Folded chromosomes from amino-acid-starved Escherichia coli DG 75 cells are to a large extent released as envelope-bound complexes which sediment more ...
Eur. J. Biochem. 65,409-414 (1976)
Significance of Folded Chromosomes Released from Amino-Acid-Starved Escherichia coli Cells Michiel MEYE,R, Marian A. DE JONG, Conrad L. WOLDRINGH, and Nanne NANNINGA Laboratory of Electron Microscopy, University of Amsterdam (Received January 5/March 8, 1976)
1. Folded chromosomes from amino-acid-starved Escherichia coli DG 75 cells are to a large extent released as envelope-bound complexes which sediment more rapidly than envelope-bound complexes from exponentially grown cells. A minor fraction (about 3 %) represents relatively slow sedimenting envelope-free nucleoids. 2. Morphological analysis of the sensitivity of amino-acid-starved cells to the action of lysozyme and/or detergents indicates that these cells are less susceptible to lysis than exponentially grown cells. This results in the production of fast sedimenting envelope-bound complexes from nondividing cells. We infer that it is not the amount of DNA, as suggested by Ryder and Smith (1974), but the low degree of envelope fragmentation that causes the high sedimentation rate. 3. After prolonged periods of starvation about 3 % of cells in the process of division persist in the population. The results indicate that these cells release their (terminated) chromosomes in the envelope-free form. At this stage it is impossible to conclude whether these chromosomes are released because of their detachment from the membrane in situ (cf. Worcel and Burgi, 1974) or because of an enhanced susceptibility of dividing cells to lysis.
Jacob et al. [l] have proposed the replicon hypothesis in which the bacterial chromosome is attached to specific sites on the cell membrane. This would permit regulation of chromosome replication and segregation of daughter chromosomes. Though the replicon hypothesis dates already from 1963, many questions on chromosome-membrane attachment still require an answer [2]. One such question is whether during the cell cycle the chromosome is permanently attached to the membrane. An indication for a temporary association of these two cell components has been presented by Worcel and Burgi [ 3 ] .This work was carried out with the use of a lysis procedure permitting the isolation of the chromosome of Escherichia coli as a highly folded structure complexed with small amounts of RNA and protein [4]. The folded chromosome can be isolated either free or attached to a portion of the cell envelope, dependent on the temperature of the lysis [3,5-71. From amino-acid-starved E. coli DG 75 cells, however, envelope-bound chromosomes could no longer be isolated, even if lysis was carried out at low temperature [ 3 ] . According to Worcel and Burgi [3] all chromosomes become detached from the cell envelope when DNA synthesis is completed in the absence of protein synthesis. They become reassociated some time after resumption of protein synthesis before the onset of DNA replication [3].
A different interpretation has been given by Ryder and Smith [S]. They showed that virtually all chromosomes isolated from amino-acid-starved E. coli TAU-bar cells remain associated with the cell envelope after completion of residual DNA synthesis [S]. Envelope-bound chromosomes prepared from aminoacid-starved cells sedimented much more rapidly than did envelope-bound chromosomes from exponentially growing cells [S]. Due to the fact that chromosome replication in the absence of protein synthesis stops just before the terminus of the chromosome [9], they concluded that the heaviness of the envelope-bound complexes is caused by twice the amount of DNA [S]. Because Worcel and Burgi [3] drew, from similar experiments, different conclusions from those of Ryder and Smith [S], we re-examined the effect of amino acid starvation on the recovery of folded chromosomes using E. coli strain K12 DG75. In this paper we have tried to clarify two questions which arose from sucrose gradient centrifugation of lysates of amino-acid-starved E. coli DG75 cells. Firstly, why do the majority of the folded chromosomes from amino-acid-starved cells sediment so fast? Secondly, is the small amount of relatively slow sedimenting envelope-free chromosomes released from a special class of cells? Our results indicate that the sedimentation properties and recoveries of both types of folded chromosomes are best understood in terms of lysis prop-
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erties of cells, and in terms of cell stage in the division cycle.
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MATERIALS AND METHODS The organism used was E. coli K12 DG75 (F-, leu-, thy-) kindly provided by Drs R. F. Heyn and A. Rorsch. The minimal salt medium, growth conditions, radioactive labelling, preparation of the crude cell lysates, fractionation of the lysates on a sucrose gradient, counting of radioactive samples and thin sectioning have been described previously [7]. Transfer of cells to a medium lacking leucine was accomplished by collecting them on a membrane filter (0.8 pm pore size; Millipore Corp., U.S.A.). After twice rinsing the filter with fresh, preheated (35 "C) medium lacking leucine the cells were resuspended in fresh medium without leucine at 35 "C and incubated further at 35 "C for 60 - 90 min.
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For the determination of length distributions cells were fixed by addition of 1% OsO4 to a final concentration of 0.1 and prepared according to the agar filtration technique of Kellenberger [lo]. The length of at least 500 cells was measured on micrographs printed to give a final magnification of 12000. For calibration a line-grating replica (spacing 1.67 pm) was used.
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RESULTS Fig. 1A shows typical sucrose gradient profiles from lysates of exponentially growing and aminoacid-starved cells. The profile of lysed amino-acidstarved cells resembles that reported by Worcel and Burgi [3]; it shows the disappearance of the envelopebound complex characteristic of exponentially growing cells and the release of a relatively slow moving envelope-free one. Worcel and Burgi [ 3 ] interpret the gradient profiles in the sense that, upon amino acid starvation, envelope-bound nucleoids are converted into envelope-free particles. If this interpretation is correct one should expect the recovery of free plus envelope-bound chromosomes sedimenting at between 1000 and 4000 S to be the same before and after amino acid starvation. Fig. 1A shows that this is not the case. From exponentially growing cells the majority of 3H-labelled DNA sediment to the bottom of the centrifuge tube (about 85%), which amount becomes even larger when lysates of starved cells are centrifuged. On an average the amount of envelopefree chromosomes represents only 3 % of the 3Hlabelled material. This percentage has been calculated from the radioactivity sedimented into the gradient, i.e. exclusive of the radioactivity that remained on the top of the gradient.
Fig. 1. Eflect of' amino acid starvation on the sedimentation velocity of folded chromosomes. E. coli cells were starved for leucine for 60 min. (A) Equal amounts of exponentially grown cells ( 0 - 4 ) and amino-acid-starved cells ( o d ) were lysed at 15 "C. I00 p1 of each lysate was applied to the gradient. Centrifugation was for 20 min on a 10 to 30% sucrose gradient as described previously [7]. Radioactivity incorporated in folded chromosomes is considered to represent the 3H-label from the bottom of the tube up to fraction number 18. Fraction number zero represents the radioactivity of the resuspended pellet. (B) As (A), but lysis has been progressed to a more complete stage as indicated by examination of the extent of disruption of cells by phase contrast microscopy. Exponentially grown cells (0-0); amino-acid0) starved cells (0-
Envelope-Bound Chromosomes from Amino-Acid-Starved Cells
Examination of lysates of amino-acid-starved cells and of exponentially growing cells by phase contrast microscopy and by electron microscopy revealed that the former lyse less easily at 15 "C than the latter. The ghosts in both types of lysates retain their overall rod
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Fig. 2. Thin-sectioned envelope remnants prepared,from amino-acid-starved cells lysrdfor 5 min at 15 " C . (A) Lysate; (B) pellet after 20 min sucrose gradient centrifugation. Magnification 60000
shape. However, with these ghosts that are present in amino-acid-starved lysates the smooth surface of the envelope is rather well preserved (Fig.2A); the vesicle-like structures which occur in ghosts of exponentially growing cells (cf. Fig. 6 in [7]) are absent.
Ghosts with a smooth surface together with DNA fibrils (Fig.2B) were also found at the bottom of the tube after centrifugation of a lysate of starved cells as shown in Fig. 1A, and are therefore considered to be the envelope-bound chromosomes of amino-acid-
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starved cells. Fig.1B shows another example of a profile obtained from lysed amino-acid-starved cells. In this case the envelope-bound complexes sediment partly as a broad band from the bottom of the tube up to the position of the slowest sedimenting envelopebound nucleoids from exponentially growing cells. We infer (Fig. 1B and 2) that it is not a large amount of DNA attached to envelope fragments that causes the relatively high sedimentation rate of envelopebound chromosomes from amino-acid-starved cells [8], but a lower degree of fragmentation of the envelope remnant into vesicle-like elements. In fact, the sucrose gradient profiles and the morphology of the ghosts obtained from lysed starved cells strongly resemble those obtained if exponentially growing cells are lysed with a ten-fold lower lysozyme concentration than the standard procedure (compare Fig.1 and 2 with Fig.7 and 8 in [7]). This again suggests that the extent to which the envelope is fragmented may influence the sedimentation rate of envelope-bound nucleoids.
Origin of Envelope-Free Chromosomes after Amino- Acid-Star vat ion Since the great majority of E. coli DG75 cells lyse rather poorly after amino-acid-starvation, the question arises whether the small fraction of envelope-free chromosomes (Fig. 1A and B) represent chromosomes that are released from a unique fraction of the cells. Therefore, length distributions were made of agar filtrations of exponentially grown cells, and aminoacid-starved cells (Fig.3). Since it is supposed that initiation of cell division is triggered by termination of DNA replication [ll], and that cells with completed rounds of DNA replication are able to finish division in the absence of protein synthesis [12,13], one would expect all dividing cells to disappear from such a cell population. Although the percentage of dividing cells indeed decreases considerably, it can be seen (Fig. 3B, Table 1) that after amino acid starvation there are still about 3 % dividing cells. These cells apparently got stuck somewhere during the process of cell division. The following observations make it very attractive to postulate that the envelope-free chromosomes from amino-acid-starved cells come from this type of cells. First, the percentage of envelope-free chromosomes released at 15 "C (Fig. 1) and the percentage of dividing cells present in such a pretreated culture (Fig. 3B, Table 1) are of the same magnitude. Second, the sedimentation behaviour of the envelopefree chromosomes released at 15 "C is slower than that of exponentially growing cells (Fig. 1 A and B). This can be expected if the chromosomes released from the dividing cells represent segregated, nonreplicating DNA molecules. Third, as is shown in Table 1, ghosts showing a visible constriction have
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Fig. 3. Length distribution defermined fkom agar filtrations of Os04-fixed preparations. (A) Exponentially grown cells; (B) cells mentioned under (A) followed by leucine starvation for 60 min. Hatched area: length distribution of cells in the process of division Table 1. Percentage of dividing cells in pellets obtained from lysates after 20 min sucrose gradient centrifugation The pelleted cells were suspended in 0.1 OsO4 and prepared by agar filtration. Exponentially grown cells were treated with solution B containing 0.4 mg/ml lysozyme for 1 mln at 4 "C, followed by lysis at 25 "C for 1 min or 2 min. Amino-acid-starved cells were treated with solution B containing 4 mg/ml lysozyme for 1 min at 4 "C, followed by lysis for 2 rnin at 15 "C Cells
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almost disappeared in the pellet of lysed amino-acidstarved cells after a standard preparative sucrose gradient run. Does the dissociation of chromosomes from the envelope reflect a release of non-replicating chromosomes in a particular stage of the cell cycle, or are dividing cells easier to open than non-dividing cells? One may thus ask whether the site where the septum is being formed is more susceptible to the action of the lysis mixture. Does this only hold for dividing cells remaining after starvation, or do dividing cells in an exponentially growing culture behave in the
M. Meyer, M. A. DeJong, C. L. Woldringh, and N. Nanninga
same way? Unfortunately, the presence of only a small amount of dividing cells in an amino-acid-starved culture makes it rather difficult to analyse this problem in thin sections. We therefore decided to make use of the resemblance of lysis of amino-acid-starved cells, and exponentially growing cells lysed with a ten-fold lower lysozyme concentration (0.4 mg/ml lysozyme in solution B; [7]) as mentioned above. In Table 1 are
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listed the percentage of dividing cells determined from agar-filtered exponentially grown cells which are lysed with 0.4mg/ml lysozyme and which are recovered from the pellet of a preparative sucrose gradient run. As compared with the control cells it is evident that the average length of lysed cells has diminished due to a slight rounding-off of the cells during lysis (data not shown). For our purpose, however, it is important that dividing cells can still be distinguished from non-dividing cells (Fig. 4). Table 1 shows that after lysis for 1 min at 25 "C the percentage of dividing cells has decreased from 17 to 7 whereas 2 min incubation at 25 "C results in a further decrease to 4 . Apparently, dividing cells from an exponentially growing culture lyse more easily than non-dividing cells. The same occurs during lysis of amino-acidstarved cells (Table 1). The most likely explanation for this different way of cell lysis is derived from thin sections made of pellets of lysates submitted to sucrose gradient centrifugation. From the thin sections (Fig.5) it is evident that the longer cells preferably show a gap in the envelope exactly in the middle of the lysed cell.
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Fig. 4. Exumpbs of Os04-jlxed cells that have been subjected to mild treatment with Iysozyme and were prepared for electron microscopy by agar fillration. Exponentially grown cells were incubated for 1 min at 4 "C with solution B [7] containing 0.4 mg/ml lysozyme, followed by lysis at 25 "C for 1 min. Micrographs were taken from the pellet (after 20 rnin sucrose gradient centrifugation) of the lysate. Magnification 7000
DISCUSSION The results presented in this paper show that the majority of complexes obtained from amino-acidstarved cells comprise relatively fast sedimentink! envelope-bound chromosomes. A very small amount
Fig. 5. Thin-sectioned envelope-bound chromosome prepared by Iysing cells with u ten-foM lower lysozynze c.oncmtrution. Cells are lysed as described in Fig. 4. Sucrose gradient centrifugation 30 min. Magnification 60000
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M. Meyer, M. A. De Jong, C. L. Woldringh, and N. Nanninga: Folded Chromosomes of Escherichia coli
(approximately 3%) is released in a relatively slow sedimenting envelope-free form. The former type of chromosomes apparently originate from cells which had not initiated a constriction (division) visible in electron microscope preparations. The latter class of chromosomes are most probably released from cells which had initiated division but could not finish it in the absence of protein synthesis. According to Ryder and Smith [8], nearly all chromosomes which have completed DNA synthesis in the absence of protein synthesis are very rapidly sedimenting envelope-bound complexes. These authors calculated a relative sedimentation coefficient for these DNA complexes of 5000 to 6000 S [8]. Like Worcel and Burgi [3], Ryder and Smith [8] explained their results in terms of events occurring during the DNA replication cycle. Ryder and Smith [8] proposed that the 5000- 6000-S particles are envelopebound chromosomes containing DNA with a mass of approximately twice that of single chromosomes. This would be in agreement with Marunouchi and Messer [9] who reported that amino acid starvation prevents the protein synthesis necessary for replication of the terminal segment of the chromosome. If this interpretation [8] is correct, one should expect that shortly after restoration of protein synthesis termination of DNA replication will result in the release of most chromosomes as envelope-free nucleoids. This is clearly not the case [3,8]. In our view, the recoveries and sedimentation rates of both types of folded chromosomes from amino-acid-starved cells are best understood if the way of lysis is considered. The bad susceptibility of amino-acid-starved cells to lysozyme treatment is best visualized by the similar sedimentation behaviour in sucrose gradients of envelope-bound complexes as compared to those of exponentially growing cells after treatment with a ten-fold smaller amount of lysozyme (Fig.7; [7]). The same information is obtained from electron micrographs of thin sections, which show that in both types of lysates ghosts do not contain vesiclelike elements (cf. [7]) but do have a rather smooth surface (Fig.2 and 5). Amino acid starvation thus causes an alteration in lysis properties, presumably because of a changed envelope composition. This results in poor lysis which yields the heavy envelope-bound complex of 5000 to 6000 S IS]. In addition, not all cells lyse just as easily. Cells which are in the process of division seem to have a weak spot at the division site, probably due to a local action of both lysozyme and other hydrolases. A weak spot at the
division site also becomes manifest after treatment of cells with penicillin [14,15]. Although this kind of differential lysis with respect to cell age occurs in both exponentially growing and starved cells, it becomes more pronounced during lysis of amino-acid-starved cells. We feel that on the basis of our observations it is premature to conclude that a replicating (or nearly terminated) chromosome is attached to a specific envelope site, and that chromosomes having a completed round of DNA replication are released from this site. In our opinion, properties of the envelope-bound complex should be understood in terms of lysis properties of the cells (cf. [7]), and in terms of the stage of the cell in the division cycle. Whether the recovery of folded genomes is affected by DNA attachment to the envelope cannot yet be judged, since the occurrence of an envelope-free complex does not imply that the DNA was free from the envelope in situ. A link between DNA and envelope could have been disrupted during lysis. We thank Mrs J. RaphaEl and Mr J. H. D. Leutscher for their excellent technical assistance, and Miss A. R. Wierdsma for helping with the English text.
REFERENCES 1. Jacob, F., Brenner, S. & Cuzin, F. (1963) Cold Spring Harbor Symp. Quant. Biol. 28,329- 347. 2. Leibowitz, P. J. & Schaechter, M. (1975) Int. Rev. Cytol. 41, 128. 3. Worcel, A. & Burgi, E. (1974) J . Mol. B i d . 82, 91-105. 4. Stonington, 0.G. &Pettijohn, D. E. (1971) Proc. Nail Acad. Sci. U.S.A. 68,6-9. 5. Delius, H. & Worcel, A. (1974) J. Mol. Biol. 82, 107-109. 6. Pettijohn, D. E., Hecht, R. M., Stonington, 0. G. & Stamato, T. D. (1973) in DNA Synthesis in vitro (Wells, R. D. & Inman, R. B., eds) pp. 145-162, University Park Press, Baltimore (Md.). 7. Meyer, M., De Jong, M. A,, Woldringh, C. L. & Nanninga, N. (1976) Eur. J . Biochem. 63, 469-475. 8. Ryder, 0. A. & Smith, D. W. (1974) J . Bacteriol. 120, 13561363. 9. Marunouchi, T. & Messer, W. (1973) J . Mol. BioL 78,211 -228. 10. Kellenberger, E. (1953) 6th Int. Congr. Microhiol. pp. 45-66. 11. Helmstetter, C. E. & Pierucci, 0. (1968) J. Bacteriol. 95, 1627- 1633. 12. Pierucci, 0. & Helmstetter, C. E. (1969) Fed. Proc. 28, 1755- 1760. 13. Kubitchek, H. E. (1974) Mol. Gen. Genet. 135, 123- 130. 94. Lederberg, J. (1957) J . Bacteriol. 73, 144. 15. Schwarz, W., Asmus, A. & Frank, H. (1969) J . Mol. Biol. 41, 419 -429.
M. Meyer, M. A. De Jong, C. L. Woldringh, and N. Nanninga Laboratorium voor Electronenmicroscopie, Universiteit van Amsterdam, Plantage Muidergracht 14, Amsterdam C, The Netherlands
