incorporated into the macromolecular fraction. The filters bearing the washed samples were dropped into vials containing a scintillation cocktail (Opti-fluor; Packard Instruments Co.) and counted in a liquid scintillation counter (Beckman LS-333). There was no ewaence for quenching of counting rates by residual trichloroacetic acid.
RESULTS AND DISCUSSION Figure 1 is representative o f the kinds o f data provided by our experiments for total m e t a b o lic a n d for catabolic pools. A t time zero, ~4Cproline was a d d e d to an overnight culture con-
176
S.R. PAl AND H.E. KUBITSCHEK
A
~ooo
~
C o.ooo ~
©oo
~
k
~
i •
II-- ;
~ ";" ~
~ooo
o+ ~oo
°
'Imo o,,~
Fig. 1. Uptake of ~4C-proline in an exponentially growing culture of E. coli THU. Labelled proline (1 ~tCi/ml) was added at time zero to a culture containing unlabelled proline (20 v.g/ml). Later, at the time shown by the vertical dashed line, unincorporated labei was removed by washing and resuspending the cells in the same unlabelled growth medium. Panel A, culture turbidity in Klett units. Panel B, relative cell count observed with the Coulter counter.
tairfing unlabelled proline (20 t~g/ml). The figure shows that both turbidity (A) and the relative cell count (B) increased exponentially and with the same doubling time (40 min) both before and after removal of extracellular radioactivity at the time shown by the vertical dashed line. This agreement between turbidity and cell numbers strongly supports the exponential growth of the culture, and the continued agreement after washing and resuspension shows that those operations had little if any effect upon the growth of the culture. As expected in figure 2, culture total radioactivity (C) increased more rapidly, as it must because steady-state conditions had not yet been obtained for uptake of label (see "Introduc-
~
~mo(h,~4
5
6
Fig. 2. Experimental conditions are given in the legend to figure 1. Panel C, increase in cell total radioactivity. Panel D, increase in culture pool radioactivity.
tion"). After removal of the extracellular radioactivity by washing and resuspen.~;.on, e,ctivities in this experiment remained reiatively constant, although in many other experiments these values were seen to decrease very slowly with time. The values for pool radioactivity (D) also increased before washing, in the same manner a~ the total radioactivity, and then remained relatively constant afterwards. The larger errors associated with the measurements of pool radioactivity reflect the fact that these data points are determined from small differences between the much larger values observed for total cell activity and macromolecular incorporation. Relative pool magnitudes were calculated as a percentage of the total amount of precursor used by the cell from the values in C and D (fig. 3) after correction for finite labelling periods. The horizontal lines are the average values obtained for the total metabolic pool (before
CATABOLIC POOLS IN ESCHERICHIA COLI
177
Table !. Dependence of relative pool size upon culture doubling time. Observed values of pool sizes are presented as percentage of the total amount of precursor material utilized by the cell.
Precursor Uracil
Doubling time (min)
No. of experiments
°7o Pool size _+ SE
23-27 42-47 108
4 5 1
6.6±0.6 6.7_+0.2 6.8
10
6.7_+0.2
3 4 5
16.3 __.0.8 15.0+0.8 14.7 _+0.6
12
15.23 0.4
Average Phosphate
35-39 41-49 50-54
Average
o
1
~
~
~
~
~
Timo (hrs)
Fig. 3. Pool radioactivity as a percentage of the total cell radioactivity for the same culture as that in figure I. Experimental conditions are given in the legend to figure 1. washing) and the catabolic pool (after washing), with values ( +_ std. error) o f 5.5 ( _+ 0.5) 070 and 4.1 ( + 0.3)070, respectively. The relatively a b r u p t transition between the labelling of the total pool and the catabolic pool in this experiment indicates rapid utilization of the precursor in the anabolic pool.
ditions, there was the question of the degree to which culture growth rate itself might affect pool size. For this ~e~son, we measured the relative magnitudes of total steady-state metabolic pools formed at different doubling times with uracil or with phosphate as precursors. The observed values, table I, show that there were no significant differences for either uracil or phosphate between the average value for all observations and those for fast, medium, and slow cultures. Th~tt is, there results do not support any variation in relative pool size with growth rate for either pool and they indicate that growth rate could have had, a~ most, a very minor effect upon pool size. Absolute magnitudes would, of course, increase with cell size.
Total metabolic pools P o o l size vs. growth rate In their original studies o f a m i n o acid pools in non-steady-state cultures of E. coli, Britten and McClure (1962) observed that pool sizes were dependent upon culture conditions and were sensitive to osmotic effects of various solvents. Although our steady-state cultures were not exposed to transient changes in culture con-
Average values for total metabolic pool sizes of several a m i n c acids and other precursors are presented in table 1I. Observed values for amino acid pools ranged from 3.0 to 5.8 070, with an ave~-a~,e value of 4.6 °7o. Metabolic pools for uracil and for glucose were even larger, 6.7 and 8.7 070,respectively. The largest pools were those for phosphate and sulphate, with values of approximately 15 and 27 070, respectively.
178
S.R. P A l A N D H.E. KUBITSCHEK
Table
Glucose Uracil Phosphate Sulphate Amino acids Glm Gly His Pro Met Val
!!. Metabolic, catabolic and anabolic pools in E. coil THU. N
Total pool (070)
N
Catabolic pool (%)
N
5 I0 12 3
8.7+0.9 6.7+0.2 15.2±0.4 26.7 _+0.3
8 3 3 --
5.8-e 1.0 3.0±0.5 8.8___0.4 --
2 --
5 6 6 7 4 5
5.8±0.4 5.0±0.7 5.1 ±0.5 5.6±0.2 3.4+0.5 3.0±0.5
6 a 4 4
5.9±0.6 1.2__.0.7 4.6:!:0.2 3.1 ±0.4 -2.1±0.6
~-
2 --
----
1 ~-
Anabolic pool (070) 1.8_+0.6 2.7 (*) 14.4_+ 1.9 __
__
2.2-~'~ 5.4 3.3 ('1 0.8+0.5
N = number of experiments. (*) Values from Kubitschek and Pal (1988).
TaMe III. Elemental contributions to metabolic pools in E. colt TltU. Eletnent
Dry wt fraction
C P S O, N, H
0.47 0.05 0.01 0.43
Total
Element Soluble mass pool ,size (07o) fraction (07o) 8.7 15 27 2.5 t°)
4.1 0.8 0.3 1.1 6.3 ('*l
Dry weigh~ data from table I in Heldal et aL (1985). Values for P and S were calculated from their table I1. (*) Calculated from the average pool size observed for amino acids, 4.6 %, assuming 55 % of the cell dry weight is protein (Ingraham et aL, 1983; Neidhardt, 1987). (**) This value does not include ~he soluble pools for nucleic acids, inorganic ions or cofaclors.
The values given in table I for the total metabolic pools can provide an estimate of the total soluble pool in E. colt. For this purpose, we have used the element dry weight fractions for E. colt of Heldal et al. (1985). As shown in table III, the dry weight fraction for elemental carbon is 0.47, so the observed elemental pool size (for glucose in table II) corresponds to a soluble C pool fraction of
4.1 07o of the cell dry weight. Cell dry weight fractions for P and S were calculated from table II in Heldal et aL (1985); together, phosphate and sulphate pools add another 1.1 07o.The contribution from O, N and H in the amino acid pools (4.6 07o average magnitude) also adds 1.1 07o assuming that 55 07o of the cell dry weight is protein (1,2). If we include the pool size of 1 07o for the inorganic ions and cofactors estimated by Ingraham et aL (1983), then the total soluble pool in E. colt is at least 7.3 07o of the total dry weight. This estimate of pool size, however, neglects the contributions of O, N and H to the soluble pools of the nucleic acid precursors, as well as the contribution of soluble pools for other macromolecules such as lipopolysaccharide and peptidoglycan. These contributions are expected to increase the total soluble pool to more than 8 07o. But these estimates also neglect the variation of pool size with cell age during the cell cycle. Earlier we found that pool sizes were small or negligible at the beginning and at the end of the cycle, at least for protein and R N A precursors, and that the midcycle pool was therefore almost twice as large as the average pool size during the cycle (Kubitschek and Pat, 1988). Those results imply that soluble pools in E. colt
CA TABOLIC POOLS I N ESCHERICHIA COLI
have a maximum value during the cycle approximately 50 % larger when the contributions of unregulated pools (see below) are taken into account. These results show that the pools in E. coil are much larger than earlier estimates. For example, Umbarger (1977) calculated the sum of the soluble pools of amino acid and nucleic acid precursors to be less than 1 % of the cell dry weight. The latest estimate by Neidhardt (1987) raised this value to 2.5 %. Neither of these estimates, however, included the contributions of the catabolic pools, which were not measured by the experimental procedures. Furthermore, even those (anabolic) pool calculations were based upon non-steady-state determinations, which may not have been fully representative of steadystate pool magnitudes.
Catabolic and anabolic pools The observed magnitudes of catabolic pools are also presented in table II. These values were smaller than those for total pools, with the exception of glutamine, for which catabolic and total pools were the same within statistical errors. This equivalence indicates the failure of the sum rule proposed earlier, Pt = Pa + Pc" This inequality was unanticipated and suggested that the sum rule does not apply to all precursor pools. For this reason, we also determined anabo!ic pool sizes for some of the other precursors, as shown in table II. Most of the anabolic pool values also were smaller than those for the corresponding total pools, but there were two cases where the magnitudes of the anabolic and the total pools were not significantly different, for phosphate and proline. The case for phosphate was especially compelling because of the large value for the soluble fraction. The failure to support pool additivity suggests that there are two different classes of pools (1) those for which the precursors pass through the pool with little or no interaction with cell produets other than the macromolecular reactions they are destined to supply and (2) pools for
179
which the magnitudes are regulated between narrow size limits. This regulation may prevent pool magnitudes from exceeding their anabolic levels, as for glutamine or, alternatively, from exceeding their catabolic levels, as for phosphorus or proline. Recently, we obtained further support for this interpretation by examining the magnitude of the phosphorus pool as a function of cell age during the division cycle. We found that the ratio of soluble to total cell phosphorous was constant during the cell cycle, and therefore independent of cell age or size (Pal and Kubitschek, 1991). These results require that the phosphorous pool per unit cell mass be maintained constant within narrow limits. In conclusion, we have provided methods for measuring magnitudes of soluble pools in cells in steady-state cultures and for distinguishing among anabolic, catabolic and total matabolic pools. These methods also distinguish between two kinds of soluble pools. For one class, unregulated pools, the magnitudes of total soluble metabolic pools were observed to equal the sum of those for their anabolic and catabolic components, i.e., the pool sum rule is obeyed.
For the second class, however, pool size was regulated within narrow limits, with the result that the total metabolic pool was no larger than its anabolic or catabolic component. Equivalence between the size of the total pool and the anabolic pool was observed with either phosphate or proline as a precursor. Evidence for manifold processes of phosphate regulation in microorganisms is well-documented (Torriani-Gorini et al., 1987) and our observation of the ageindependence of the soluble pool fraction is not inconsistent with the role of phosphate in the regulation of many other cell processes. The regulatory behaviour of the proline as an osmoprotectant in E. coli (Le Rudulier et al., 1984). The apparent equivalence between catabolic and total pool values for glutamine might be related to the involvement of glutamine in the regtllation of nitrogen utiliz,~uon by glutamine synthetase. Our observations that uracil and phosphate precursor pool size~ were independent of growth
180
S.R. P A l A N D H.E. K U B I T S C H E K
rate were unexpected becau.~e RNA is known to increase with growth rate more rapidly than cell dry weight in E. coil (Dennis and Bremer, 1974). Our results indicate, nevertheless, that relative pool s~:e fractions are independent o f growth rate both for metabolically regulated pools (phosphorous) and for those for which there is no evidence o f regulation (uracil). Because different growth rates were obtained by using a variety o f different media, these results indicate that relative soluble pool magnitudes do not depend upon the composition o f the medium. The results for uracil are especially compelling here because the large range o f growth rates with this precursor were obtained by growing cultures in very different kinds o f media, from r~utrient broth to glycerol minimal medium. Furthermore, because mean cell size increases with growth rate (Schaechter et aL, 1958; Kubitschek, 1974), relative soluble pool magnitudes are independent, or nearly so, o f cell volume. The finding that total pool size in E. coil is much larger thap therefore determined, is of special significance. This result is consistent with a much greater role for soluble pools than has been apparent from earlier studies. As neither the anabolic or catabolic pools can be neglected, it is clear that the organization o f the cell depends strongly upon the contribution o f both components, and any model o f cell growth or its chemical composition must take into account the contribution o f both pools. Thus, these results indicate that previous models based only upon the increase in macromolecular incorporation during the division cycle, as for example the " C o n t i n u m M o d e l " (Cooper, 1979), are incomplete because they fail to include the behaviour o f pool fractions that are now known to be too large to be neglected.
Acknowledgments This work was supported by Public Health Service grant ROI A121954 from the National Institutesof Health and by the US Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38.
Masses cataboliques dans les cellules de Escherlchla coil Des m6thodes sont d~crites, permettant de mesurer les fractions solubles dans les cultures en fonction de la courbe de croissance et de distinguer les masses cataboliques, anaboliques et totales dans les cellules bact6riennes. Ces m6thodes sont appliqu6es pour mesurer les quantit6s d'acides amines divers et d'autres m~tabolites chez Escherichia coll. On observe que les masses totales des m6tabolites et le taux de croissance sont ind6pendants au cours de la phase stationnaire. De plus, la masse totale appara~t plus importante que les estimations ant~rieures le montraient, lesquelles ne tenaient pas compte de la contribution des masses cataboliques. La valeur moyenne du mat6riel soluble total ~t la phase exponemielle de la croissance est 6valu~e ~ 8-9 ~/a du poids cellulaire see; enfin les valeurs des masses m6taboliques peuvent ~tre deux fois plus importante au milieu de la phase de croissance 6tant donn6 l'augmentation 6vidente des masses de prot6ines et d'ARN au cours du cycle cellulaire. Mots-clds: Croissance bact~rienne, Escherichia coil; M6taboiisme, Nutrition, Masses cataboliques et anaboliques, Assimilation.
References Britten, R.J. & McCarthy, B.J. (1962), The synthesis of ribosomes in E. coli.-ll. Analysis of the kinetics of tracer incorporation in growing cells. Biophys. J., 2, 49~55. Britten, R.J. & McClure, P.T. (1962), The amino acid pool in E. coll. Bact. Rev.. 26, 292-335. Cooper, S. (1979), A unifying model for the GI period of prokaryotes and eukaryotes. Nature (Lond.), 280, 17-19. Dennis, P.P. & Bremer, H. (1974), Macromolecularcompositionduring steadystate growth of Escherichia coil B/r. J. Bact., 119, 270-281. Heldal, M., Norland, S. & Tumyr, O. (1985), X-ray microanalytic method for measurement of dry matter and elementalcontent of individualbacteria.Appl. environ. Microbiol., 50, 1251-1257. Ingraham, J.L., Maaloe, O. & Neidhardt, F.C. (1983), Growth of the bacterial cell. Sinauer Associates,Sunderland, Mass. Koch, A.L. (1979), Microbialgrowth in low concentration of nutrients, in "strategiesof microbial life in extreme environments" (M. shilo) (pp. 261-279), Verlag Chemie, Weinheim. Kubitschek, H.E. (1968), Constancy of uptake during the cell cycle in Escherichia coil Biophys. J., 8, 1401-1412.
C A T A B O L I C P O O L S I N ESCHERICHIA COLI Kubitschck, H.E. (1974), Constancy of the ratio of DNA to cell volume in steady-state cultures of E. coli B/r. Biophys. J., 14, 119-122. Kubitsehek, H.E. & Pai, S.R. (1988), Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth. Z Bact., 170, 431-435. Le Rudulier, D., Strom, A.R., Dandekar, A.N., Smith, L.T, & Valentine, R.C. (1984), Molecular biology of osmoregulation. Science, 224, 1064-1067. Mosteller, R.D., Goldstein, R.V. & Nishimoto, K.R. (1980), Metabolism of individual proteins in exponentially growing Escherichia coli. J. bioL Chem., 255, 2524-2532. Neidhardt, F.C. (1987), Chemical composition of Escherichia coli. in "'Escherichia coli and Salmonella typhimurium, Cellular and molecular biology" (F.C. Neidhardt et al.) (pp. 3-6). American Society for Microbiology, Washington, D.C. Pai, S.R. & Kubitschek, H.E. (1991), The phosphate pool in Escherichia coli THU, FEMS Microbiol. Letters, 81, 49-51. Pine, M.J. (1970), Steady-state measurement of the turnover of amino acid in the cellular protein of growing Escherichia coil: existence of two kinetically distinct reactions. J. Bact. 103, 207-215.
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Roberts, _~ B., Abelson, R.H., Cowie, D.B., Bolton, E.T. & Britten, R.J. (1955), Studies of biosynthesis in Escherichia coli. Publication 607, Carnegie Institute of Washington Publications, Washington, D.C. Schaechter, M.O., Maaloe, O. & Kjeldgaard, N.O. (1958), Dependency upon medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. gen. MicrobioL, 19, 592-606. Summerton, S.E. (1976), Measurements of the pool size and synthesis rate of the metabolically unstable fraction of RNA in Escherichio co!i by ~ method independent of hybridization efficiency and unaffected by precursor zompartmentation. J. tool Biol., 200, 127-140. Torriani-Gorini, A., Rothman, F.G., Silver, S., Wright, A. & Yagil, E. (1987), Phosphate metabolism and cellular regulation in microorganism. American Society for lqicrobio!ogy, Wasl-,mgton, D.C. Umbarger, H.E. (1977), A one-semester project for the immersion of graduate students in metabolic pathway.,. Biochem. Edu.-a:., 5, 67-71. Willetts, N.S. (1967), Intracellular protein breakdown in growing cells of Escherichia coli. Biochem. J,, 103, 462-466.
Res. Microbiol. 1992, 143, 173-181
Catabolic pools in Escherichia coli S.R. Pai(*) and H.E. Kubitschek Biological, Environmental and Medical Research Division, Argonne National Laboratory, Argonne, IL 60439-4833 (USA) SUMMARY
Methods are described for measuring soluble pool magnitudes in steady-state or exponentially growing cultures, and for distinguishing between anabolic, catabolic and total metabolic pools within cells. These methods were applied to the measurement of pool magnitudes for several amino acids and other precursors in Escherichia coli THU. Our results support the independence of the magnitudes of total metabolic pools and growth rate in steady-state cultures. Our results also show that the total metabolic pool size is much larger than previously published estimates, which failed to include the contribution of catabolic pools. The average value of the total soluble material in exponentialphase cells is estimated to be 8 to 9 % of the cell dry mass; pool values could be almost twice as large during midcycle because of the known increase in the magnitudes of protein and RNA precursor pool during the cell cycle.
Key-words: ~acterial growth, Escherichia coil; Metabolism, Nutrition, Anabolic and catabolic pools, Incorporation.
INTRODUCTION Cell dry mass in Escherichia coil consists mainly of macromolecular compounds that cannot be extracted with trichloroacetic acid, along with a much smaller fraction of soluble material located in metabolic pools (Roberts et aL, 1955; Umbarger, 1977; Ingraham et al., 1983; Neidhardt, 1987). In addition to ions, cofactors and vitamins, these metabolic pools consist of two major components, anabolic pools of chemical precursors newly transported into the cell or resident as soluble intermediates of synthesis, and catabolic pools, p~oducts arising from degradation of macromolecular compounds.
Earlier measurements of pool magnitudes provided estimates only of their anabolic components, as few, if any, studies determined the magnitudes of catabolic pools in E. coll. This was, perhaps, to be expected because, although several different kinds of experimental approaches were used, all were designed to examine precursor pool formation in the ab~c~ce of macromolecular degradation (Roberts et aL, 1955; Dennis and Bremer, 1974; Umbarger, 1977; Summerton, 1976; Ingraham et al., 1983 ; Neidhardt, 1987). The occurrence of catabolic pool~ in E. coli is amply supported by numerous studies of pro-
Submitted May 28, 1991, accepted August 2, 1991. (*) Correspondingauthor and presentaddress: Departmentof Pathobiology,Auburn University,Auburn, AL 36849-5519(USA).
174
S.R. P A l AND H.E. KUB1TSCHEK
teolytic degradation. Observed rates of degradation of individual proteins are initially low and diminish further with time. When a culture is pulse-treated with a radioactive amino acid, about 5 % of the radioactivity is released as free amino acid dering the first minute (Pine, 1970). This initial release may not constitute normal proteolysis, but a part of protdn synthesis or the failure to complete protein synthesis (A. L. Koch, 1979).
A~ = A / ( I - e - t / z ) , where t is the time after adding the label and x is the time for the cell population to increase by the factor e (Britten and McCarthy, 1962). In terms of the doubling time T, A ~t = A / ( 1 - 2 - t / T ) ) . The term in parentheses describes the relative accumulation of labelled material in the presence of total incorporated precursor, both labelled and unlabelled.
Degradation rates may ~hen become reduced to values as low as 0.5 to !.0 % per hour after several hours in glucose cultures (Pine, 1970; Willets, 1967) although individual proteins have widely different half-lives for degradation (Mosteller et el., 1980) and some degrade even more slowly. Nevertheless, total protein breakdown can still be significant; in chemostat-grown cultures labelled with valine, alr,:~ost 60 % of the label was released as acid-soluble components during a period of 3 days (Willets, 1967). This kind of evidence cle~.rly indicates the presence of catabolic pools, whose magnitude would also depend upon the efficiency of reincorporation of breakdown products into macromolecules.
Anabolic pools
We describe here experimental approaches that distinguish between anabolic and catabolic pools by using cultures labelled in the steadystate or experimental growth phase. In order to maintain steady growth after labelling~ the growth medium must first contain the unlabeiled precursor because de novo addition of even a very small amount of labelled precursor can qualitatively alter growth or transport kinetics (Kubitschek, ~968). Pool magnitudes are determined as the difference between paired culture samples: the first is washed with unlabelled growth medium to determine the total cellular rad--:.(.~activity, A, and the second is extracted with ice-cold trichloracetic acid before washing to determine the radioactivity of the pool is A - B and the fraction of the labelled precursor is (A - B)/A. Of greater interest, however, is the magnitude of the pool in terms of the total amount of precursor used by the cell after an indefinitely long labelling period. To good approximation, this maximal activity is given by
After addition of label, the radioactivity of the anabolic pool increases within a few minutes to a constant plateau value per cell (see for example Britten and McClure, 1962; fig. 3). As this time, the relative magnitude of the pool in terms of the total amount of precursor used by the cell is given by Pa = _A .-_.~B (1 --2-t/T). A
Catabolic pools
When the radioactivity of the anabolic pool reaches its maximum value, the rate of incorporation of precursor label into macromolecules also becomes maximum. The subsequent breakdown of labelled macromolecules then leads to an increase in the size of the labelled catabolic pool, which, in turn, might require several generations to reach its saturation value, Pc. Measurement of the metabolic pool at that time provides the sum Pt of the magnitudes of thc anabolic and catabolic pools, Pt = Pa + Pc, when these pools are formed independently. The magnitude of the catabolic pool can then be determined by the difference between the values of the total metabolic pool and the anabolic pool. Alternatively, the magnitude of the catabolic pool can be determined directly by labelling a culture for several generations and then washing the culture free of the extracellular label. The residual anabolic pool is rapidly used, after which the measured values then correspond to those for the catabolic pool. The catabolic pool then diminishes only very slowly, at a rate of the order of 1% per ho~r. Furthermore, this slow decrease in the magnitude of the catabolic pool is matched by the decrease in the labelled macromolecular fraction. Thus, the magnitude of the catabolic pool is given to good approximation by Pc = (A - B)/A. The criterion that Pa and Pt have reached their saturation values is provided by the constancy of these values during the experiment.
CATABOLIC POOLS IN ESCHERICHIA COLI In this paper, we report the use of these approaches to determine the magnitudes of some total metabolic and catabolic pools in E. coli. la a few ca~e.% magnitudes of anabolic pools were determh~ed for comparison.
MATERIALS AND METHODS Culture conditions E. coli 15 THU (thy his ura), CGSC strain 4908, was used because earlier studies with this strain had already provided accurate measurements of anabolie pool sizes for several amino acids. Cultures (30 ml) were grown to exponential phase in Erlenmyer flasks under conditions described earlier (Kubitschek and Pal, 1988), except that sucrose usually was omitted from the growth medium. In addition, growth was always carried out in the presence of an unlabelled precursor added to a concentration of 5 to 20 ~tg/ml. The average doubling time was 40 min. For some measurements of phosphorous and uracil pools, cultures were grown in nutrient both (Difco) or Casamino acids were added to 0 . 1 % to provide rapidly growing cultures. Growth rates also were sometimes increased, less markedly, by addition of methionine to a concentration of 40 ~g/ml. Occasionally, an inefficiently utilized carbon source such as glycerol was used to provide a very slowly growing culture. Growth was monitored as the increase in turbidity observed with a "Klett-Summersoa" colorimeter with a "Kodak 66" filter, or occasionally, as the increase in cell count as measured with a "Coulter" counter. Experiments were carried out with exponential-phase cultures inoculated the previous day.
Radioactive materials
H ,~32po4, 207 Ci/mg P; H232SO4, 43 Ci/mg S; L-(U-~'C)-glutamine, 200 mCi/mmol, were all from ICN Radiochemicals, Irvine, CA. (2-~4C)-uracil, 50 mCi/mmol; L-(U-TC)-histidine, 336 mCi/mmol, were from Amersham, Arlington Hts., IL. L-(U-laC)-valine, 250 mCi/mmol, was from Becton-Dickinson, Oxnard, CA. D-(U-14C)-glucose, 14.4 mCi/mmol; (U-t4C)glycine, 113 mCi/mmol; L-(35S)-methionine, 800 Ci/mmol; L-(U-t4C)-proline, 1 mCi/mg, were from New England Nuclear, Boston, MA.
175
Labelling and ~o_moi|ng Cultures were always labelled durin~ exponential phase growth. Labelled precursors were added to concentrations of 0.01 to 0.I ~tCi/mL Labelling and sampling protocols depended upon the kind of pool under study. For anabolic pools, radioactivity was added when cultures reached 1.2 x l0 s cells/ml (10 Klett units). After about 10 min, 0.1-mi samples were removed at 5-min intervals for a period of 30 min to determine total and macromolecular cellular radioactivity. Earlier e×l~eriments had shown that anabolic pools in our cultures became saturated within approximately 10 min (Kubitschek and Pai, 1988). To produce more fully labelled metabolic pools, cultures were labelled at lov,er call concentrations (1-2 × 107/ml) for periods of about 3 to 4 generations, providing cell activities that were 90 % or more of their maximum values. Samples were removed at intervals of 15 to 30 rain. The continued presence of the label assured that the anabolic pool remained fully labelled. Cells with labelled catabolic pools and unlabetled anabolic pools were prepared by washing and resuspending cultures in unlabelled growth medium following determination of the total metabolic pools through the method just described. Cultures were filtered over a nitrocellulose membrane filter, immediately washed with unlabelled medium as described earlier (Kubitschek and Pai, 1988) and suspended to volume.
Cell and pool radioactivity Culture samples (0.1 ml) were always taken in pairs. As described earlier in detail (Kubitschek and Pai, 1988), samples were washed in unlabelled growth medium to determine total cell radioactivity, or first extracted with ice-cold 10 % trichloroacetic acid and then washed to determine radioactivity incorporated into the macromolecular fraction. The filters bearing the washed samples were dropped into vials containing a scintillation cocktail (Opti-fluor; Packard Instruments Co.) and counted in a liquid scintillation counter (Beckman LS-333). There was no ewaence for quenching of counting rates by residual trichloroacetic acid.
RESULTS AND DISCUSSION Figure 1 is representative o f the kinds o f data provided by our experiments for total m e t a b o lic a n d for catabolic pools. A t time zero, ~4Cproline was a d d e d to an overnight culture con-
176
S.R. PAl AND H.E. KUBITSCHEK
A
~ooo
~
C o.ooo ~
©oo
~
k
~
i •
II-- ;
~ ";" ~
~ooo
o+ ~oo
°
'Imo o,,~
Fig. 1. Uptake of ~4C-proline in an exponentially growing culture of E. coli THU. Labelled proline (1 ~tCi/ml) was added at time zero to a culture containing unlabelled proline (20 v.g/ml). Later, at the time shown by the vertical dashed line, unincorporated labei was removed by washing and resuspending the cells in the same unlabelled growth medium. Panel A, culture turbidity in Klett units. Panel B, relative cell count observed with the Coulter counter.
tairfing unlabelled proline (20 t~g/ml). The figure shows that both turbidity (A) and the relative cell count (B) increased exponentially and with the same doubling time (40 min) both before and after removal of extracellular radioactivity at the time shown by the vertical dashed line. This agreement between turbidity and cell numbers strongly supports the exponential growth of the culture, and the continued agreement after washing and resuspension shows that those operations had little if any effect upon the growth of the culture. As expected in figure 2, culture total radioactivity (C) increased more rapidly, as it must because steady-state conditions had not yet been obtained for uptake of label (see "Introduc-
~
~mo(h,~4
5
6
Fig. 2. Experimental conditions are given in the legend to figure 1. Panel C, increase in cell total radioactivity. Panel D, increase in culture pool radioactivity.
tion"). After removal of the extracellular radioactivity by washing and resuspen.~;.on, e,ctivities in this experiment remained reiatively constant, although in many other experiments these values were seen to decrease very slowly with time. The values for pool radioactivity (D) also increased before washing, in the same manner a~ the total radioactivity, and then remained relatively constant afterwards. The larger errors associated with the measurements of pool radioactivity reflect the fact that these data points are determined from small differences between the much larger values observed for total cell activity and macromolecular incorporation. Relative pool magnitudes were calculated as a percentage of the total amount of precursor used by the cell from the values in C and D (fig. 3) after correction for finite labelling periods. The horizontal lines are the average values obtained for the total metabolic pool (before
CATABOLIC POOLS IN ESCHERICHIA COLI
177
Table !. Dependence of relative pool size upon culture doubling time. Observed values of pool sizes are presented as percentage of the total amount of precursor material utilized by the cell.
Precursor Uracil
Doubling time (min)
No. of experiments
°7o Pool size _+ SE
23-27 42-47 108
4 5 1
6.6±0.6 6.7_+0.2 6.8
10
6.7_+0.2
3 4 5
16.3 __.0.8 15.0+0.8 14.7 _+0.6
12
15.23 0.4
Average Phosphate
35-39 41-49 50-54
Average
o
1
~
~
~
~
~
Timo (hrs)
Fig. 3. Pool radioactivity as a percentage of the total cell radioactivity for the same culture as that in figure I. Experimental conditions are given in the legend to figure 1. washing) and the catabolic pool (after washing), with values ( +_ std. error) o f 5.5 ( _+ 0.5) 070 and 4.1 ( + 0.3)070, respectively. The relatively a b r u p t transition between the labelling of the total pool and the catabolic pool in this experiment indicates rapid utilization of the precursor in the anabolic pool.
ditions, there was the question of the degree to which culture growth rate itself might affect pool size. For this ~e~son, we measured the relative magnitudes of total steady-state metabolic pools formed at different doubling times with uracil or with phosphate as precursors. The observed values, table I, show that there were no significant differences for either uracil or phosphate between the average value for all observations and those for fast, medium, and slow cultures. Th~tt is, there results do not support any variation in relative pool size with growth rate for either pool and they indicate that growth rate could have had, a~ most, a very minor effect upon pool size. Absolute magnitudes would, of course, increase with cell size.
Total metabolic pools P o o l size vs. growth rate In their original studies o f a m i n o acid pools in non-steady-state cultures of E. coli, Britten and McClure (1962) observed that pool sizes were dependent upon culture conditions and were sensitive to osmotic effects of various solvents. Although our steady-state cultures were not exposed to transient changes in culture con-
Average values for total metabolic pool sizes of several a m i n c acids and other precursors are presented in table 1I. Observed values for amino acid pools ranged from 3.0 to 5.8 070, with an ave~-a~,e value of 4.6 °7o. Metabolic pools for uracil and for glucose were even larger, 6.7 and 8.7 070,respectively. The largest pools were those for phosphate and sulphate, with values of approximately 15 and 27 070, respectively.
178
S.R. P A l A N D H.E. KUBITSCHEK
Table
Glucose Uracil Phosphate Sulphate Amino acids Glm Gly His Pro Met Val
!!. Metabolic, catabolic and anabolic pools in E. coil THU. N
Total pool (070)
N
Catabolic pool (%)
N
5 I0 12 3
8.7+0.9 6.7+0.2 15.2±0.4 26.7 _+0.3
8 3 3 --
5.8-e 1.0 3.0±0.5 8.8___0.4 --
2 --
5 6 6 7 4 5
5.8±0.4 5.0±0.7 5.1 ±0.5 5.6±0.2 3.4+0.5 3.0±0.5
6 a 4 4
5.9±0.6 1.2__.0.7 4.6:!:0.2 3.1 ±0.4 -2.1±0.6
~-
2 --
----
1 ~-
Anabolic pool (070) 1.8_+0.6 2.7 (*) 14.4_+ 1.9 __
__
2.2-~'~ 5.4 3.3 ('1 0.8+0.5
N = number of experiments. (*) Values from Kubitschek and Pal (1988).
TaMe III. Elemental contributions to metabolic pools in E. colt TltU. Eletnent
Dry wt fraction
C P S O, N, H
0.47 0.05 0.01 0.43
Total
Element Soluble mass pool ,size (07o) fraction (07o) 8.7 15 27 2.5 t°)
4.1 0.8 0.3 1.1 6.3 ('*l
Dry weigh~ data from table I in Heldal et aL (1985). Values for P and S were calculated from their table I1. (*) Calculated from the average pool size observed for amino acids, 4.6 %, assuming 55 % of the cell dry weight is protein (Ingraham et aL, 1983; Neidhardt, 1987). (**) This value does not include ~he soluble pools for nucleic acids, inorganic ions or cofaclors.
The values given in table I for the total metabolic pools can provide an estimate of the total soluble pool in E. colt. For this purpose, we have used the element dry weight fractions for E. colt of Heldal et al. (1985). As shown in table III, the dry weight fraction for elemental carbon is 0.47, so the observed elemental pool size (for glucose in table II) corresponds to a soluble C pool fraction of
4.1 07o of the cell dry weight. Cell dry weight fractions for P and S were calculated from table II in Heldal et aL (1985); together, phosphate and sulphate pools add another 1.1 07o.The contribution from O, N and H in the amino acid pools (4.6 07o average magnitude) also adds 1.1 07o assuming that 55 07o of the cell dry weight is protein (1,2). If we include the pool size of 1 07o for the inorganic ions and cofactors estimated by Ingraham et aL (1983), then the total soluble pool in E. colt is at least 7.3 07o of the total dry weight. This estimate of pool size, however, neglects the contributions of O, N and H to the soluble pools of the nucleic acid precursors, as well as the contribution of soluble pools for other macromolecules such as lipopolysaccharide and peptidoglycan. These contributions are expected to increase the total soluble pool to more than 8 07o. But these estimates also neglect the variation of pool size with cell age during the cell cycle. Earlier we found that pool sizes were small or negligible at the beginning and at the end of the cycle, at least for protein and R N A precursors, and that the midcycle pool was therefore almost twice as large as the average pool size during the cycle (Kubitschek and Pat, 1988). Those results imply that soluble pools in E. colt
CA TABOLIC POOLS I N ESCHERICHIA COLI
have a maximum value during the cycle approximately 50 % larger when the contributions of unregulated pools (see below) are taken into account. These results show that the pools in E. coil are much larger than earlier estimates. For example, Umbarger (1977) calculated the sum of the soluble pools of amino acid and nucleic acid precursors to be less than 1 % of the cell dry weight. The latest estimate by Neidhardt (1987) raised this value to 2.5 %. Neither of these estimates, however, included the contributions of the catabolic pools, which were not measured by the experimental procedures. Furthermore, even those (anabolic) pool calculations were based upon non-steady-state determinations, which may not have been fully representative of steadystate pool magnitudes.
Catabolic and anabolic pools The observed magnitudes of catabolic pools are also presented in table II. These values were smaller than those for total pools, with the exception of glutamine, for which catabolic and total pools were the same within statistical errors. This equivalence indicates the failure of the sum rule proposed earlier, Pt = Pa + Pc" This inequality was unanticipated and suggested that the sum rule does not apply to all precursor pools. For this reason, we also determined anabo!ic pool sizes for some of the other precursors, as shown in table II. Most of the anabolic pool values also were smaller than those for the corresponding total pools, but there were two cases where the magnitudes of the anabolic and the total pools were not significantly different, for phosphate and proline. The case for phosphate was especially compelling because of the large value for the soluble fraction. The failure to support pool additivity suggests that there are two different classes of pools (1) those for which the precursors pass through the pool with little or no interaction with cell produets other than the macromolecular reactions they are destined to supply and (2) pools for
179
which the magnitudes are regulated between narrow size limits. This regulation may prevent pool magnitudes from exceeding their anabolic levels, as for glutamine or, alternatively, from exceeding their catabolic levels, as for phosphorus or proline. Recently, we obtained further support for this interpretation by examining the magnitude of the phosphorus pool as a function of cell age during the division cycle. We found that the ratio of soluble to total cell phosphorous was constant during the cell cycle, and therefore independent of cell age or size (Pal and Kubitschek, 1991). These results require that the phosphorous pool per unit cell mass be maintained constant within narrow limits. In conclusion, we have provided methods for measuring magnitudes of soluble pools in cells in steady-state cultures and for distinguishing among anabolic, catabolic and total matabolic pools. These methods also distinguish between two kinds of soluble pools. For one class, unregulated pools, the magnitudes of total soluble metabolic pools were observed to equal the sum of those for their anabolic and catabolic components, i.e., the pool sum rule is obeyed.
For the second class, however, pool size was regulated within narrow limits, with the result that the total metabolic pool was no larger than its anabolic or catabolic component. Equivalence between the size of the total pool and the anabolic pool was observed with either phosphate or proline as a precursor. Evidence for manifold processes of phosphate regulation in microorganisms is well-documented (Torriani-Gorini et al., 1987) and our observation of the ageindependence of the soluble pool fraction is not inconsistent with the role of phosphate in the regulation of many other cell processes. The regulatory behaviour of the proline as an osmoprotectant in E. coli (Le Rudulier et al., 1984). The apparent equivalence between catabolic and total pool values for glutamine might be related to the involvement of glutamine in the regtllation of nitrogen utiliz,~uon by glutamine synthetase. Our observations that uracil and phosphate precursor pool size~ were independent of growth
180
S.R. P A l A N D H.E. K U B I T S C H E K
rate were unexpected becau.~e RNA is known to increase with growth rate more rapidly than cell dry weight in E. coil (Dennis and Bremer, 1974). Our results indicate, nevertheless, that relative pool s~:e fractions are independent o f growth rate both for metabolically regulated pools (phosphorous) and for those for which there is no evidence o f regulation (uracil). Because different growth rates were obtained by using a variety o f different media, these results indicate that relative soluble pool magnitudes do not depend upon the composition o f the medium. The results for uracil are especially compelling here because the large range o f growth rates with this precursor were obtained by growing cultures in very different kinds o f media, from r~utrient broth to glycerol minimal medium. Furthermore, because mean cell size increases with growth rate (Schaechter et aL, 1958; Kubitschek, 1974), relative soluble pool magnitudes are independent, or nearly so, o f cell volume. The finding that total pool size in E. coil is much larger thap therefore determined, is of special significance. This result is consistent with a much greater role for soluble pools than has been apparent from earlier studies. As neither the anabolic or catabolic pools can be neglected, it is clear that the organization o f the cell depends strongly upon the contribution o f both components, and any model o f cell growth or its chemical composition must take into account the contribution o f both pools. Thus, these results indicate that previous models based only upon the increase in macromolecular incorporation during the division cycle, as for example the " C o n t i n u m M o d e l " (Cooper, 1979), are incomplete because they fail to include the behaviour o f pool fractions that are now known to be too large to be neglected.
Acknowledgments This work was supported by Public Health Service grant ROI A121954 from the National Institutesof Health and by the US Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38.
Masses cataboliques dans les cellules de Escherlchla coil Des m6thodes sont d~crites, permettant de mesurer les fractions solubles dans les cultures en fonction de la courbe de croissance et de distinguer les masses cataboliques, anaboliques et totales dans les cellules bact6riennes. Ces m6thodes sont appliqu6es pour mesurer les quantit6s d'acides amines divers et d'autres m~tabolites chez Escherichia coll. On observe que les masses totales des m6tabolites et le taux de croissance sont ind6pendants au cours de la phase stationnaire. De plus, la masse totale appara~t plus importante que les estimations ant~rieures le montraient, lesquelles ne tenaient pas compte de la contribution des masses cataboliques. La valeur moyenne du mat6riel soluble total ~t la phase exponemielle de la croissance est 6valu~e ~ 8-9 ~/a du poids cellulaire see; enfin les valeurs des masses m6taboliques peuvent ~tre deux fois plus importante au milieu de la phase de croissance 6tant donn6 l'augmentation 6vidente des masses de prot6ines et d'ARN au cours du cycle cellulaire. Mots-clds: Croissance bact~rienne, Escherichia coil; M6taboiisme, Nutrition, Masses cataboliques et anaboliques, Assimilation.
References Britten, R.J. & McCarthy, B.J. (1962), The synthesis of ribosomes in E. coli.-ll. Analysis of the kinetics of tracer incorporation in growing cells. Biophys. J., 2, 49~55. Britten, R.J. & McClure, P.T. (1962), The amino acid pool in E. coll. Bact. Rev.. 26, 292-335. Cooper, S. (1979), A unifying model for the GI period of prokaryotes and eukaryotes. Nature (Lond.), 280, 17-19. Dennis, P.P. & Bremer, H. (1974), Macromolecularcompositionduring steadystate growth of Escherichia coil B/r. J. Bact., 119, 270-281. Heldal, M., Norland, S. & Tumyr, O. (1985), X-ray microanalytic method for measurement of dry matter and elementalcontent of individualbacteria.Appl. environ. Microbiol., 50, 1251-1257. Ingraham, J.L., Maaloe, O. & Neidhardt, F.C. (1983), Growth of the bacterial cell. Sinauer Associates,Sunderland, Mass. Koch, A.L. (1979), Microbialgrowth in low concentration of nutrients, in "strategiesof microbial life in extreme environments" (M. shilo) (pp. 261-279), Verlag Chemie, Weinheim. Kubitschek, H.E. (1968), Constancy of uptake during the cell cycle in Escherichia coil Biophys. J., 8, 1401-1412.
C A T A B O L I C P O O L S I N ESCHERICHIA COLI Kubitschck, H.E. (1974), Constancy of the ratio of DNA to cell volume in steady-state cultures of E. coli B/r. Biophys. J., 14, 119-122. Kubitsehek, H.E. & Pai, S.R. (1988), Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth. Z Bact., 170, 431-435. Le Rudulier, D., Strom, A.R., Dandekar, A.N., Smith, L.T, & Valentine, R.C. (1984), Molecular biology of osmoregulation. Science, 224, 1064-1067. Mosteller, R.D., Goldstein, R.V. & Nishimoto, K.R. (1980), Metabolism of individual proteins in exponentially growing Escherichia coli. J. bioL Chem., 255, 2524-2532. Neidhardt, F.C. (1987), Chemical composition of Escherichia coli. in "'Escherichia coli and Salmonella typhimurium, Cellular and molecular biology" (F.C. Neidhardt et al.) (pp. 3-6). American Society for Microbiology, Washington, D.C. Pai, S.R. & Kubitschek, H.E. (1991), The phosphate pool in Escherichia coli THU, FEMS Microbiol. Letters, 81, 49-51. Pine, M.J. (1970), Steady-state measurement of the turnover of amino acid in the cellular protein of growing Escherichia coil: existence of two kinetically distinct reactions. J. Bact. 103, 207-215.
181
Roberts, _~ B., Abelson, R.H., Cowie, D.B., Bolton, E.T. & Britten, R.J. (1955), Studies of biosynthesis in Escherichia coli. Publication 607, Carnegie Institute of Washington Publications, Washington, D.C. Schaechter, M.O., Maaloe, O. & Kjeldgaard, N.O. (1958), Dependency upon medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. gen. MicrobioL, 19, 592-606. Summerton, S.E. (1976), Measurements of the pool size and synthesis rate of the metabolically unstable fraction of RNA in Escherichio co!i by ~ method independent of hybridization efficiency and unaffected by precursor zompartmentation. J. tool Biol., 200, 127-140. Torriani-Gorini, A., Rothman, F.G., Silver, S., Wright, A. & Yagil, E. (1987), Phosphate metabolism and cellular regulation in microorganism. American Society for lqicrobio!ogy, Wasl-,mgton, D.C. Umbarger, H.E. (1977), A one-semester project for the immersion of graduate students in metabolic pathway.,. Biochem. Edu.-a:., 5, 67-71. Willetts, N.S. (1967), Intracellular protein breakdown in growing cells of Escherichia coli. Biochem. J,, 103, 462-466.
