Escherichia coli During the Division Cycle

Transcription

1 JOURNAL OF BACTERIOLOGY, Mar. 1983, p /83/ $0.00/0 Copyright C 1983, American Society for Microbiology Vol. 153, No. 3 Labeling Pattern of Major Penicillin-Binding Proteins of Escherichia coli During the Division Cycle FRANS B. WIENTJES,l* TOM J. M. OLIJHOEK, ULI SCHWARZ,1AND NANNE NANNINGA Department of Biochemistry, Max-Planck-Institut fur Virusforschung, Tubingen, Federal Republic of Germany,1 and Department of Electron Microscopy and Molecular Cytology, University ofamsterdam, Amsterdam, The Netherlands Received 1 November 198/Accepted 7 December 198 Escherichia coli cells were synchronized by the elutriation technique. The pattern of penicillin-binding proteins (PBPs) in synchronously growing cells was determined with an iodinated derivative of ampicillin in intact cells as well as in isolated membranes. This was done under nonsaturating conditions as well as under conditions in which the PBPs were saturated with [151]ampicillin. No evidence was found for fluctuations in the PBP pattern: the PBPs seem to be present in a constant ratio throughout the division cycle. The E. coli cells exert their control on shape maintenance and cell wall growth apparently not on the level of concentration of PBPs in the cell but rather on activation of existing components. 187 Bacteria possess a set of proteins that bind penicillin covalently. These penicillin-binding proteins (PBPs) play roles in cell wall synthesis and regulation of cell growth and division. In Escherichia coli six PBPs were originally found with [14C]benzylpenicillin (30) which were numbered 1 to 6 according to their locations in polyacrylamide gels. By the use of other electrophoresis conditions (9, 31) and of an iodinated derivative of ampicillin (6), some additional bands were discovered; thus, the total number of PBPs in E. coli may be around 10. From several studies in recent years, it is known that some of the PBPs play specific roles in cell wall metabolism and shape maintenance of the E. coli cell. The most clear examples are PBPs and 3. PBP was shown to be necessary for maintenance or production of the rod form of the cells or both (15, 7, 30), whereas PBP 3 was assigned a function in cell division (7, 31). PBP 3 may be directly involved in murein synthesis during septum formation (, 13). Also, for some other PBPs, roles are suggested in murein synthesis (PBPs la and lb) (14, ), transpeptidation (PBP 4) (5), or regulation of septum formation (PBP 5) (19-1). No such roles have been ascribed to the other known PBPs (1c, 6, 7, and 8). The activity of the enzymes involved in murein metabolism must underlie spatial and temporal control. New murein is incorporated into the sacculus of E. coli predominantly in growth zones: one in the center of the cell where elongation takes place and an additional one in dividing cells at the future site of septum formation (5, 3). Furthermore, the rate of murein biosynthesis oscillates during the division cycle, being maximal before division (11, 4). Also, several murein hydrolase activities have been shown to oscillate during the cell cycle (1, 9). It is not known whether the cells control the enzymes involved in murein metabolism by controlling their presence in time (i.e., by switching the synthesis of the enzymes on and off), their presence in space (i.e., a defined location of the proteins), or by controlling their activities only (i.e., by activation and deactivation). If the first possibility were true, for instance, one would expect PBP 3 tp be present mainly at the time of septation, whereas PBP would be found to be increased when the cells elongate. For a better understanding of the way in which the cell wall growth ofe. coli is regulated, we decided to investigate the PBP pattern in synchronously growing E. coli cells. We did not find evidence that any of the PBPs is present exclusively or mainly in one part of the division cycle. This makes it unlikely that the cells exert their control on murein metabolism on the level of enzyme synthesis. MATERIALS AND METHODS Bacterial strain, growth conditions, and synchronization procedure. The E. coil strain PA309 (K-1, F- lys thr leu trp his thy argh thi lacy mala mtl mel tona supe str) from the collection of Y. Hirota was used. The bacteria were grown in batch cultures at 30 C in minimal citrate medium containing the appropriate requirements and 0.4,uCi of a 3H-amino acid mixture (Radiochemical Centre, Amersham, England; about

2 188 WIENTJES ET AL. 100 Ci/mmol) per ml. The doubling time was 70 to 75 min under these conditions. Synchronously growing cells were obtained by the elutriation method (7) as modified by Olijhoek et al. (4). Briefly, 50 ml of an exponentially growing culture was harvested at an optical density of 0.4 (Sorvall RCB centrifuge; 5 min at 10,000 x g). The cells were resuspended in 5 ml of cold growth medium and injected into the Beckman elutriator rotor system at 1.8 mumin. The rotor speed was kept constant at 5,80 rpm. Elution was with ice-cold growth medium at.6 ml/min and was continued until a total of 100 ml was collected. This suspension contained about 5 x 107 small cells per ml. These cells grew synchronously for more than two generations when replaced at 30 C in a shaking water bath. Cell numbers were measured with a Coulter Counter. Elutriation was checked by Coulter Counter volume distributions determined before and after elutriation. Preparation of membranes. The samples taken during synchronous growth of the bacteria were centrifuged as described above. The cells were washed once with ice-cold 10 mm Tris-maleate buffer (ph 7.4), resuspended in 1 ml of this buffer, and opened by sonication (Branson sonifier; microtip), two times for 1 min each time, in an ice bath. Phase-contrast microscopy showed that in all samples 95% of the cells were disrupted. Intact cells were removed by centrifugation (Eppendorf minicentrifuge; 0.5 min). Membrane fragments were isolated by centrifugation through a layer of 30%o (wt/vol) sucrose in 50 mm potassium phosphate buffer (ph 7.0) (MSE ultracentrifuge; 100,000 x g at 4 C). The pelleted membranes were resuspended in 0 jjl of 50 mm phosphate buffer and used immediately. Penicillin-binding assay. Ampicillin (obtained from Bayer) was iodinated with the Bolton and Hunter reagent (Radiochemical Centre) as described by Schwarz et al. (6). The p-hydroxy-[1"i]phenylpropionylampicillin (briefly, [15I]ampicillin) was purified by gel filtration on Bio-Gel P, concentrated by lyophilization, and dissolved to the desired concentration in 50 mm phosphate buffer immediately before use. The penicillin-binding assay was done essentially as described by Spratt (8), except that the membrane protein concentration was lower. Unless otherwise stated, the same protein amount was applied to each test mixture to minimize differences in test conditions. For this purpose, bacteria were grown in the presence of 3H-amino acids (see above), and an equal amount of 3H radioactivity was applied to each assay. The total volume of the assay was 0 IL, in which 10 to 0 1Lg of membrane proteins was present (or the corresponding amount of intact cells) except when the penicillinbinding assay was performed under saturating conditions; in that case, about 10 times less protein was applied (see below). Each test tube contained 10 ILCi of [1-I]ampicillin, and incubation was for 10 min at 30'C. The reaction was stopped by the addition of an excess of cold phosphate buffer. Intact cells were washed once with this buffer and then boiled in sodium dodecyl sulfate-containing sample buffer for gel electrophoresis. Labeled membrane fragments were washed free of unbound [1"Ijampicillin by sucrose cushion centrifugation as described above and were then resuspended in sample buffer and boiled. Polyacrylamide gel electrophoresis was performed on slab gels (10% acrylamide) as described by Lugtenberg et al. (17). Acrylamide, N,N'-methylenebisacrylamide, and N,N,N',N'-tetramethylethylenediamine were obtained from Serva. Sodium dodecyl sulfate was from BDH. Gels were stained with Coomassie brilliant blue R50 (Merck), destained electrophoretically, dried, and exposed for 1 to 10 days with Kodak XAR-5 X-ray film and a Dupont-Cronex intensifying screen. The films were scanned with a Joyce-Loebl microdensitometer, and quantitation was done by measuring the peak surfaces with a planimeter. RESULTS J. BACTERIOL. Constant ratio of PBPs in synchronously growing cells. The experiments described in this section were done with intact cells. Working with cells instead of membranes has the advantage of easier handling and smaller losses. However, the accessibility of [151]ampicillin to the PBPs might be different in intact cells than in isolated membranes. Therefore, the experiments were repeated with isolated membranes to allow comparison with the results obtained with intact cells; no clear-cut differences were observed (data not shown). Centrifugal elutriation was chosen as the synchronization method because it yields relatively high amounts of small cells without the need of dense materials such as sucrose or Ficoll. Routinely, 5 x 109 cells were obtained in one run and divided over a number of duplicate samples taken during two successive generations. For successful labeling of the PBPs in intact cells, high cell concentrations were needed. The concentration in the assay mixture was about 1010 cells per ml (or about 1 mg of membrane protein per ml when membrane fragments were used). In pilot experiments, it was established that during growth of the cells, the amount of label in all PBPs increased with cell size (not shown). The experiments were repeated in a more controlled way in which care was taken to apply equal amounts of protein to the test mixtures, as described above. Figure 1 shows an example of the pattern of PBPs labeled in intact cells, together with the densitometer tracing used for the quantification. The minor PBPs become visible after long exposure time (not shown here). PBPs la and lb were not separated in our gel system. Between PBPs la/b and, a band appears which is seen only with iodinated 3-lactams (PBP lc) (6). Sometimes some other bands are seen (between PBP 3 and 4 and below PBP 8) but, owing to their faint and irreproducible character, they are not considered here. Losses of material during washing procedures could not be completely avoided, leading to differences in the amount of material applied to the gels. Instead of expressing the PBP amounts

3 VOL. 153, lc la/b bottom X:4 v ; /ASI / I PBPs IN SYNCHRONIZED E. COLI 189 top FIG. 1. PBP pattern after labeling of intact E. coli cells with [15I]ampicillin. Approximately x 108 cells were taken from a synchronously growing culture at the time of division. The cells were washed and labeled with 10,uCi of [151]ampicillin for 10 min at 30 C in a total volume of 10,ul. For further experimental details, see the text. The gel was exposed for days. The positions of the visible PBPs are indicated. The minor PBPs became visible after longer exposure. as optical density units, the relative intensities of the PBPs were calculated from the total amount in the sample. The values are therefore in percentages of [15 ]ampicillin bound to the particular PBP in relation to the total amount of label in the sample. Figure shows the result of a series of experiments with intact cells. The values are averages of four to six determinations (two different experiments) with the standard deviation. Despite the high standard error inherent in the test, the data in Fig. clearly show that none of the labeled PBPs is predominantly present at any stage of the division cycle. To show the absence of shifts in the PBP pattern more clearly, the cells were grouped into elongating cells and dividing cells. The relative intensities of the PBPs in these cell classes are given in Table 1. No differences are seen between cells which grow in length and cells which are involved in division. This observation is particularly noteworthy for PBPs and 3, for which such differences would have been expected Ȯne might argue that the ratio of the PBPs being constant, the total amount of PBPs (measured as [15I]ampicillin bound) might fluctuate. This appeared not to be so: the total bound radioactivity, measured as the total peak area in the densitometer tracings, was proportional to the amount of protein in the samples (measured by densitometry of the negatives of the photographed gels). When membranes were isolated from synchronously growing cells, similar results were obtained, except that the variation among the values was larger, which is probably caused by irreproducible protein losses during sonication. The fact that the results obtained with membranes are consistent with those obtained with intact cells justifies our conclusion that the ratio of at least most labeled PBPs is constant during the division cycle of E. coli. Saturation of PBPs with [1'I]ampicilHin. Owing to the high specific activity of the [151]ampicillin (about,000 Ci/mmol), its chemical concentration in the test was very low, too low to saturate the PBPs; thus, under our conditions, only part of the PBP molecules were labeled. To exclude the possibility that this might obscure eventual fluctuations in the ratio of the PBPs, we tried to saturate the PBPs with [15IJampicillin. On the basis of Spratt's data (8), it was estimated that about 1 mci of 15I would saturate the PBPs in E. coli membranes in the classical penicillin-binding assay (ca. 00,ug of membrane protein). Since such amounts of radioactivity are inconvenient for routine experiments, the mem-

4 190 WIENTJES ET AL.. I.1 0 la : S.C a_v,i s la/b 5 6-~~~~~~~~~ ti+t i flj{fl..7 sf _0... ifttti+++t4 _1..*.@... * FIG.. D t thus (min) time (min) Relative intensities of PBPs after labeling of intact cells with [1"I]ampicillin. In a series of experiments, intact cells from synchronous cultures were labeled with [15I]ampicillin, and the percentage of radioactivity in each PBP band was measured as described in the text. Average values standard deviation are shown. S.C., Synchronization curve; the numbers 1 through 8 refer to the different PBPs. brane protein amount was reduced to,ug, and it was shown (Fig. 3) that the PBPs were saturated by about 10,uCi of ['5I]ampicillin under these conditions. Figure 3A shows an X-ray film Is of such a saturation experiment, and Fig. 3B shows the saturation curves for three PBPs. The ^ saturation curves of the other PBPs were similar, except that the film had to be exposed for a 30 shorter (PBP 5) or longer (minor PBPs) period, 0 respectively. This result was obtained regardless,0 of whether the experiment was done with intact cells or with membranes. In both cases, PBP 5 S a ~was the predominant band, in accordance with 6 Z the fact that it is the most abundant PBP in the 4: E. coli membrane (8). * The PBP pattern of synchronously growing E. coli cells was 40 determined under the saturating 0 s conditions described above. The results were 01. similar to those of Fig. ; with intact cells as well *o as with isolated membranes, no changes in the relative intensities of the PBPs were observed (not shown). 4 J. BACTERIOL. DISCUSSION At present, there is no method available to measure the rate of synthesis of the various PBPs during the division cycle, and all we can do is determine their amount through specific binding of,3-lactams. This was done in this study by measuring the ampicillin-binding capacity. The standard deviations of the data we obtained are rather high, but the method is sufficiently accurate to detect major changes in the PBP pattern. We estimate that for the PBPs which TABLE 1. Comparison of relative PBP intensities in elongating and dividing E. coli cellsa Type of cell PBP (%) (no. of determinations) la/b lc Elongating ± ± ± ± ± ± ± 1. cells (9) (0-50 min) Dividing cells ± ± ± ± ± ±. 7.3 ± 1.3 (10) (60-80 min) Elongating ± ± ± ± ± ± ± ± 0.8 cells (5) ( min) Dividing cells ± ± ± ± ± ± ± ± 0.5 (5) (10-140min)_ a E. coli was grown synchronously, and at time intervals, intact cells were labeled with [15Ilampicillin. The percentages of [(15IJampicillin bound to the different PBPs were calculated for elongating cells and dividing cells, respectively. Although some cells will have started the division process before an actual increase in cell number is observed, as an approximation "elongating cells" are defined by the periods in which the cell number remains constant, whereas "dividing cells" are defined by the periods in which the cell number increases (compare the synchronization curve in Fig. ). Values are given as mean percentages ± standard deviation, with the number of determinations in parentheses.

5 VOL. 153, 1983 PBPs IN SYNCHRONIZED E. COLI 191 A a ease_ B 1{ v.e I= '10@ i'/ 6 1 D - I 81 St t m iz Tm pci [5 I] ampicillin FIG. 3. Saturation of PBPs in E. coli with [15I]ampicillin. Membranes of E. coli (,ug of membrane protein) were labeled with increasing amounts of [15I]ampicillin (1 to 0.Ci). (A) X-ray film, exposed for 3 days. (B) Saturation curve for PBPs 1, 3, and 7. appear as "major" PBPs in our system (PBPs 3, 5, and 7, [Fig. ]), fluctuations of 10% of their intensities could have been detected. For the "minor" PBPs (PBPs la/b, 6, and 8), this would be 0%, and for the "very minor" ones (PBPs lc,, and 4), about 30% owing to the increasing inaccuracy of the quantification when the bands are faint. Especially for PBP 4 one should be careful in drawing a definitive conclusion. Our results show that when there is any fluctuation of the labeled PBPs during the division cycle, these fluctuations are small. This is apparent from the graphical representation in Fig. as well as from the data in Table 1, in which elongating and dividing cells are directly compared. The absence of oscillations is especially striking for PBPs and 3 (and remarkably clear in the latter case [Fig. ]). For these proteins, an oscillation could be expected on the basis of their roles in elongation and septation, respectively (15, 7, 30, 31). PBP 5 is also worth mentioning in this context because this protein is

6 19 WIENTJES ET AL. a carboxypeptidase (3) which seems to have a regulating function in cell division (19-1). Our results are partly in conflict with those of Buchanan (4), who reported that PBP was decreased in minicells as compared with rods, which suggests that this PBP is decreased in the elongating part of the cell. However, as suggested by Buchanan, these results might have been affected by the sonication procedure, because longer sonication times were needed for disruption of normal cells. We conclude that the concentration of the 15I-labeled PBPs in E. coli is constant during the division cycle and that murein synthesis is probably not regulated by controlling the amount of PBPs. This agrees with the results of Lutkenhaus et al. (18), who studied the protein composition of synchronously growing E. coli cells on two-dimensional polyacrylamide gels and concluded that the individual proteins of E. coli were synthesized continuously during the cell cycle. This points to the concept of an E. coli cell which increases in mass in an approximately exponential way (3, 6) and also increases the rate of synthesis of the individual proteins in an exponential way, resulting in a constant concentration of the proteins in the cell (although not all proteins may behave in the same way; compare reference 3). When the cells do not control their cell wall growth by regulating the amount of the enzymes involved, the question remains, how do the cells control their shape and growth. Are the activities of the enzymes under control rather than their synthesis? Hakenbeck and Messer (9) found an oscillation in murein hydrolase activity during the life cycle of E. coli, and several authors reported changes in the carboxypeptidase activities (however, with conflicting results; compare references 1 and 0). Furthermore, Hoffmann et al. (11) as well as Olijhoek et al. (4) found that the rate of murein synthesis was oscillating, being maximal before division and dropping off thereafter. Such a control might be exerted on the level of substrate availability in the sense that barriers might exist between enzyme and substrate (compare the barrier suggested by Hartmann et al. [10] between murein hydrolases and the murein). Alternatively, the murein structure might play a role, i.e., the murein might have a different composition in longitudinal growth zones, septal regions, and metabolically less active regions. However, the latter assumption is in conflict with the observation that conversion of rodlike cells to spherical cells by mecillinam or by a temperature-sensitive PBP (15) is reversible. Other possibilities can be envisioned. Regulatory factors such as ppgpp may play a role in control of murein metabolism (1). Also, the J. BACTERIOL. inward growth of murein during division and the initiation of new longitudinal growth zones are triggered by changes in macroscopic parameters such as osmotic pressure or tension on the cell wall (16). It is also conceivable that the enzymes involved in cell wall metabolism function under topological control. This would mean, for example, that PBP 3 is exclusively or mainly present in the region of the cell where the polar cap is formed, whereas PBPs la, lb, and would be located at the longitudinal growth zones. Studies are being initiated in our laboratories to investigate the topographical distribution of several of the relevant enzymes over the cell envelope of E. coli. ACKNOWLEDGMENTS We thank Johan Leutscher and Joop Woons for help with the drawings, Christine van Wijngaarden for typing the manuscript, and Vicky Kastner and Piet de Boer for technical assistance in some of the experiments. This work was supported by NATO research grant (N.N. and U.S.) and by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (A.J.M.O.). LITERATURE CITED 1. Beck, B. D., and J. T. Park Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-1 as measured in toluene-treated cells. J. Bacteriol. 16: Botta, A. G., and J. T. Park Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation. J. Bacteriol. 145: Boyd, A., and I. B. Holland Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r. Cell 18: Buchanan, C. E Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane. J. Bacteriol. 145: de Pedro, M. A., and U. Schwarz Heterogeneity of newly inserted and preexisting murein in the sacculus of Escherichia coli. Proc. Natl. Acad. Sci. 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