Cell. Vol. 18, 287-296,

October

1979,

Copyright

0 1979

by MIT

Regulation of the Synthesis Cell Cycle of E. coli B/r

of Surface

A. Boyd* Department University University Leicester

Trentini, 1966). Individual cell mass is thought to be produced exponentially during balanced growth, since the rates of synthesis of stable RNA and protein increase continuously throughout the cell cycle (Ecker and Kokaisl, 1969; Dennis, 1971; Churchward and Holland, 1976). In contrast, it has been claimed that the increase in bacterial length and volume proceeds at a constant rate which doubles at some point in the cell cycle, as measured in synchronously growing cultures (Kubitschek, 1968; Ward and Glaser, 1971; Donachie, Begg and Vicente, 1976); this mode of growth is referred to as a linear pattern. A linear pattern of cell elongation has also been inferred from both an analysis of length distributions in exponentially growing cultures (Cullum and Vincente, 1978) and cell age-dependent variations in density which are consistent with a linear increase in volume coupled with exponential accumulation of mass (Poole, 1977). As proposed previously by several investigators, any linear increase in length and volume of an elongating rod-shaped cell must arise because the cell surface, or a major component of the surface, is laid down at a constant rate (Previc, 1970; Pritchard, 1974; Pierrucci, 1978; Rosenberger et al., 1978). The surface or envelope of E. coli is a complex threelayered structure consisting of an inner or cytoplasmic membrane, a rigid layer of murein or peptidoglycan and an outer membrane at the cell surface (reviewed by Inouye, 1975). Since the rigid peptidoglycan layer or sacculus determines cell surface area and volume, this layer is the best candidate for linear growth, and there is some evidence to support this idea (Hoffman, Messer and Schwarz, 1972; Koppes, Overbeeke and Nanninga, 1978). We believe, however, that a definitive study of peptidoglycan growth remains to be carried out. An important point which must be borne in mind when considering exponential and linear patterns of growth for components of a single cell is that the relative amounts of two such components will only fluctuate between narrow limits; the theoretical maximum variation in relative amounts is only 6%. There would therefore seem to be no compelling reason why the plastic membrane layers of the cell envelope should be constrained to grow in step with the peptidoglycan sacculus. Nevertheless, Churchward and Holland (1976) reported that protein accumulates linearly in the whole envelope fraction of E. coli during the cell cycle, and similar results have been described for Bacillus subtilis (Sargent, 1975) and Rhodopseudomonas sphaeroides (Fraley, Leuking and Kaplan, 1977). In eucaryotes, measurements of surface growth are usually restricted to the accumulation of surface components over the cell cycle rather than to their actual rates of synthesis. Some of these studies indicate that, as in bacteria, plasma membrane proteins [for example, in mouse cells in culture (Pasternak et al., 1974) and several cytochromes in S. cerevisiae

and I. 6. Holland of Genetics of Leicester Road LEl 7RH, England

Summary We have studied the biogenesis of the envelope of E. coli B/r by measuring the synthesis of protein in separated inner and outer membranes during the cell cycle. While total protein and bulk inner membrane protein were synthesized continuously and at an exponentially increasing rate throughout the cycle, bulk outer membrane protein was synthesized at a constant rate throughout the cycle with an abrupt doubling in rate occurring lo-15 min before division. A similar pattern was observed when the rate of synthesis of an individual protein, the 36SK outer membrane protein, was measured directly in total cell lysates. Neither thymine starvation nor changes in gene dosage of exponential cultures affected the synthesis of outer membrane protein, indicating that the doubling in rate is not controlled by a gene duplication mechanism. Other findings, however, further indicate that outer membrane protein synthesis is regulated in some way. Thus the concentration of 36.5K porin per unit surface area remained constant as the surface area/volume ratio varied widely with growth rate. We also obtained direct evidence for an overall limitation on the rate of synthesis of bulk outer membrane proteins; when a new class of outer membrane proteins was induced, the rate of synthesis of other surface proteins was correspondingly reduced. On the basis of these results, we discuss a model in which the linear growth of outer membrane protein results from a limitation of outer membrane polypeptide synthesis at the translational level, reflecting the linear expansion of the underlying peptidoglycan layer in the envelope. Introduction We have sought to characterize some aspects of surface growth in E. coli, with the expectation that knowledge of the pattern of growth of the cell envelope in the cell cycle will provide a basis for an understanding of bacterial growth and may, in particular, enable identification of those growth processes that must be modulated during septation and cell division. E. coli cells are cylindrical rods with rounded ends; at any one growth rate these rods have a fixed diameter and grow solely by elongation (Marr, Harvey and * Present address: Department of Biology 6-022. fornia at San Diego, La Jolla. California 92093.

University

of Cali-

Protein in the

Cell 268

(Cottrell et al., 1975)] all accumulate continuously over the cell cycle. Other studies, however, indicate that in Tetrahymena or in animal cells in culture, particular phases of the cycle are associated with high rates of synthesis or even with the specific appearance of certain proteins in the plasma membrane (reviewed by Bluemink and de Laat, 1977). In this paper we report that the linear accumulation of cell envelope protein in E. coli B/r is, in fact, restricted specifically to the outer membrane fraction. Moreover, direct measurement of the synthesis of a major surface protein demonstrated that the rate of synthesis (not merely accumulation in the envelope) is also linear. We present evidence which indicates that the pattern of synthesis of outer membrane protein may be due to an overall limitation on the capacity of the bacterial cell for synthesis of this class of protein. We also report that the cell cycle pattern of synthesis appears to be unrelated to the DNA replication cycle.

ber should fall rapidly and the cycle should then repeat, as shown in Figures 1 and 3. Each eluted bacterium should contain half the radioactivity of its parent, and therefore the radioactivity per cell in the eluted fractions is expected to decay exponentially (provided the molecule is not appreciably turned over), with a slope equal to the generation time, in molecules that were synthesized at an exponentially increasing rate in the culture initially labeled. Similarly, molecules synthesized linearly during the cell cycle, with a step-

Results Synthesis of Outer Membrane Protein in the Cell Cycle The method used to study the bacterial cell cycle was the filter elution technique of Helmstetter (1967). A bacterial culture in balanced growth is pulse-labeled with a radioactive precursor of the macromolecule of interest and the labeled culture is fractionated according to the age of cells at the time of labeling, as described below. The amount of radioactivity per bacterium in each age fraction is then taken as a measure of the rate of macromolecular synthesis at that particular cell age. It is important to note that all manipulations of the culture occur after radioactive labeling is completed; the procedure does not involve the generation of synchronous cultures. Age fractionation is achieved as follows. Pulse-labeled bacteria are bound to a nitrocellulose filter which is then inverted and eluted with a continuous flow of fresh medium. The bacteria continue to grow and divide on the filter. When a filter-bound cell divides, it sheds a daughter cell into the eluate; those bacteria that are about to divide when labeled and bound to the filter will almost immediately contribute a daughter to the eluate, while bacteria that were newborn in the original culture will shed a daughter after one generation has elapsed. Thus the earliest eluate fractions correspond to points late in the cell cycle, and subsequent fractions correspond to progressively earlier points in the cycle. Since in an exponentially growing culture of bacteria there are twice as many newborn cells as there are cells about to divide (Powell, 1956), the number of bacteria appearing in the eluate should increase and reach a maximum after one generation, whereupon the num-

L. ___1_ e. . .

.

l

.*

.

l

l

0

.

I.Mern. 0 . . l

e

.

0

Figure 1. Membrane Protein Synthesis in the Cell Cycle E. coli LEBl6 growing exponentially in proline/alanine medium (+20 pg/ml thymine) was pulse-labeled with “C-leucine for 5 min and fractionated on an age basis by the filter elution technique. Cell number and acid-precipitable radioactivity were determined in various cell fractions in each eluted sample. For membrane fractions the data were presented as a ratio of “C/3H. where 3H is derived from the 3H-leucine-labeled culture, added as internal standard before the preparation of the membranes. Cell age increases from right to left in the figure. The bacterial number profile deviates considerably from the theoretical age distribution curve (Powell. 1956), presumably because of the variation in individual cell doubling times (Schaechter et al., 1962.).

Surface 289

Growth

in E. coli

wise increase in rate, will show a step-wise decay in the elution profile. Figure 1 shows the results of a filter elution experiment in which the rates of synthesis of cell protein fractions were measured during the cell cycle of E. coli B/r LEB16. The data in Figure 1 clearly show a continuous increase in the rate of synthesis of total cell protein with cell age, as reported previously (Dennis and Young, 1975; Churchward and Holland, 1976). The envelope fraction was separated into inner and outer membranes and the rate of synthesis of the cytoplasmic membrane protein fraction was also found to increase continuously with cell age. In contrast, the rate of synthesis of outer membrane protein (3-5% of total protein) remained constant throughout the cycle, with an abrupt increase in rate occurring late in the cycle, close to the time of termination of DNA replication under these conditions. The timing of the latter was deduced from the data of Meacock and Pritchard (1975), who used the same strain and culture conditions used here. The rate of synthesis of outer membrane protein relative to that of cytoplasmic membrane protein is also plotted in the figure, and displays an age-dependent variation which confirms the interpretation of the rate data. To extend this observation of a linear accumulation of outer membrane protein, a further experiment was performed to determine the cell age-dependence of the actual rate of synthesis of an individual outer membrane protein rather than its mere appearance in the envelope. The protein chosen was the major surface porin, molecular weight 36.5K (Rosenbusch, 1974; Bavoil, Nikaido and von Meyenberg, 1977). Measurement of the rate of synthesis was achieved by cutting out and counting the radioactivity in the cognate band from an SDS-PAGE analysis of total cell lysates prepared from eluate fractions. As shown in Figure 2, this polypeptide forms the most heavily staining band in a gel analysis of total cell protein of E. coli LEB16 grown in proline-alanine medium. The 36.5K porin is present in about 1 O5 copies per cell, is located exclusively in the outer membrane and constitutes 35% of total outer membrane protein in proline-alanine-grown cells (our unpublished data). From analysis of total cell lysates of mutant strains lacking this protein (lyer, Darby and Holland, 19771, we conclude that at least 70% of the material in the 36.5K gel band is specific to the 36.5K porin. Another major band analyzed in this experiment is formed by the cytoplasmic protein, elongation factor (EF)-Tu, molecular weight 44K. There are about 7 X lo4 copies of EF-TU per cell in slowly growing E. coli B/r (Pedersen et al., 1978), so it seems probable that most of the material in the 44K gel band is specific to EF-Tu. The data shown in Figure 3 demonstrate that the rate of synthesis of EF-TU increased continuously during the cycle, in parallel with total cell protein; in

i

4’

60

, Pi-

57

OM

slot: Figure

2. Stained

i Gel of SDS Lysates

Cultures of E. coli LEB18 were lysed 11% polyacrylamide gel. The major lysates are indicated on the left, and of standard proteins and major lysate membrane fraction from E. coli LEBl8 position of the major 36SK porin.

2

3

of E. coli B/r with SDS and analyzed in an identifiable proteins in whole the molecular weight (x 1 Om3) proteins on the sides. An outer is also included to indicate the

contrast, the 36.5K porin was synthesized at a constant rate which doubled 10 min before division. This interpretation of the rate measurements is once again confirmed by the plot of relative rate of synthesis (36.5K/44K). Our calculations indicate that the linear synthesis of the 36.5K protein, which constitutes about one third of total outer membrane protein synthesis, would not be sufficient to generate the linear profile shown in Figure 1 if other proteins were synthesized exponentially. In addition, we reported previously (Churchward and Holland, 1976) that other individual outer membrane proteins were synthesized linearly. All these results therefore suggest that the majority of proteins of the outer membrane (but not of the cytoplasmic membrane) are synthesized at a constant rate in the

Cell 290

oddition

01

dipyridyl

Total protein

d’

365K

Pain

-I

v-Tu

.a

H .--‘*a- . 1 w Figure Cycle

3. Rates

of Synthesis

of Individual

Polypeptides

‘d

in the Cell

Samples fractionated by age were prepared and analyzed as in Figure 1. In addition, the rate of synthesis of elongation factor EF-TU and the 365K porin was determined by cutting out the corresponding gel band after analysis of whole cell lysates by SDS-PAGE and counting the rates of ‘%-leucine (pulse label) to ‘H-leucine (internal standard). The data are presented as a relative rate as in Figure 1. The arrow in the middle curve indicates the midpoint of the step in rate of synthesis of the 36.5K porin; the lower arrows indicate the average time of division (on left) and termination of DNA replication (right) in the cell cycle measured from the half heights of the fall in the cell number curve and rate of DNA synthesistfrom Meacock and Pritchard, 1975). respectively.

.f

/9

/

cell cycle, which doubles approximately 10 min before division, close to termination of DNA replication. Limitation on Overall Synthesis of Outer Membrane Protein A hypothesis that can explain the cell cycle pattern of synthesis of outer membrane protein is that there is some factor uniquely involved in the synthesis of these proteins whose availability limits overall synthesis. In this view, the cell cycle doubling in rate would merely reflect a discrete doubling in the availability of this limiting factor. To test this hypothesis the synthesis of a new class of outer membrane proteins was induced and the effects of this induction on the rates of synthesis of bulk outer membrane protein and the 36.5K porin were measured. The predictions of a hypothesis of overall limitation are that the rate of synthesis of bulk outer membrane protein should not increase

,/ 0 Figure

20 4. Effects

LO time lminutcsl of Dipyridyl on Outer Membrane

Protein

Synthesis

An exponentially growing culture of LEE1 6 was pulse-labeled for 2 min at intervals with %-methionine; at an A,= = 0.15 dipyridyl (final concentration 50 FM) was added and pulse-labeling continued. Samples were mixed with a constant amount of ‘H-leucine-labeled bacteria grown in dipyridyl and the relative rates of synthesis (35S/3H) were determined in various cell fractions or in specific gel bands after SDS-PAGE. (a) 61K protein (fed protein) in the outer membrane; (b) 36.5K protein in total cell lysates; (c) 36.5K protein in outer membrane preparation: fd) total outer membrane protein; (e) total cytoplasmic membrane protein; tf) total cell protein; (g) mass (A.&.

under these conditions, but that the rate of synthesis of other outer membrane proteins should be correspondingly reduced as the new class of proteins com-

Surface 291

Growth

in E. coli

petes for a share of the limiting factor. In the experiment summarized in Figure 4, induction of outer membrane proteins was achieved by use of the compound cY,a’-dipyridyl (DP), which chelates ferric ions and thus reduces the amount of available iron in growth medium. E. coli responds to this type of iron deficit with an increase in the rate of synthesis of a group of outer membrane proteins involved in iron uptake; these proteins are the fed protein (81 K), the tonA protein (76K) and the cir protein (74K) (Hancock, Hankte and Braun, 1976). The addition of DP (50 FM) had a brief effect on growth after which the former rate was rapidly resumed, and this effect was reflected in the rate of synthesis of total protein in the culture. Data relating to synthesis of bulk membrane fractions, the 36.5K porin and the fed protein are plotted as rates of synthesis relative to that of total cell protein. The relative rate of synthesis of the fed protein, measured in outer membrane fractions, was increased 14 fold by DP treatment. In contrast, the treatment reduced the relative rate of synthesis of the 36.5K porin measured in both outer membrane fractions and cell lysates; as before, the latter measurements indicate that the rate of synthesis of the porin was indeed reduced, not merely the rate of insertion into the outer membrane. In contrast to these effects on individual outer membrane proteins, the relative rate of synthesis of bulk outer membrane protein was not significantly affected during the experiment. The brief increase in relative rate of synthesis immediately after DP addition probably reflects a differential insensitivity of outer membrane proteins to inhibition of transcription because of the relatively long half-lives of outer membrane protein mRNAs (Hirashima, Childs and Inouye, 1973). DP treatment also increased the proportion of pulselabeled outer membrane protein present in the 74K81 K region of an SDS gel from 3 to 17%. This induction is lower than that found for the individual 81 K polypeptide, a difference which probably reflects the presence in that region of the gel of polypeptides whose synthesis is not induced. It can be calculated from these data that if there is indeed an overall limitation on synthesis of outer membrane protein, and if all proteins compete on an equal footing, then the relative rate of synthesis of the porin would be expected to fall by 15%. In fact, the maximum reduction in relative rate during the experiment was approximately 20%. The results of this experiment are therefore in complete agreement with the predictions of the limitation hypothesis.

Pritchard, 1974; Pierrucci, 1978). One prediction of any model of cell growth that postulates tight coupling between chromosome replication and the rate of synthesis of the cell surface is that inhibition of DNA synthesis in an exponentially growing bacterial culture should fix every cell at its particular rate of synthesis (either pre- or post-doubling) and should thus fix the rate of synthesis in the culture as a whole. To test this hypothesis, a culture of LEB16 (a thymine auxotroph) was starved of thymine to block DNA synthesis and the rate of synthesis of membrane proteins was measured as shown in Figure 5. After the removal of thymine, which immediately reduces the rate of DNA synthesis to ~4% in this strain (data not shown), growth continued for about one mass doubling with a detectable deviation from exponential growth after about half a mass doubling. Because of this, conclusions can only be drawn from the early

Lack of Coupling between Outer Membrane Protein Synthesis and DNA Replication It has been suggested that if growth of the E. coli surface is indeed a linear process, then the underlying trigger for the rate doubling could be the replication of a specific region of the chromosome (Previc, 1970;

Figure

I , -THY.

0

20

LO 60 80 100 720 UO TIME

5. Envelope

Protein

Synthesis

(MIN) during

Thymine

Starvation

An exponential culture of strain LEB16 (supplemented with 20 pg/ml thymine) was pulse-labeled for 4 min at intervals with ‘“C-leucine before and after removal of thymine. which completely blocks DNA replication in this strain. The isotope ratio “C/3H was determined as before in outer membranes tom), inner membrane (im) or total protein, and plotted as a measure of the rate of synthesis in these fractions.

Cell 292

period of the experiment when the rate of total protein synthesis continued to increase exponentially. During this period of growth in the absence of DNA synthesis, the rate of synthesis of both outer and cytoplasmic membrane protein continued to increase in parallel with that of total protein. There is therefore no indication from this experiment that chromosome replication is necessary for any increase in the rate of synthesis of outer membrane protein. As an alternative test of the hypothesis that replication of a specific gene controls surface growth, experiments were performed in which the supply of thymine to LEB16 was not completely removed, but merely limited. It is well established that lowering the concentration of exogenous thymine supplied to certain thymine auxotrophs of E. coli, including LEB16, causes a decrease in replication velocity (Meacock and Pritchard, 1975) without affecting culture growth rate. Since there is no effect on the rate of initiation of replication under these conditions, there is an alteration in the steady-state pattern of gene concentrations in a culture (the gene concentration is the number of copies of a gene per unit cell mass; Pritchard, 1974). Chandler and Pritchard (1975) have demonstrated that the rates of processes that are limited by gene concentration are altered in a predictable fashion by the thymine limitation; the magnitude of such an effect depends on the chromosomal location of the gene in question, being zero for a gene at the replicative origin and maximal (36%reduction) for a gene at the terminus of replication. This technique thus provides another means of testing the hypothesis that the rate of synthesis of outer membrane protein is linked to the number of copies of some gene. Thus if the cell cycle doubling in rate of synthesis of outer membrane protein is causally linked to the duplication of a particular gene, then the differential rate of synthesis of outer membrane protein (rate of synthesis of outer membrane protein per rate of mass increase) should change in parallel with the concentration of the hypothetical “controlling” gene. The results of experiments in which various cell parameters were measured for LEB16 growing in the presence of 2 or 20

Table

1. Effect

of Thymine

Concentration

on Membrane

DNA Mass (Arbitrary Units)

Protein

Growth Rate Dependence of Cellular Levels of 36SK Porin E. coli shows a marked shape change with increase in growth rate, becoming shorter and fatter, with a consequent fall in the surface area-to-mass ratio (Schaechter, Maalae and Kjeldgaard, 1958). To extend our study of the regulation of synthesis of outer membrane protein and of the 365K porin in particular, we therefore determined cellular levels of the porin over a 4 fold range of culture growth rates. Over this range, surface/volume varies more than 2 fold. The levels of porin per unit cell mass are plotted in Figure 6, together with values of cell surface/volume calculated from measurements of dimensions of E. coli B/ r at different growth rates (Ft. F. Rosenberger, personal communication). The two parameters, porin/ mass and surface area/volume, show a markedly similar growth rate dependence, indicating (as discussed below) that the porin is maintained at a constant concentration/unit surface area at all growth rates. Discussion We have demonstrated that the linear accumulation of envelope protein in the cell cycle of E. coli B/r previously reported by Churchward and Holland (1976) is, in fact, restricted to the outer membrane protein-that is, the sarkosyl-insoluble fraction. This pattern of synthesis may be related to the postulated linear growth of the cell surface (Pritchard, 1974; Pierrucci, 1978; Rosenberger et al., 1978). There is some experimental evidence that the peptidoglycan sacculus also grows linearly (Hoffman et al., 1972; Koppes et al.,

Synthesis Outer Membrane/ Total Protein”

RNA @g/A4

20

0.91

1 .o

107

28.5

1.07

1 .o

1 .o

2

0.91

0.88

108

27.9

1.23

1 .Ol

0.98

Level

Growth Rate (Doublings/Hr)

(pg/

Inner Membrane/ Total Protein’

Cell Mass A.&t OS Ceil

Thymine @g/ml)

Protein Am)

pg/ml thymine in proline-alanine medium are presented in Table 1. The former concentration of thymine is low enough to be rate-limiting for DNA synthesis in LEB16 (Meacock and Pritchard, 1975), and this is confirmed in the DNA/mass measurement. Nevertheless, there was no change in the differential rate of synthesis of outer membrane protein at the two thymine concentrations.

a Separated by Sarkosyl fractionation. Two cultures of strain LEE1 6 in balanced growth were labeled for six generations with “C-leucine to an A 450 = 0.1. Samples were removed at intervals and mixed with 3H-leucine-labeled cells as internal standard, and the membranes were isolated. The quotient “C/3H in a membrane fraction divided by ‘% in total protein was used to calculate proportions of membrane protein and was expressed relative to that from cultures in 20 pg/ml thymine. Protein and RNA contents were determined by Folin and by absorbance at 260 rnp, respectively; DNA/mass ratios were determined from parallel cultures labeled for several generations in the presence of ‘H-thymine.

Surface 293

Growth

in E. coli

051

Figure

6. Effect

IOI Dcublings

of Growth

158 per

201

251

h

hr

Rate on Porin Content

of E. coli B/r

E. coli LEB18 was grown in the presence of “C-leucine in media with a variety of carbon sources, and the growth rate (doublings per hour) was determined. At A450 = 0.2, whole cell lysates ware prepared and analyzed in duplicate by SDS-PAGE, and the proportion of total cellular radioactivity present as 36SK porin was determined by cutting out and counting the cognate band. The ratio of porin/mass (filled circles) was then calculated from direct measurement of protein/Ads,, unit in cultures grown at the different growth rates. A regression line is drawn through the mean values of surface area/ volume of E. coli B/r (open circles) at different growth rates. P/M values (in arbitrary units) are plotted so that the mean of all P/M values lies on the S/V regression line. The slope of the regression line through the P/M values (not shown) is not significantly different (at the 95% level of confidence) from the line drawn. The dimensions of E. coli B/r at different growth rates (supplied by R. F. Rosenberger) were determined by electron microscopy of pre-fixed bacteria as described by Woldringh et al. (1977).

1978) and since the outer membrane has an intimate structural relationship with the sacculus (Braun, 1975; Yu and Mizushima, 1977; Lugtenberg et al., 1977; Maclachlan, 1978; Sonntag et al., 1978) it may be desirable for some structural reason for the synthesis of these surface layers to be matched. In this case synthesis of both surface components might be subject to a common regulatory mechanism. Alternatively, outer membrane synthesis might be directly constrained by the growth of the sacculus. We found no evidence to support the theory that the doubling in rate of synthesis of outer membrane protein is triggered by the replication of a terminally located gene. Thus, during thymine starvation, the rate of synthesis of outer membrane protein continued to increase in the absence of DNA synthesis. Our data also show no effect upon outer membrane protein synthesis in an experiment in which the number of copies per unit cell mass of terminally located genes was decreased in balanced growth by thymine limitation. We therefore conclude that there is no link between the DNA replication cycle and the pattern of synthesis of outer membrane protein. This conclusion is in general agreement with recent findings that there is no causal link between overall surface growth and DNA replication (Donachie et al., 1976; Segg and Donachie, 1978; Zaritsky and Woldringh, 1978; Pritchard, Meacock and Orr, 1978).

It was previously found (Churchward and Holland, 1976) from measurements of the unfractionated whole envelope that the rate of synthesis of at least some surface proteins doubled in early to mid-cycle. This is in contrast to the timing reported here for the outer membrane fraction (5-15 min before division in three separate experiments). Since the same strain and growth medium were used in both studies, the reason for this discrepancy is not clear, although the separation of the inner (exponential) and outer (linear) membrane components of the envelope in this study may have resulted in a more accurate measurement. Moreover, the current result is now more in line with previous reports of the doubling in rate of peptidoglycan synthesis (Hoffman et al., 1972; Koppes et al., 1978) or length extension (Donachie et al., 1976) 1 O20 min before division. We have demonstrated that in an exponentially growing culture the capacity of cells for synthesis of outer membrane proteins is saturated (Figure 4); this indicates the existence of a factor which varies discontinuously during the cell cycle, the availability of which limits the overall rate of synthesis of these proteins. While transcriptional control by such a factor is feasible, the relative longevity of mRNA molecules for a number of outer membrane proteins (Inouye, 1975) makes the existence of such a mechanism improbable. There are two possible constraining mechanisms that could operate at the level of translation, which arise directly from specific features of outer membrane structure and biosynthesis. First, since it is clear that surface proteins are synthesized on membrane-bound polysomes (Randall, Hardy and Josefsson, 1978) the availability of hypothetical membrane sites involved specifically in translation and translocation of outer membrane proteins could limit the overall rate of synthesis of these proteins. In this view, the cell cycle doubling in rate would be determined by synchronous duplication of membrane sites at a specific cell age. Second, the constraining mechanism could be a direct structural limitation on the rate of synthesis of outer membrane protein, determined by the rate of growth of the peptidoglycan. In this case the sacculus is envisaged as a foundation or template upon which outer membrane proteins are assembled; the rate of expansion of this foundation layer would determine the rate of provision of “vacant space” for the assembly of outer membrane protein. The cell cycle doubling in rate would thus directly reflect an underlying linear pattern of synthesis of the peptidoglycan. This hypothesis does not exclude the existence of special sites of recognition and translocation for nascent polypeptides but does suggest that their number is not the rate-limiting factor, thus removing the need for the discontinuous synthesis of the sites themselves. This may be important since Smit and Nikaido (1978) have

Cell 294

presented data which indicated that antibody-labeled major porin appears at several hundred discrete sites randomly dispersed over the bacterial surface rather than at a distinct growth zone. A specific prediction of this “structural” hypothesis is that there should be a strong correlation between cellular levels of outer membrane protein and cell surface area under all conditions. The data presented above demonstrate that over a 4 fold range of growth rates, the cellular level of the 36.5K porin (expressed as porin/cell mass) and cell surface area/volume show a remarkably similar growth rate dependence. Assuming that mean cell density is independent of growth rate (Kubitschek, 1974), these data are consistent with a constant amount of porin per unit surface area over this range of growth rates. Since the porin constitutes a large proportion of bulk outer membrane protein (35% in proline-alanine medium) and since physiological variations in the proportion of total outer membrane protein represented by the porin are likely to be small, these data may also be taken, at a first approximation, as supporting the idea of a strong correlation between cell surface area and total outer membrane protein. We have, however, noted one exception to this rule. As indicated above, reducing the thymine concentration supplied to the thymine auxotroph LEB16 had no effect on the differential rate of outer membrane protein synthesis. Previous studies (Pritchard, 1974) and present experiments have shown that under such conditions, in addition to gene dosage changes, bacteria become shorter and fatter and thus the surface-to-mass ratio of this strain is reduced. Our calculations therefore indicate that the ratio of outer membrane protein to surface area at the low thymine concentration was increased by about 15% without any effect on growth rate. It now seems probable, however, that the shape changes which occur in thymine-limited cultures are due to perturbation of the level of thymidine-linked sugars, which in turn affects the biosynthesis of two major surface components, lipopolysaccharide and peptidoglycan, leading to increased fragility of the envelope (Ohkawa, 1977; Zaritsky and Woldringh, 1978; Pritchard et al., 1978). Although the result obtained may indicate that outer membrane protein synthesis can be uncoupled to some extent from growth of the surface as a whole, uncertainties about the precise effect of lowering the thymine concentration in such experiments make this particular aspect of the results difficult to evaluate. We conclude that the most coherent interpretation of our data is that the cell cycle pattern of synthesis of outer membrane protein is related to the overall linear growth of the cell surface. The reasons for linear surface growth are nevertheless obscure. It is possible that the molecular nature of the rigid peptidoglycan dictates that the sacculus should be synthesized at a constant rate at growth zones (Schwartz et al., 19751,

and it has also been suggested (Previc, 1970; Pritchard, 1974) that the fluctuations in relative rates of formation of cell surface and cell mass (that is, linear and exponential rates, respectively) may provide a mechanism by which the process of cell division itself can be initiated. In further studies we hope to investigate the possible role of linear synthesis of the outer membrane protein in septum formation. Experimental

Procedures

Media and Growth of Bacteria E. coli s/r, strain LEB16 (/acZ, strA. thyA. drm) and its thy+ derivative, LEBl8. were usually grown by shaking in a minimal salts medium with proline and alanine (each at a final concentration of 0.04% w/v) as carbon source (Churchward and Holland, 1976). The generation time in this medium was 65-70 min at 37V. To vary the growth rate of strain LEB18. proline and alanine were replaced by alanine (0.04% w/v). glycerol (0.2% w/v). casamino acids (1%). or glucose (0.4% w/v) plus casamino acids (1% w/v). For growth of strain LEB16. all media were supplemented with 2 or 20 pg/ml thymine as appropriate. To ensure balanced exponential growth of bacteria, cultures containing about 1 O* stationary phase bacteria per ml were grown through at least six generations before use. Removal of thymine from growing cultures was achieved by filtration through a nitocellulose filter (Sartorius, 0.45 p pore size) followed by washing and resuspension in prewarmed medium lacking thymine. Bacterial Cell Numbers Samples of bacteria fixed by mixing with an equal volume of filtered 0.9% (w/v) saline plus 0.6% (w/v) formaldehyde were further diluted in saline and counted in a Model 6 Coulter Counter fitted with a 30 pm orifice. Macromolecular Composition of Bacteria Samples of culture (l-2 Asso units) were precipitated perchloric acid and fractionated, and RNA and protein determined as described by von Meyenberg (1971).

with 0.2 M content were

Age Fractionation of Bacterial Cultures Exponentially growing cultures were pulse-labeled with 50 PCi “Cleucine for 5 min as described by Churchward and Holland (1976). Age fractionation was then carried out at 37°C by the filter elution method of Helmstetter (1987). The basis of the fractionation procedure is described in the text. Measurement of Protein Synthesis TO determine the rate of synthesis of total bacterial protein in age fractionation experiments. 1 .O ml eluate fractions (1 OS-1 0’ bacteria) from the membrane filter were mixed with an equal volume of 10% (w/v) TCA. and the amount of acid-precipitable “C-leucine was determined. For measurement of protein synthesis in membrane fractions, 5 or 10 ml eluate fractions were immediately chilled and mixed with chloramphenicol (final concentration 250 pglml) and membranes were prepared as described below. The radioactive content was then determined after precipitation with ice-cold 10% TCA or by dissolving in Biosolv (Beckman Instruments Inc.) and counting in an aqueous scintillation fluid. In experiments with exponentially growing batch cultures. cultures were grown to A,50, 0.1-0.2, and 1 .O ml samples were removed at intervals and pulse-labeled for 2 or 4 min with “C-leucine (0.6 gCi; 311 mCi/mmole) or with ??-methionine (5 pCi: 17 pCi/pg). Radioactivity was then determined by TCA precipitation or by placing thoroughly washed samples directly into a Biosolv-based scintillation fluid. Preparation of Bacterial Envelopes Envelopes were prepared from sonic lysates as described previously (Churchward and Holland, 1976). except that MgSO. was omitted

Surface 295

Growth

in E. coli

from the wash buffer; each sample usually contained 1 O’-10’ cells mixed prior to sonication. with 2 X 10” unlabeled bacteria (grown to A.,% = 0.5 in a parallel culture).

labeled carrier

Separation of Outer and Cytoplasmic Membranes by Sarkosyl Washed envelope pellets were resuspended in 100 Al of 0.5% (w/v) Sarkosyl NL97 and incubated for 30 min at room temperature (Filip et al.. 1973). The outer membrane, which remains insoluble after this procedure, was recovered by centrifugation (39.000 rpm for 2 hr) in a type 40 Beckman rotor: the inner membrane protein, which constitutes about 60% of total envelope protein in E. coli B/r. remains in the supernatant. Inner and outer membranes prepared in this way (A. Boyd and I. B. Holland, manuscript in preparation) have protein profiles identical to those prepared by the less convenient density gradient procedure (Osborn et al., 1972), with the Sarkosyl-soluble fraction containing a maximum of 13% of protein derived from the outer membrane. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis (SDSPAGE) The basic procedure was that of Laemmli (1970). with an 11% (w/v) acrylamide separating gel and a 5% stacking gel. The acrylamide monomer:dimer ratio was 37.51, or 44:0.3 when better resolution of polypeptides in the 70-90K range was required. Electrophoresis was carried out at a constant current of 25 mA per gel; other details have been described by Churchward and Holland (1976). In some experiments, stained radioactive gels were dried down and individual bands were cut out, dissolved in NCS tissue solubilizer (Amersham/Searle. Ardington Heights, Illinois) and counted in scintillation fluid (Ames. 1974). Use of an Internal Standard of ‘H-Leucine-Labeled Bacteria To minimize errors in the measurement of radioactive amino acids in cell fractions and individual gel bands, a method was devised to eliminate the need for reproducible quantitative recovery from sample to sample. A 30 ml exponentially growing culture (AIM = 0.1) was labeled for two generations in the presence of 200 pCi ‘H-leucine (53 Ci/mmole); unlabeled leucine (20 F/ml final concentration) was then added and the cells were chilled. After determining the level of TCA-precipitable radioactivity, a constant volume was added to samples of bacteria labeled in various experiments with either 35S-methionine or ‘?Z-leucine immediately prior to preparation of cell lysates or envelopes. The ratio of “C or 35S to ‘H in any sample was thus a measure of the relative amounts of 14C or 35S in the whole of the original sample. Counting was performed using the “C?H setting of a Packard Model 3255 liquid scintillation spectrophotometer. The ratio of 3H:‘4C or 35S was usually not less than 1 0:l , ensuring that the spillover correction was less than 10% of the total ‘H-leucine radioactivity. Preparation of Cell Lysates for SDS-PAGE Samples (lo’-10’ bacteria per ml) were mixed with 3H-leucinelabeled bacteria (about 10’ per sample) to provide carrier material and an internal standard. The bacteria were harvested and washed twice with ice-cold 10 mM sodium phosphate buffer (pH 7.2); the washed pellet was then resuspended in 50 Al of buffer and mixed with an equal volume of electrophoresis buffer (0.0625 M Tris (pH 6.8). 20% w/v glycerol, 4% w/v SDS, 5% w/v mercaptoethanol). The samples were boiled for 5-l 0 min to complete lysis and stored at -2O’C; samples were always reboiled immediately prior to electrophoresis. Chemicals Radioisotopes were obtained from the Radiochemical Center, Amersham: Sarkosyl NL97 from Geigy Ltd (England); all materials for electrophoresis were from BioRad Laboratories (Watford, England), except acrylamide (from Kodak Ltd., Liverpool, England, then further purified by activated charcoal); o,a-dipyridyl was from Sigma Laboratories (London).

Acknowledgments We gratefully acknowledge the support of an MRC Research Studentship (to A.B.) throughout this project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

June

1, 1979;

revised

July 4. 1979

References Ames, amide

G. F. (1974). Resolution of bacterial proteins by polyacrylgel electrophoresis on slabs, J. Biol. Chem. 249, 634-644.

Bavoil. P.. Nikaido. H. and von Meyenburg. K. (1977). Pleiotropic transport mutants of Eschenchia co/i lack porin, a major outer membrane protein. Mol. Gen. Genet. 158, 23-33. Begg. K. J. and Donachie, W. D. (1978). Changes in cell size and shape in thymine-requiring Escherichia co/i associated with growth in low concentrations of thymine. J. Bacterial. 133, 452-458. Bluemink. J. G. and de Laat. S. W. (1977). Plasma membrane assembly as related to cell division. In Cell Surface Reviews, 4, G. Poste and G. L. Nicolson. eds. (Amsterdam: North-Holland), pp. 403449. Braun. V. (1975). Covalent lipoprotein from the outer Escherichia co/i. Biochim. Biophys. Acta 4 15, 335-377.

membrane

of

Chandler, M. G. and Pritchard, R. H. (1975). The effect of gene concentration and relative gene dosage on gene output in Escherichia co/i. Mol. Gen. Genet. 738, 127-l 41. Churchward, G. G. and Holland, during the cell cycle in Escherichia

I. B. (1976). Envelope synthesis co/i. J. Mol. Biol. 705. 245-261.

Coltrell. S. F., Rabinowitz. M. and Getz, G. S. (1975). Cytochrome synthesis in synchronous cultures of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 250. 4087-4094. Cullum. J. and Vicente. M. (1978). Cell growth in Escherichia co/i. J. Bacterial. 134, 330-337. Dennis, P. P. (1971). Regulation of stable richia co/i. Nature New Biol. 232, 43-46.

and length distribution

RNA synthesis

in Esche-

Dennis, P. P. and Young, R. F. (1975). Regulation of ribosomal protein synthesis in Escherichia co/i B/r. J. Bacterial. 721, 994-999. Donachie, W. D.. Begg, K. J. and Vincente, M. (1976). cell growth and cell division. Nature 264, 328-333.

Cell length,

Ecker. R. E. and Kokaisl. G. (1969). Synthesis of protein, ribonucleic acid and ribosomes by individual bacterial cells in balanced growth. J. Bacterial. 98, 1219-l 226. Filip, C.. Fletcher, G.. Wulff. J. L. and Earhart. C. F. (1973). Solubilization of the cytoplasmic membrane of Eschenchra co/i by the ionic detergent sodium lauryl sarcosinate. J. Bacterial. 115, 717-722. Fraley. R. T.. Lueking. D. R. and Kaplan, S. (1977). lntracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Polypeptide insertion into growing membrane. J. Biol. Chem. 253, 458-464. Hancock, R. E. W.. Hankte. K. and Braun. V. (1976). Iron transport in Escherichia co/i K12: involvement of the colicin B receptor and of a citrate-inducible protein. J. Bacterial. 127. 1370-l 375. Helmstetter. C. E. (1967). Rate of DNA synthesis during cycle of Escherichia cob B/r. J. Mol. Biol. 24, 417-427.

the division

Hirashima, A., Childs. G. and Inouye. M. (1973). Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia co/i. J. Mol. Biol. 79, 273-389. Hoffman, B.. Messer, W. and Schwarz. U. (1972). cap formation in the life cycle of Escherichia Structure 1, 29-37. Inouye. proteins

Regulation of polar co/i. J. Supramol.

M. (1975). Biosynthesis and assembly of outer membrane of Escherichia co/i. In Membrane Biogenesis, A. Tzagoloff,

Cell 296

ed. (New

York:

Plenum

Press).

Bacillus

PP. 351-391.

lyer, R.. Darby. V. and Holland, I. B. (1977). Alterations in the outer membrane proteins of Escherichia co/i B/r associated with the presence of the R plasmid rRM98. FEES Letters 85. 127-132. Koppes, L. J. H.. Overbeeke, N. and Nanninga. N. (1978). DNA replication pattern and cell wall growth in Escherichia co/i PAT84. J. Bacterial. 133, 1053-l 061. Kubitschek. H. E. (1968). Biophys. J. 8. 792-804.

Linear

cell

growth

in Escherichia

co/;.

Kubitschek, H. E. (1974). Constancy of the ratio of DNA to cell volume in steady state growth of E. coli B/r. Biophys. J. 14. 119-l 23. Laemmli. assembly

U. K. (1970). Cleavage of structural proteins during the of the head of bacteriophage T4. Nature 227. 680-685.

Lugtenberg. B., Bronstein, H., van Selm. H. and Peters, Ft. (1977). Peptidoglycan-associated outer membrane proteins in gram negative bacteria. Biochim. Biophys. Acta 465, 571-578. Machlachian. A. D. (1978). The double helix coiled-coil structure of murein lipoprotein from Escherichia co/i. J. Mol. Biol. 122. 493-506. Marr, A. G.. Harvey, R. J. and Trentini. W. C. (1966). Growth division in Escherichia co/i. J. Bacterial. 91, 2388-2389. Meacock. P. A. and Pritchard, R. H. (1975). Relationship chromosome replication and cell division in a thymineless Escherichia co/i B/r. J. Bacterial. 122. 931-942.

and cell between mutant of

Ohkawa. T. (1977). Abnormal metabolism of thymidine nucleotides in UV-irradiated dTDPG pyrophosphorylase-deficient mutants of E. co/i. Biochim. Biophys. Acta 4 76, 190-202. Osborn. M. J., Gander, J. E.. Parisi. E. and Carson, J. (1972). Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterisation of cytoplasmic and outer membrane. J. Biol. Chem. 247, 3962-3972. Pasternak, C. A., Sumner, M. C. 8. and Collin. R. C. L. S. (1974). Surface changes during the cell cycle. In Cell Cycle Controls, G. M. Padilla. I. L. Cameron and A. Zimmerman, eds. (New York: Academic Press), pp. 117-l 24. Pedersen, S.. Bloch. P. L.. Reeh, S. and Neidhardt. F. C. (1978). Patterns of protein synthesis in E. coli: a catalog of the amount of 140 individual proteins at different growth rates. Cell 14, 179-l 90. Pierrucci, 0. (1978). Dimensions of Escherichia rates: model for envelope growth. J. Bacterial.

co/i at various 135. 559-574.

growth

Poole, R. K. (1977). cycle of Escherichia synchronous cultures 186.

Fluctuation in buoyant density during the cell co/i K12: significance for the preparation of by age selection. J. Gen. Microbial. 98, 177-

Powell, G. 0. (1956). with special reference 492-511.

Growth rate and generation time of bacteria to continuous culture. J. Gen. Microbial. 15,

Previc. E. P. (1970). Biochemical determination of bacterial morphology and the geometry of cell division. J. Theoret. Biol. 27, 471-497. Pritchard, R. f-l. (1974). On the growth Phil. Trans. Roy. Sot. B 267. 303-336.

and form

of a bacterial

cell.

Pritchard, R. H.. Meacock. P. A. and Orr. E. (1978). Diameter of cells of a thermosensitive dnaA mutant of Escherichia CO/I cultivated at intermediate temperatures. J. Bacterial. 735. 575-580. Randall. L. L.. Hardy, S. J. S. and Josefsson. L. G. (1978). Precursors Of three exported proteins in Escherichia co/;. Proc. Nat. Acad. Sci. USA 75, 1209-l 212. Rosenberger. R. F., Grover, (1978). Control of microbial 244-245.

N. B., Zaritsky. surface growth

A. and Woldringh, by density. Nature

C. L. 271,

Rosenbusch, J. P. (1974). Characterization of the major outer membrane protein from Escherichia co/i: regular arrangement on the paptidoglycan and unusual dodecyl sulphate binding, J. Biol. Chem. 249, 8019-8029. Sargent.

M. G. (1975).

Control

of membrane

protein

synthesis

in

subt;;;s.

Biochim.

Biophys.

Acta 406. 564-574

Schaechter. M., Maalae, 0. and Kjeldgaard. N. 0. (1958). ency on medium and temperature of cell size and chemical sition during balanced growth of Salmonella typhimurium. Microbial. 19, 592-606.

DependcompoJ. Gen.

Schaechter. M.. Wilkinson, J. P.. Hood, J. R. and Koch, A. L. (1962). Growth, cell and nuclear division in some bacteria. J. Gen. Microbial. 29. 421-434. Schwartz. U.. Ryter. A.. Rambach, A., Hellio. R. and Hirota, Y. (1975). Process of cellular division in Escherichia co/i: differentiation of growth zones in the sacculus. J. Mol. Biol. 98, 749-759. Smit. J. and Nikaido, H. (1978). Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on protein insertion sites and growth of the cell surface of Salmonella typhimurium. J. Bacterial. 135. 687-702. Sonntag. I., Schwarz. H.. Hirota. Y. and Henning, V. (1978). Cell envelope and shape of Escherichia co/i: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. J. Bacterial. 136, 280-285. von Meyenburg. K. (1971). Transport limited growth of Escherichia co/i. J. Bacterial. 107, 878-888. Ward, C. B. and Glaser. D. A. (1971). Correlation growth and rate of DNA synthesis in Escherichia Acad. Sci. USA 68, 1061-l 064.

rates in a mutant

between co/i B/r.

rate of cell Proc. Nat.

Woldringh. C. L.. de Jong, M. A., Van den Berg, W. and Koppes, L. (1977). Morphological analysis of the division cycle of two Escherichia co/i substrains during slow growth. J. Bacterial. 731, 270-279. Yu. F. and Mizushima, S. (1977). Stimulation by lipopolysaccharide of the binding of outer membrane proteins O-8 and O-9 to the peptidoglycan layer of Escherichia co/i K12. Biochem. Biophys. Res. Commun. 74. 1397-l 402. Zaritsky, A. and Woldringh. C. L. (1978). Chromosome rate and cell shape in Escherichia co/i: lack of coupling. 135. 581-587.

replication J. Bacterial.