OF BACTERIOLOGY, Mar. 1978, p. 1492-1500 0021-9193/78/0133-1492$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 133, No. 3
JOURNAL
Printed in U.S.A.
Inhibition of Escherichia coli Division by Protein X GIUSEPPE SATTAt AND ARTHUR B. PARDEEtt* Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540
Received for publication 23 August 1977
We propose that protein X provides the connection between damage to Escherichia coli DNA and inhibition of septation and cell division. This connection is needed to guarantee that each new bacterium receives a complete DNA copy. We present several new experiments here which demonstrate that the degree to which septation is inhibited following damage to DNA is correlated with the amount of protein X that is produced. Rifampin selectively blocks protein X production. This drug was shown to allow cells whose DNA had been damaged by nalidixic acid to resume septation. Several mutants formed septa-less filaments and also produced protein X at 42°C; rifampin both inhibited their production of protein X and permitted them to form septa and divide. Essentially complementary results were obtained with a dnaA mutant which at 42°C stopped making DNA, did not produce protein X, and continued to divide; added bleomycin degraded DNA, induced protein X, and inhibited septation. These results, as well as previous observations, are all consistent with the proposal that protein X is produced as a consequence of DNA damage and is an inhibitor of septation. We suggest that septation could require binding of a single-stranded region of DNA to a septum site in the membrane. Protein X could block this binding by combining with the DNA. This control could provide an emergency mechanism in addition to the usually proposed coordination in which completion of DNA synthesis creates a positive effector for a terminal step of septation. Or it could be the sole coordinating mechanism, even under unperturbed growth conditions.
Cell division of Escherichia coli normally occurs only after completion of a round of DNA replication (26). The nature of the control mechanism is unknown; current hypotheses are based on the idea that termination of DNA replication initiates the production of a positive effector protein essential for cell division (19, 35). Most of the data supporting this view only show correlations between inhibition of DNA synthesis and inhibition of septation. For instance, nalidixic acid arrests DNA synthesis and damages the DNA (10) and arrests septation (8). As Witkin (34) earlier proposed, damage to DNA could produce an inhibitor of cell division. Protein X of 40,000 daltons is produced upon inhibition of DNA replication by several drugs, including nalidixic acid (15, 23, 29, 31). Several temperaturesensitive mutants overproduce protein X and form filaments. The protein is not produced in temperature-sensitive DNA synthesis mutants or in recA or lexA mutants after inhibition of DNA replication, whereas these mutants continue to divide without DNA synthesis, forming DNA-less cells (4, 12, 14). These results strongly t Present address: Istituto di Microbiologia dell' Universita di Genova, 16132 Genova, Italy. tt Present address: Sidney Farber Cancer Institute, Boston, MA 02115.
suggest a relationship between protein X and the processes of DNA synthesis and cell division. Other observations led to our suggestion that protein X induction is related to the so-called "S.O.S. functions" of cell recovery and mutation, which are induced in wild-type cells when DNA synthesis is inhibited or DNA is degraded. In particular, protein X binds to single-stranded DNA. It is formed as about 3% of total cell protein following DNA synthesis arrest or damage and is almost absent in undamaged cells (6). It is mostly in the cytoplasm, although 10% is associated with the membrane fraction (4, 8). Protein X was proposed to protect singlestranded DNA from degradation by recBC nuclease (7). It has recently been shown to be the product of the recA gene (6, 23). Several further experimental approaches to the question of the relation between DNA replication, protein X, and cell division are now available and are applied here. We have recently shown that rifampin, at a concentration which reduces total protein synthesis by only 25%, strongly inhibits induction of protein X following nalidixic acid addition (G. Satta, L. J. Gudas, and A. B. Pardee, submitted for publication). Under these conditions it enhances septation. Temperature-sensitive dnaA mutants continue
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to divide at the nonpermissive temperature following completion of DNA replication (11, 27, 28, 33), and other temperature-sensitive mutants form filaments and overproduce protein X (4); rifampin's effects correlate DNA metabolism and protein X in relation to cell division. All conditions that we have tested which specifically modify protein X induction also inversely modify cell division. We propose that when DNA is damaged protein X is induced, and the latter acts as a negative effector of septation. Possibly it binds to single-stranded DNA and displaces it from the septum site in the membrane.
MATERIALS AND METHODS The strains used have been described elsewhere (4, 8, 15) and are listed in Table 1. Cells were grown in M9 medium supplemented as required. General methods have been described (8). Microscopic observations were carried out at room temperature at a magnification of 1,000. Culture samples were placed on microscope slides, a drop of melted 0.5% Noble agar solution was added to the sample, and a cover slip was pressed onto the mixture. Vaseline or paraffin was placed around the cover slip to prevent evaporation. Photomicrographs were obtained with a phase-contrast objective and Kodak high-contrast copy film. RESULTS
The general plan of these experiments was to test whether a variety of situations that damage DNA, or produce different amounts of protein X, also cause a corresponding degree of inhibition of septation. A new tool for these studies is the selective inhibition of protein X production by rifampin (Satta et al., submitted for publication). We found here that rifampin also permits septation in cells whose ability to divide has been inhibited by nalidixic acid. When we measured the change in cell number in the presence TABLE 1. Bacterial strains and requirements Strain
Source
E. coli B/r E. coli DM511
D. Mount
E. coli DM936
D. Mount
E. coli WP 44S E. Witkin B. Shapiro E. coli CRT 4610 S. typhimurium B. G. Spratt 11G
Klebsiella pneumoniae
G. Satta
Genotype
lexA3 tsl-l arg his leu pro thr thi lexA3 tsl-1 recAl arg his leu pro thr thi tif-1 uvrA try arg dnaA leu thy thi dnaA metA22 tryB2 ilv-90 xyl-1 fla-66 strA210 malAU tre-12 Hi-b H2enx dnaA enuD thr leu
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of nalidixic acid (Fig. 1) over the period, an increase of 25% was observed. Addition of rifampin in combination with nalidixic acid permitted an approximate 80% increase in the number of cells by 6 h; thus rifampin decreased the inhibition of septation by nalidixic acid. Addition of chloramphenicol with nalidixic acid did not have the same effect on septation, although protein synthesis was inhibited to a similar degree. As observed in the microscope, cells growing continuously in the presence of 30 fig of nalidixic acid per ml elongated slowly and formed short but not long filaments, even after several hours. Cells in the presence of both nalidixic acid and rifampin were shorter; the second drug diminished the inhibition of cell division by the first. After 6 h of incubation in the presence of the two drugs, numerous cells were observed with reduced amounts of DNA, and many of the cells lacked DNA completely (as noted previously with dnaB mutants [11, 13]). After nalidixic acid-treated cells were washed and suspended in fresh medium, they started elongating much faster than in the presence of the inhibitor. Filaments were formed whose maximal length before starting septation appeared to be proportional to the duration of incubation with nalidixic acid. Cells treated for 1 h with nalidixic acid and allowed 4 h to recover had decreased in size, but those inhibited for 2 h elongated and did not divide much after 4 h further. If cells were first incubated in the presence of both nalidixic acid and rifampin and then in fresh medium without drugs to permit recovery, the cells showed some septa, even if they were grown for 3 h and allowed 5 h of subsequent incubation. Thus, rifampin improved recovery of the nalidixic acid-treated
cells. Rifampin did not interfere with the inhibition of DNA synthesis by nalidixic acid, as shown by [3H]thymidine uptake measurements; nor was the relatively slight inhibition of total protein synthesis responsible, because when chloramphenicol, at a concentration similarly inhibitory, replaced rifampin, none of the above effects was observed. Cultures treated as above with nalidixic acid, or with nalidixic acid and chloramphenicol, lost little viability in 1 h, but lost 99% viability by 3 h. By comparison, cultures treated with nalidixic acid plus rifampin lost only 80% viability in 3 h. This result is the opposite of the viability decrease obtained with rifampin following UV irradiation (Satta et al., submitted for publication). Transitory delay of cell division. The protein X that accumulates during nalidixic acid
treatment should have to be inactivated before
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OD
0
10 m
4, D
0
2
X
E
5 C
a 4)
U
u
I II 1
2
4
6
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FIG. 1. Effect of rifampin or chloramphenicol on cell division of E. coli treated with nalidixic acid. A culture of E. coli Blr exponentially growing in glucose M9 medium at 37°C was subdivided into four parts. One (0) was incubated without any additional treatment; the second (Cl) received 30 pg of nalidixic acid per ml; the third (A) received 4 pg of rifampin per ml plus 30 pg of nalidixic acid per ml; and the fourth (a) received 1.5 pg of chloramphenicol per ml plus 30 pg of nalidixic acid per ml. IHJthymidine (2 pCi/ml) was added to all, and at intervals samples were taken to count the cell number with a Coulter Counter (-) and to evaluate the 3H incorporated (---).
the cells can form septa, according to our hypothesis. A delay should exist after removing nalidixic acid and before the cells start to divide. Less protein X would be formed in the presence of rifampin, and hence this delay should be shortened. E. coli treated with nalidixic acid, washed free of the drug, and suspended in fresh medium showed a lag before the start of division which increased with the duration of incubation (Fig. 2). This lag was reduced if the cells were treated with rifampin in addition to nalidixic acid. Chloramphenicol did not reduce the lag. The different lag times cannot be accounted for by different survivals of the cells, since after 1 h of treatment there were almost no viability losses of these cultures. The results are thus consistent with the prediction, but elimination of this proposed inhibitory effect cannot simply be explained by dilution or disappearance of protein X, which turns over very slowly (15). Effect of rifampin on conditional filament-forming mutants. The combination of rifampin with temperature-sensitive, filament-
forming mutants (4) can be used as a further test of our hypothesis. E. coli strains 44S, DM936, and DM511 share the common property at 420C of not septating and of producing an amount of protein X greater than at the permissive temperature (30°C). Very few other changes could be seen in electrophoretic patterns of cytoplasmic or membrane proteins at 420C (4). The mutants continue to synthesize nornal amounts of DNA at 420C (20, 25). They appear to be "constitutive," i.e., to bypass the DNA degradation requirement for protein X induction. They are therefore useful for testing the relation of protein X and cell division independent of DNA damage. In addition, rifampin specifically inhibited the appearance of protein X in each of these three strains following a shift to 420C (data not shown), as was shown previously for E. coli B treated with nalidixic acid (Satta et al., submitted for publication). The shift to 420C blocked cell division of the three strains, most effectively with strain DM936 (Fig. 3). For all three strains, rifampin reversed
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0~~~~~~~~~ El
1
2.
1
3
2
3
h a u-r s
FIG. 2. Resumption of cell division. Nalidixic acid (30 pg/ml) was added to a culture exponentially growing in M9 medium, and the culture was split into three parts. One (5) was incubated without additional treatment; one (U) received 1.5 pig of chloramphenicol per ml; and one (A) received 4 pug of rifampin per ml. (A) After 30 min of incubation, a sample of each culture was taken and suspended in fresh medium. (B) After an additional 30 min, the remaining part of the three cultures was similarly treated. Cell counts were then made with the Coulter Counter at intervals.
05
0 4 2
E
0 3 0
3E
0
0.2
M in utes
FIG. 3. Effect of rifampin on mass increase and cell division of strains (a) DM936, (b) WP 44S, and (c) DM511 growing at a nonpermissive temperature (42°C). Rifampin was added at 0 min, and temperature was ), and mass increase was measured shifted at 30 min. Cell counts were made with the Coulter Counter ( by optical density (---- -). (0) Control sample, (A) rifampin sample.
this division inhibition and allowed cells to continue to divide at roughly the same rate as at 30°C. This effect of rifampin would seem to depend more on its ability to inhibit protein X production than on its slight inhibition of total
protein synthesis, since chloramphenicol, at a comparable inhibitory level as before, did not stimulate cell division. Rifampin and dnaA mutants. Further experiments were done to relate DNA synthesis,
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protein X induction, and cell division by using a dnaA mutant. Shifting DNA synthesis mutants to the nonpermissive temperature inhibited DNA initiation, and synthesis stopped after completion of the ongoing round of replication. The DNA mutants from several different species continue to divide and form anucleate cells after DNA synthesis stops (11, 26, 33). Protein X is not induced after DNA replication ceases (8). Thus dnaA mutants at 420C develop responses of DNA synthesis, protein X production, and septation that are the opposite of those of the filament-forming mutants described above. The rate of protein X synthesis was determined at both the permissive and nonpermissive temperatures; it was not induced even as late as 1 h after DNA synthesis stopped, nor did nalidixic acid cause it to appear (Fig. 4) (8). This lack of production of protein X after termination of DNA synthesis may well be a general characteristic of dnaA mutants, since the same results were obtained with Salmonella typhimurium 11G, a dnaA mutant (data not shown). DNA synthesis stopped after about an hour at 42°C (Fig. 5). Cell division at 420C continued at the normal rate for about 1 h, until DNA synthesis terminated, and then became slower but did not stop. This continued cell division appears to be correlated with the absence of protein X. Bleomycin is a drug that can damage DNA, even in the resting dnaA cells, and it induces protein X (8). Bleomycin should therefore inhibit the residual division of the DNA mutant at 42°C. As shown in Fig. 5, with nalidixic acid the cells divided only slightly more slowly than did the untreated mutant cells at 420C, but bleomycin completely inhibited this residual division. The possibility that bleomycin blocks cell division by inhibiting protein synthesis can be excluded because the optical density and the rate of incorporation of [3H]leucine into trichloroacetic acid-precipitable material were little influenced. Also, the cells were viable; after bleomycin was washed away and the cells were transferred to the permissive temperature, they restarted division almost normally, after a short lag.
DISCUSSION Protein X is produced in wild-type E. coli after DNA is damaged. In addition to its role in DNA repair (4, 7; Satta et al., submitted for publication), many prior data (Table 2) support a role for this protein as an inhibitor of cell septation. We present here additional correlations consistent with this idea. (i) In wild-type cells treated with nalidixic acid, which damages replicating DNA (10), thus inducing protein X and blocking septation, rifampin diminishes pro-
a
b
c
d
f
e
-
-
.-..
..,_ Protein x
jI
am tNW
_
W
.:,
FIG. 4. Electrophoreticpatterns of total membrane and cytoplasmic proteins of E. coli CRT4610 grown at nonpermissive temperatures. Samples of 10 ml of the various cultures described in the legend to Fig. 5 were taken after 90 min ofgrowth at the nonpermissive temperature and pulsed with 20 ICi of 13H]methionine per ml for 5 min. Cell collection, membrane and cytoplasmic proteinpreparation, and electrophoresis were performed. (a) to (c) Total membranes Qf strain CRT4610, grown (a) without antibiotic, (b) with added nalidixic acid, or (c) with added bleomycin; (d) to (t) cytoplasmic proteins of the above samples, respectively.
tein X production and also restores septation. (ii) In temperature-sensitive filament-forming mutants at 420C, protein X is produced and septation does not occur. Rifampin reverses this effect by permitting cell division at 420C. (iii) In dnaA mutants, after DNA synthesis terminates at 420C, the cells do not make protein X and continue to form septa. In these mutants, bleo-
VOL. 133, 1978
INHIBITION OF E. COLI DIVISION BY PROTEIN X
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2 0 OD
E c
0.5
m
0
0
3a
0 0
C.)
0.2
2,,
2 40 1 80 1 20 u t e s FIG. 5. Effect of nalidixic acid or bleomycin on residual cell division of a dnaA mutant. A culture of E. coli CRT4610 growing exponentially at its pernissive temperature in M9 medium was shifted to its nonpermissive temperature. After 60 min, the culture was split into three parts. One part (0) was reincubated without any treatment; one (l) received 30 pg of nalidixic acid per ml; and one (A) received 15 pg of bleomycin per ml. Samples were taken at intervals for cell counts using the Coulter Counter, and for DNA synthesis (x).
-30
0
60
Mi n
mycin damages DNA, induces protein X formation, and blocks septation. In all these cases, the appearance of protein X and the inhibition of septation correlate; therefore, protein X might well be the inducible septum inhibitor proposed by Witkin (34). A few further correlations may be mentioned. While thymidine starvation inhibits septation of wild-type E. coli, lexA mutants continue to septate and to form anucleate cells (12). The lexA mutants produce little or no protein X. They behave in these respects like recA mutants (14). At least two dnaB mutants, which stop DNA synthesis immediately at the nonpermissive temperature, continue to form septa (11, 13). These mutants therefore have properties similar to dnaA mutants. We suggest that simply arresting DNA synthesis does not prevent septation, e.g., by preventing chromosome completion. Rather, damage to DNA seems itself to arrest septation; this is in view of the report of Hill and Fangman (10) that dnaB mutants do not degrade their DNA extensively, in contrast to cells exposed to UV irradiation or to nalidixic acid. Furthermore, some dnaA and dnaB mutants do not produce protein X, in contrast to irradiated nalidixic acid-treated or thymidinestarved wild-type cells. Some results obtained with cells carrying multiple mutations do not agree with our model. For
instance, tim mutants, which are derived from the filament-forming tif mutants through a second mutation, no longer form filaments at 42°C; like tif mutants, they do produce protein X at this temperature (4). Possibly the inhibitor (protein X) is unable to function owing to modification of the target to which the protein binds, or because of its more rapid inactivation. Indeed, multiple mutations can produce almost any combination of DNA synthesis or arrest, DNA distribution (seen in filaments as one lump or evenly spaced bodies), and septation or filamentation (11). In general, attempts to interpret the basis for properties of cells carrying several mutations are very uncertain. In the commonly invoked model relating DNA synthesis and septation in undamaged cells, it is assumed that both processes occur through most of the cell cycle, but that completion of DNA synthesis is required to produce a positive signal (a protein) that permits the final stages of septation (19, 35). Our observations suggest an additional control mechanism, activated when a variety of agents damages DNA. Protein X is induced and could act as a negative signal to arrest septation. This mode of control might only apply to emergency conditions. But this control alone might also be essential in normal cells, provided such cells start DNA synthesis and septation simultaneously but require a
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TABLE 2. Summary of the relation between induction ofprotein X and inhibition of cell division Determination
ProtCeindX Cen division induced
inhibited
dNA dNgaddRfrnes
derddRfen(s
Chemical and physical treatment of wild-type strains Nalidixic acid Mitomycin Bleomycin 5-Diazouracil UV irradiation Thymine starvation
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes
Yes Yes Yes
Treatment of mutants RecA- with nalidixic acid RecA- thymine starved LexA- with nalidixic acid
No No No
No No No
Yes Yes Yes
LexA- thymine starved
No
No
Yes
DNA temperature-sensitive mutants at nonpermissive temperatures E. coli MX 74T2 ts27 S. typhimurium 11G dnaA
No No
No No
Yes No
No
No
No
No
No
No
Yes
Yes
Yes
Yes Yes Yes Yes
Yes Yes Yes No
No No No
K pneumoniae Mir M7 dnaA K. pneumoniae Mir M7 + nalidixic acid K. pneumoniae + bleomycin Division temperature-sensitive mutants at nonpermissive conditions E. coli DM936 recA tsl E. coli DM511 tsl E. coli WP 44S tif E. coli tim-1
longer period to complete septation than DNA synthesis. This suggestion is in accord with the continued septation of mutants such as dnaA after they complete their chromosomes (30). Finally, we ask by what molecular mechanism protein X could block septation. One functional property of this protein on which to base an explanation is its ability to bind to singlestranded DNA (8). We propose an explanation based on the original hypothesis of Jacob et al. (16) that the DNA origin is attached to the cell membrane, an idea supported in a recent review (22). DNA also binds to perhaps 20 other sites on the membrane (1). In addition to the original suggestion, which was designed to account for equipartition of chromosomes between daughter cells, Helmstetter (9) suggested that the DNA binding at a special region of the membrane signals the initiation of DNA synthesis. We propose here that the same attachment of
?
Yes Yes
(15) (15) (8) (15) (15) (15) (14) (14)
(4; Satta et al., submitted for publication) (4, 12)
(15) Satta and Pardee, unpublished data Satta and Pardee, unpublished data Satta and Pardee, unpublished data Satta and Pardee, unpublished data
(4, 25) (4, 25) (4, 20) (4)
the chromosome origin is also requisite for the formation of a septum, perhaps through allosteric modification of a protein in the septum site (Fig. 6). Several proteins in the membrane may be related to growth and septation; these include the two main lipoproteins (17), penicillin-dependent proteins (32), and protein D, whose synthesis is blocked by nalidixic acid (5). Furthermore, DNA binding proteins have been identified in the membrane (5, 21). We propose that a single-stranded region of the DNA loops out and attaches to a specific site on the membrane to activate septation and initiate DNA synthesis. Protein X can bind to single-stranded DNA (8). It could thereby compete with a septum activating site for DNA attachment, as indicated in Fig. 6. It does not seem unreasonable that the very large quantity of protein X made after DNA damage could interact stoichiometrically with single-stranded regions of DNA to
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FIG. 6. Model for inhibition of septation by protein X. The top drawing represents DNA origin-membrane attachment, just before septum initiation. In the absence ofprotein X (lower left drawing), the second strand attaches symmetrically to a new site (A), thereby allowing septation to proceed. In the presence of protein X (lower right drawing), the second strand is bound by protein X and is thereby prevented from combining with the septum site. Continued binding of single-stranded DNA to this site is assumed to be required for septum formation.
modify their functions and reactions. Another soluble DNA binding protein has been reported to block DNA degradation and modify repair in vitro (24). Although the structure of the E. coli chromosomal origin is not known, singlestranded loops in DNA have been proposed (3). The model suggests that the origin should have a palindromic base sequence; although this has not been determined for E. coli, palindromic sequences have been reported at or near the origin of simian virus 40 (18; K. N. Subramanian and S. M. Weissman, Fed. Proc. 36:654, 1977). A role of palindromic sequences in the replication of chromosome ends in eucaryotes has also been proposed (2). ACKNOWLEDGMENTS This work was aided by Public Health Service grant CA11595 from the National Cancer Institute. We thank L. J. Gudas and J. L. Hamlin for helpful discussions.
LITERATURE CITED 1. Abe, M., C. Brown, W. G. Hendrickson, D. H. Boyd, P. Clifford, R. H. Cote, and M. Schaechter. 1977.
The release of E. coli DNA from membrane complexes by single strand endonucleases. Proc. Natl. Acad. Sci. U.S.A. 74:2756-2760. 2. Cavalier-Smith, T. 1974. Palindromic base sequences and replication of eukaryotic chromosome ends. Nature (London) 250:467470.
3. Frenster, J. H. 1976. Selective control of DNA helix openings during gene regulation. Cancer Res.
36:3394-3398. 4. Gudas, L. J. 1976. The induction of protein X in DNA repair and cell division mutants of Escherichia coli. J. Mol. Biol. 104:567-587. 5. Gudas, L. J., R. James, and A. B. Pardee. 1976. Evidence for the involvement of an outer membrane protein in DNA initiation. J. Biol. Chem. 251:3470-3479. 6. Gudas, L. J., and D. W. Mount. 1977. Identification of the recA (tif) gene product of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 74:5280-5284. 7. Gudas, L. J., and A. B. Pardee. 1975. Model for regulation of Escherichia coli DNA repair functions. Proc.
Natl. Acad. Sci. U.S.A. 72:2330-2344. 8. Gudas, L. J., and A. B. Pardee. 1976. DNA synthesis inhibition and the induction of protein X in Escherichia coli. J. Mol. Biol. 101:459-477. 9. Helmstetter, C. E. 1974. Initiation of chromosome replication in E. coli. J. Mol. Biol. 84:1-19, 21-36. 10. Hill, W. E., and W. L. Fangman. 1973. Single-strand breaks in deoxyribonucleic acid and viability loss during deoxyribonucleic acid synthesis inhibition in Escherichia coli. J. Bacteriol. 116:1329-1335. 11. Hirota, Y., A. Ryter, and F. Jacob. 1968. Thermosensitive mutants affected in the processes of DNA synthesis and cellular division. Cold Spring Harbor Symp. Quant. Biol. 33:677-693. 12. Howe, W. E., and D. W. Mount. 1975. Production of cells without deoxyribonucleic acid during thymidine starvation of lexA- cultures of Escherichia coli K-12. J. Bacteriol. 124:1113-1121. 13. Inouye, M. 1969. Unlinking of cell division from deoxyribonucleic acid replication in a temperature-sensitive deoxyribonucleic acid synthesis mutant of Escherichia coli. J. Bacteriol. 99:842-850.
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14. Inouye, M. 1971. Pleiotropic effect of the recA gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid replication. J. Bacteriol. 106:539-542. 15. Inouye, M., and A. B. Pardee. 1970. Changes in membrane proteins and their relation to deoxyribonucleic acid synthesis and cell division of Escherichia coli. J. Biol. Chem. 245:5813-5819. 16. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:329-348. 17. James, R. 1975. Identification of an outer membrane protein of Escherichia coli, with a role in the coordination of deoxyribonucleic acid replication and cell elongation. J. Bacteriol. 124:918-929. 18. Jay, E., R. Roychoudhury, and R. Wu. 1976. Nucleotide sequence with elements of an unusual two-fold rotational symmetry in the region oforigin ofreplication of SV40 DNA. Biochem. Biophys. Res. Commun. 69:678-686. 19. Jones, N. C., and W. D. Donachie. 1973. Chromosome replication, transcription and control of cell division in Escherichia coli. Nature (London) New Biol. 243:100-103. 20. Kirby, E. P., F. Jacob, and D. A. Goldthwait. 1967. Prophage induction and filament formation in a mutant strain of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 58:1903-1910. 21. Kohiyama, M., R. Kollek, W. Goebel, and A. Kepes. 1977. Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid. J. Bacteriol. 129:658-667. 22. Leibowitz, P. J., and M. Schaechter. 1975. The attachment of the bacterial chromosome to the cell membrane. Int. Rev. Cytol. 41:1-28. 23. McEntee, K., J. E. Hesse, and W. Epstein. 1976. Identification and radiochemical purification of the recA protein of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 73:3979-3983. 24. MacKay, V., and S. Linn. 1976. Selective inhibition of the DNase activity of the recBC enzyme by the DNA binding protein from Escherichia coli. J. Biol. Chem. 251:3716-3719. 25. Mount, D. W., A. C. Walker, and C. Kosel. 1973.
J. BACTERIOL. Suppression of kx mutations affecting deoxyribonucleic acid repair in Escherichia coli K-12 by closely linked thermosensitive mutations. J. Bacteriol. 116:950-956. 26. Pardee, A. B., P. C. Wu, and D. R. Zusman. 1973. Bacterial division and the cell envelope, p. 357412. In L. Leive (ed.), Bacterial membranes and walls. Marcel Dekker, New York. 27. Sargent, M. G. 1975. Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis. J. Bacteriol. 123:1218-1234. 28. Satta, G., P. Canepari, R. Fontana, and L. Callegari. 1974. Envelope protein alteration in a conditional mutant of Klebsiellapneumoniae with pH dependent morphology and temperature dependent division. Ann. Microbiol. (Paris) 125B:259-273. 29. Sedgwick, S. G. 1975. UV-inducible protein associated with error-prone repair in E. coli B. Nature (London) 255:349-350. 30. Shannon, K. P., and R. J. Rowbury. 1972. Alteration of the ratio of cell division independent of the rate of DNA synthesis in a mutant of Salmonella typhimurium. Mol. Gen. Genet. 115:122-125. 31. Siccardi, A. G., B. M. Shapiro, Y. Hirota, and F. Jacob. 1971. On the process of cellular division in Escherichia coli. IV. Altered protein composition and turnover of the membranes of thermosensitive mutants defective in chromosomal replication. J. Mol. Biol. 56:475-490. 32. Spratt, B. G. 1975. Distinct penicillin binding proteins involved in the division, elongation and shape of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 72:2999-3003. 33. Spratt, B. G., and R. J. Rowbury. 1970. A mutant in the initiation of DNA synthesis in Salmonella typhimurium. J. Gen. Microbiol. 64:127-138. 34. Witkin, E. AL 1967. The radiation sensitivity of Escherichia coli B: a hypothesis relating filament formation and prophage induction. Proc. Natl. Acad. Sci. U.S.A. 57:1275-1279. 35. Zaritsky, A., and R. H. Pritchard. 1973. Changes in cell size and shape associated with changes in the replication time of the chromosome of Escherichia coli. J. Bacteriol. 114:824-837.
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Printed in U.S.A.
Inhibition of Escherichia coli Division by Protein X GIUSEPPE SATTAt AND ARTHUR B. PARDEEtt* Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540
Received for publication 23 August 1977
We propose that protein X provides the connection between damage to Escherichia coli DNA and inhibition of septation and cell division. This connection is needed to guarantee that each new bacterium receives a complete DNA copy. We present several new experiments here which demonstrate that the degree to which septation is inhibited following damage to DNA is correlated with the amount of protein X that is produced. Rifampin selectively blocks protein X production. This drug was shown to allow cells whose DNA had been damaged by nalidixic acid to resume septation. Several mutants formed septa-less filaments and also produced protein X at 42°C; rifampin both inhibited their production of protein X and permitted them to form septa and divide. Essentially complementary results were obtained with a dnaA mutant which at 42°C stopped making DNA, did not produce protein X, and continued to divide; added bleomycin degraded DNA, induced protein X, and inhibited septation. These results, as well as previous observations, are all consistent with the proposal that protein X is produced as a consequence of DNA damage and is an inhibitor of septation. We suggest that septation could require binding of a single-stranded region of DNA to a septum site in the membrane. Protein X could block this binding by combining with the DNA. This control could provide an emergency mechanism in addition to the usually proposed coordination in which completion of DNA synthesis creates a positive effector for a terminal step of septation. Or it could be the sole coordinating mechanism, even under unperturbed growth conditions.
Cell division of Escherichia coli normally occurs only after completion of a round of DNA replication (26). The nature of the control mechanism is unknown; current hypotheses are based on the idea that termination of DNA replication initiates the production of a positive effector protein essential for cell division (19, 35). Most of the data supporting this view only show correlations between inhibition of DNA synthesis and inhibition of septation. For instance, nalidixic acid arrests DNA synthesis and damages the DNA (10) and arrests septation (8). As Witkin (34) earlier proposed, damage to DNA could produce an inhibitor of cell division. Protein X of 40,000 daltons is produced upon inhibition of DNA replication by several drugs, including nalidixic acid (15, 23, 29, 31). Several temperaturesensitive mutants overproduce protein X and form filaments. The protein is not produced in temperature-sensitive DNA synthesis mutants or in recA or lexA mutants after inhibition of DNA replication, whereas these mutants continue to divide without DNA synthesis, forming DNA-less cells (4, 12, 14). These results strongly t Present address: Istituto di Microbiologia dell' Universita di Genova, 16132 Genova, Italy. tt Present address: Sidney Farber Cancer Institute, Boston, MA 02115.
suggest a relationship between protein X and the processes of DNA synthesis and cell division. Other observations led to our suggestion that protein X induction is related to the so-called "S.O.S. functions" of cell recovery and mutation, which are induced in wild-type cells when DNA synthesis is inhibited or DNA is degraded. In particular, protein X binds to single-stranded DNA. It is formed as about 3% of total cell protein following DNA synthesis arrest or damage and is almost absent in undamaged cells (6). It is mostly in the cytoplasm, although 10% is associated with the membrane fraction (4, 8). Protein X was proposed to protect singlestranded DNA from degradation by recBC nuclease (7). It has recently been shown to be the product of the recA gene (6, 23). Several further experimental approaches to the question of the relation between DNA replication, protein X, and cell division are now available and are applied here. We have recently shown that rifampin, at a concentration which reduces total protein synthesis by only 25%, strongly inhibits induction of protein X following nalidixic acid addition (G. Satta, L. J. Gudas, and A. B. Pardee, submitted for publication). Under these conditions it enhances septation. Temperature-sensitive dnaA mutants continue
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to divide at the nonpermissive temperature following completion of DNA replication (11, 27, 28, 33), and other temperature-sensitive mutants form filaments and overproduce protein X (4); rifampin's effects correlate DNA metabolism and protein X in relation to cell division. All conditions that we have tested which specifically modify protein X induction also inversely modify cell division. We propose that when DNA is damaged protein X is induced, and the latter acts as a negative effector of septation. Possibly it binds to single-stranded DNA and displaces it from the septum site in the membrane.
MATERIALS AND METHODS The strains used have been described elsewhere (4, 8, 15) and are listed in Table 1. Cells were grown in M9 medium supplemented as required. General methods have been described (8). Microscopic observations were carried out at room temperature at a magnification of 1,000. Culture samples were placed on microscope slides, a drop of melted 0.5% Noble agar solution was added to the sample, and a cover slip was pressed onto the mixture. Vaseline or paraffin was placed around the cover slip to prevent evaporation. Photomicrographs were obtained with a phase-contrast objective and Kodak high-contrast copy film. RESULTS
The general plan of these experiments was to test whether a variety of situations that damage DNA, or produce different amounts of protein X, also cause a corresponding degree of inhibition of septation. A new tool for these studies is the selective inhibition of protein X production by rifampin (Satta et al., submitted for publication). We found here that rifampin also permits septation in cells whose ability to divide has been inhibited by nalidixic acid. When we measured the change in cell number in the presence TABLE 1. Bacterial strains and requirements Strain
Source
E. coli B/r E. coli DM511
D. Mount
E. coli DM936
D. Mount
E. coli WP 44S E. Witkin B. Shapiro E. coli CRT 4610 S. typhimurium B. G. Spratt 11G
Klebsiella pneumoniae
G. Satta
Genotype
lexA3 tsl-l arg his leu pro thr thi lexA3 tsl-1 recAl arg his leu pro thr thi tif-1 uvrA try arg dnaA leu thy thi dnaA metA22 tryB2 ilv-90 xyl-1 fla-66 strA210 malAU tre-12 Hi-b H2enx dnaA enuD thr leu
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of nalidixic acid (Fig. 1) over the period, an increase of 25% was observed. Addition of rifampin in combination with nalidixic acid permitted an approximate 80% increase in the number of cells by 6 h; thus rifampin decreased the inhibition of septation by nalidixic acid. Addition of chloramphenicol with nalidixic acid did not have the same effect on septation, although protein synthesis was inhibited to a similar degree. As observed in the microscope, cells growing continuously in the presence of 30 fig of nalidixic acid per ml elongated slowly and formed short but not long filaments, even after several hours. Cells in the presence of both nalidixic acid and rifampin were shorter; the second drug diminished the inhibition of cell division by the first. After 6 h of incubation in the presence of the two drugs, numerous cells were observed with reduced amounts of DNA, and many of the cells lacked DNA completely (as noted previously with dnaB mutants [11, 13]). After nalidixic acid-treated cells were washed and suspended in fresh medium, they started elongating much faster than in the presence of the inhibitor. Filaments were formed whose maximal length before starting septation appeared to be proportional to the duration of incubation with nalidixic acid. Cells treated for 1 h with nalidixic acid and allowed 4 h to recover had decreased in size, but those inhibited for 2 h elongated and did not divide much after 4 h further. If cells were first incubated in the presence of both nalidixic acid and rifampin and then in fresh medium without drugs to permit recovery, the cells showed some septa, even if they were grown for 3 h and allowed 5 h of subsequent incubation. Thus, rifampin improved recovery of the nalidixic acid-treated
cells. Rifampin did not interfere with the inhibition of DNA synthesis by nalidixic acid, as shown by [3H]thymidine uptake measurements; nor was the relatively slight inhibition of total protein synthesis responsible, because when chloramphenicol, at a concentration similarly inhibitory, replaced rifampin, none of the above effects was observed. Cultures treated as above with nalidixic acid, or with nalidixic acid and chloramphenicol, lost little viability in 1 h, but lost 99% viability by 3 h. By comparison, cultures treated with nalidixic acid plus rifampin lost only 80% viability in 3 h. This result is the opposite of the viability decrease obtained with rifampin following UV irradiation (Satta et al., submitted for publication). Transitory delay of cell division. The protein X that accumulates during nalidixic acid
treatment should have to be inactivated before
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OD
0
10 m
4, D
0
2
X
E
5 C
a 4)
U
u
I II 1
2
4
6
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FIG. 1. Effect of rifampin or chloramphenicol on cell division of E. coli treated with nalidixic acid. A culture of E. coli Blr exponentially growing in glucose M9 medium at 37°C was subdivided into four parts. One (0) was incubated without any additional treatment; the second (Cl) received 30 pg of nalidixic acid per ml; the third (A) received 4 pg of rifampin per ml plus 30 pg of nalidixic acid per ml; and the fourth (a) received 1.5 pg of chloramphenicol per ml plus 30 pg of nalidixic acid per ml. IHJthymidine (2 pCi/ml) was added to all, and at intervals samples were taken to count the cell number with a Coulter Counter (-) and to evaluate the 3H incorporated (---).
the cells can form septa, according to our hypothesis. A delay should exist after removing nalidixic acid and before the cells start to divide. Less protein X would be formed in the presence of rifampin, and hence this delay should be shortened. E. coli treated with nalidixic acid, washed free of the drug, and suspended in fresh medium showed a lag before the start of division which increased with the duration of incubation (Fig. 2). This lag was reduced if the cells were treated with rifampin in addition to nalidixic acid. Chloramphenicol did not reduce the lag. The different lag times cannot be accounted for by different survivals of the cells, since after 1 h of treatment there were almost no viability losses of these cultures. The results are thus consistent with the prediction, but elimination of this proposed inhibitory effect cannot simply be explained by dilution or disappearance of protein X, which turns over very slowly (15). Effect of rifampin on conditional filament-forming mutants. The combination of rifampin with temperature-sensitive, filament-
forming mutants (4) can be used as a further test of our hypothesis. E. coli strains 44S, DM936, and DM511 share the common property at 420C of not septating and of producing an amount of protein X greater than at the permissive temperature (30°C). Very few other changes could be seen in electrophoretic patterns of cytoplasmic or membrane proteins at 420C (4). The mutants continue to synthesize nornal amounts of DNA at 420C (20, 25). They appear to be "constitutive," i.e., to bypass the DNA degradation requirement for protein X induction. They are therefore useful for testing the relation of protein X and cell division independent of DNA damage. In addition, rifampin specifically inhibited the appearance of protein X in each of these three strains following a shift to 420C (data not shown), as was shown previously for E. coli B treated with nalidixic acid (Satta et al., submitted for publication). The shift to 420C blocked cell division of the three strains, most effectively with strain DM936 (Fig. 3). For all three strains, rifampin reversed
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0~~~~~~~~~ El
1
2.
1
3
2
3
h a u-r s
FIG. 2. Resumption of cell division. Nalidixic acid (30 pg/ml) was added to a culture exponentially growing in M9 medium, and the culture was split into three parts. One (5) was incubated without additional treatment; one (U) received 1.5 pig of chloramphenicol per ml; and one (A) received 4 pug of rifampin per ml. (A) After 30 min of incubation, a sample of each culture was taken and suspended in fresh medium. (B) After an additional 30 min, the remaining part of the three cultures was similarly treated. Cell counts were then made with the Coulter Counter at intervals.
05
0 4 2
E
0 3 0
3E
0
0.2
M in utes
FIG. 3. Effect of rifampin on mass increase and cell division of strains (a) DM936, (b) WP 44S, and (c) DM511 growing at a nonpermissive temperature (42°C). Rifampin was added at 0 min, and temperature was ), and mass increase was measured shifted at 30 min. Cell counts were made with the Coulter Counter ( by optical density (---- -). (0) Control sample, (A) rifampin sample.
this division inhibition and allowed cells to continue to divide at roughly the same rate as at 30°C. This effect of rifampin would seem to depend more on its ability to inhibit protein X production than on its slight inhibition of total
protein synthesis, since chloramphenicol, at a comparable inhibitory level as before, did not stimulate cell division. Rifampin and dnaA mutants. Further experiments were done to relate DNA synthesis,
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protein X induction, and cell division by using a dnaA mutant. Shifting DNA synthesis mutants to the nonpermissive temperature inhibited DNA initiation, and synthesis stopped after completion of the ongoing round of replication. The DNA mutants from several different species continue to divide and form anucleate cells after DNA synthesis stops (11, 26, 33). Protein X is not induced after DNA replication ceases (8). Thus dnaA mutants at 420C develop responses of DNA synthesis, protein X production, and septation that are the opposite of those of the filament-forming mutants described above. The rate of protein X synthesis was determined at both the permissive and nonpermissive temperatures; it was not induced even as late as 1 h after DNA synthesis stopped, nor did nalidixic acid cause it to appear (Fig. 4) (8). This lack of production of protein X after termination of DNA synthesis may well be a general characteristic of dnaA mutants, since the same results were obtained with Salmonella typhimurium 11G, a dnaA mutant (data not shown). DNA synthesis stopped after about an hour at 42°C (Fig. 5). Cell division at 420C continued at the normal rate for about 1 h, until DNA synthesis terminated, and then became slower but did not stop. This continued cell division appears to be correlated with the absence of protein X. Bleomycin is a drug that can damage DNA, even in the resting dnaA cells, and it induces protein X (8). Bleomycin should therefore inhibit the residual division of the DNA mutant at 42°C. As shown in Fig. 5, with nalidixic acid the cells divided only slightly more slowly than did the untreated mutant cells at 420C, but bleomycin completely inhibited this residual division. The possibility that bleomycin blocks cell division by inhibiting protein synthesis can be excluded because the optical density and the rate of incorporation of [3H]leucine into trichloroacetic acid-precipitable material were little influenced. Also, the cells were viable; after bleomycin was washed away and the cells were transferred to the permissive temperature, they restarted division almost normally, after a short lag.
DISCUSSION Protein X is produced in wild-type E. coli after DNA is damaged. In addition to its role in DNA repair (4, 7; Satta et al., submitted for publication), many prior data (Table 2) support a role for this protein as an inhibitor of cell septation. We present here additional correlations consistent with this idea. (i) In wild-type cells treated with nalidixic acid, which damages replicating DNA (10), thus inducing protein X and blocking septation, rifampin diminishes pro-
a
b
c
d
f
e
-
-
.-..
..,_ Protein x
jI
am tNW
_
W
.:,
FIG. 4. Electrophoreticpatterns of total membrane and cytoplasmic proteins of E. coli CRT4610 grown at nonpermissive temperatures. Samples of 10 ml of the various cultures described in the legend to Fig. 5 were taken after 90 min ofgrowth at the nonpermissive temperature and pulsed with 20 ICi of 13H]methionine per ml for 5 min. Cell collection, membrane and cytoplasmic proteinpreparation, and electrophoresis were performed. (a) to (c) Total membranes Qf strain CRT4610, grown (a) without antibiotic, (b) with added nalidixic acid, or (c) with added bleomycin; (d) to (t) cytoplasmic proteins of the above samples, respectively.
tein X production and also restores septation. (ii) In temperature-sensitive filament-forming mutants at 420C, protein X is produced and septation does not occur. Rifampin reverses this effect by permitting cell division at 420C. (iii) In dnaA mutants, after DNA synthesis terminates at 420C, the cells do not make protein X and continue to form septa. In these mutants, bleo-
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2 0 OD
E c
0.5
m
0
0
3a
0 0
C.)
0.2
2,,
2 40 1 80 1 20 u t e s FIG. 5. Effect of nalidixic acid or bleomycin on residual cell division of a dnaA mutant. A culture of E. coli CRT4610 growing exponentially at its pernissive temperature in M9 medium was shifted to its nonpermissive temperature. After 60 min, the culture was split into three parts. One part (0) was reincubated without any treatment; one (l) received 30 pg of nalidixic acid per ml; and one (A) received 15 pg of bleomycin per ml. Samples were taken at intervals for cell counts using the Coulter Counter, and for DNA synthesis (x).
-30
0
60
Mi n
mycin damages DNA, induces protein X formation, and blocks septation. In all these cases, the appearance of protein X and the inhibition of septation correlate; therefore, protein X might well be the inducible septum inhibitor proposed by Witkin (34). A few further correlations may be mentioned. While thymidine starvation inhibits septation of wild-type E. coli, lexA mutants continue to septate and to form anucleate cells (12). The lexA mutants produce little or no protein X. They behave in these respects like recA mutants (14). At least two dnaB mutants, which stop DNA synthesis immediately at the nonpermissive temperature, continue to form septa (11, 13). These mutants therefore have properties similar to dnaA mutants. We suggest that simply arresting DNA synthesis does not prevent septation, e.g., by preventing chromosome completion. Rather, damage to DNA seems itself to arrest septation; this is in view of the report of Hill and Fangman (10) that dnaB mutants do not degrade their DNA extensively, in contrast to cells exposed to UV irradiation or to nalidixic acid. Furthermore, some dnaA and dnaB mutants do not produce protein X, in contrast to irradiated nalidixic acid-treated or thymidinestarved wild-type cells. Some results obtained with cells carrying multiple mutations do not agree with our model. For
instance, tim mutants, which are derived from the filament-forming tif mutants through a second mutation, no longer form filaments at 42°C; like tif mutants, they do produce protein X at this temperature (4). Possibly the inhibitor (protein X) is unable to function owing to modification of the target to which the protein binds, or because of its more rapid inactivation. Indeed, multiple mutations can produce almost any combination of DNA synthesis or arrest, DNA distribution (seen in filaments as one lump or evenly spaced bodies), and septation or filamentation (11). In general, attempts to interpret the basis for properties of cells carrying several mutations are very uncertain. In the commonly invoked model relating DNA synthesis and septation in undamaged cells, it is assumed that both processes occur through most of the cell cycle, but that completion of DNA synthesis is required to produce a positive signal (a protein) that permits the final stages of septation (19, 35). Our observations suggest an additional control mechanism, activated when a variety of agents damages DNA. Protein X is induced and could act as a negative signal to arrest septation. This mode of control might only apply to emergency conditions. But this control alone might also be essential in normal cells, provided such cells start DNA synthesis and septation simultaneously but require a
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TABLE 2. Summary of the relation between induction ofprotein X and inhibition of cell division Determination
ProtCeindX Cen division induced
inhibited
dNA dNgaddRfrnes
derddRfen(s
Chemical and physical treatment of wild-type strains Nalidixic acid Mitomycin Bleomycin 5-Diazouracil UV irradiation Thymine starvation
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes
Yes Yes Yes
Treatment of mutants RecA- with nalidixic acid RecA- thymine starved LexA- with nalidixic acid
No No No
No No No
Yes Yes Yes
LexA- thymine starved
No
No
Yes
DNA temperature-sensitive mutants at nonpermissive temperatures E. coli MX 74T2 ts27 S. typhimurium 11G dnaA
No No
No No
Yes No
No
No
No
No
No
No
Yes
Yes
Yes
Yes Yes Yes Yes
Yes Yes Yes No
No No No
K pneumoniae Mir M7 dnaA K. pneumoniae Mir M7 + nalidixic acid K. pneumoniae + bleomycin Division temperature-sensitive mutants at nonpermissive conditions E. coli DM936 recA tsl E. coli DM511 tsl E. coli WP 44S tif E. coli tim-1
longer period to complete septation than DNA synthesis. This suggestion is in accord with the continued septation of mutants such as dnaA after they complete their chromosomes (30). Finally, we ask by what molecular mechanism protein X could block septation. One functional property of this protein on which to base an explanation is its ability to bind to singlestranded DNA (8). We propose an explanation based on the original hypothesis of Jacob et al. (16) that the DNA origin is attached to the cell membrane, an idea supported in a recent review (22). DNA also binds to perhaps 20 other sites on the membrane (1). In addition to the original suggestion, which was designed to account for equipartition of chromosomes between daughter cells, Helmstetter (9) suggested that the DNA binding at a special region of the membrane signals the initiation of DNA synthesis. We propose here that the same attachment of
?
Yes Yes
(15) (15) (8) (15) (15) (15) (14) (14)
(4; Satta et al., submitted for publication) (4, 12)
(15) Satta and Pardee, unpublished data Satta and Pardee, unpublished data Satta and Pardee, unpublished data Satta and Pardee, unpublished data
(4, 25) (4, 25) (4, 20) (4)
the chromosome origin is also requisite for the formation of a septum, perhaps through allosteric modification of a protein in the septum site (Fig. 6). Several proteins in the membrane may be related to growth and septation; these include the two main lipoproteins (17), penicillin-dependent proteins (32), and protein D, whose synthesis is blocked by nalidixic acid (5). Furthermore, DNA binding proteins have been identified in the membrane (5, 21). We propose that a single-stranded region of the DNA loops out and attaches to a specific site on the membrane to activate septation and initiate DNA synthesis. Protein X can bind to single-stranded DNA (8). It could thereby compete with a septum activating site for DNA attachment, as indicated in Fig. 6. It does not seem unreasonable that the very large quantity of protein X made after DNA damage could interact stoichiometrically with single-stranded regions of DNA to
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FIG. 6. Model for inhibition of septation by protein X. The top drawing represents DNA origin-membrane attachment, just before septum initiation. In the absence ofprotein X (lower left drawing), the second strand attaches symmetrically to a new site (A), thereby allowing septation to proceed. In the presence of protein X (lower right drawing), the second strand is bound by protein X and is thereby prevented from combining with the septum site. Continued binding of single-stranded DNA to this site is assumed to be required for septum formation.
modify their functions and reactions. Another soluble DNA binding protein has been reported to block DNA degradation and modify repair in vitro (24). Although the structure of the E. coli chromosomal origin is not known, singlestranded loops in DNA have been proposed (3). The model suggests that the origin should have a palindromic base sequence; although this has not been determined for E. coli, palindromic sequences have been reported at or near the origin of simian virus 40 (18; K. N. Subramanian and S. M. Weissman, Fed. Proc. 36:654, 1977). A role of palindromic sequences in the replication of chromosome ends in eucaryotes has also been proposed (2). ACKNOWLEDGMENTS This work was aided by Public Health Service grant CA11595 from the National Cancer Institute. We thank L. J. Gudas and J. L. Hamlin for helpful discussions.
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The release of E. coli DNA from membrane complexes by single strand endonucleases. Proc. Natl. Acad. Sci. U.S.A. 74:2756-2760. 2. Cavalier-Smith, T. 1974. Palindromic base sequences and replication of eukaryotic chromosome ends. Nature (London) 250:467470.
3. Frenster, J. H. 1976. Selective control of DNA helix openings during gene regulation. Cancer Res.
36:3394-3398. 4. Gudas, L. J. 1976. The induction of protein X in DNA repair and cell division mutants of Escherichia coli. J. Mol. Biol. 104:567-587. 5. Gudas, L. J., R. James, and A. B. Pardee. 1976. Evidence for the involvement of an outer membrane protein in DNA initiation. J. Biol. Chem. 251:3470-3479. 6. Gudas, L. J., and D. W. Mount. 1977. Identification of the recA (tif) gene product of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 74:5280-5284. 7. Gudas, L. J., and A. B. Pardee. 1975. Model for regulation of Escherichia coli DNA repair functions. Proc.
Natl. Acad. Sci. U.S.A. 72:2330-2344. 8. Gudas, L. J., and A. B. Pardee. 1976. DNA synthesis inhibition and the induction of protein X in Escherichia coli. J. Mol. Biol. 101:459-477. 9. Helmstetter, C. E. 1974. Initiation of chromosome replication in E. coli. J. Mol. Biol. 84:1-19, 21-36. 10. Hill, W. E., and W. L. Fangman. 1973. Single-strand breaks in deoxyribonucleic acid and viability loss during deoxyribonucleic acid synthesis inhibition in Escherichia coli. J. Bacteriol. 116:1329-1335. 11. Hirota, Y., A. Ryter, and F. Jacob. 1968. Thermosensitive mutants affected in the processes of DNA synthesis and cellular division. Cold Spring Harbor Symp. Quant. Biol. 33:677-693. 12. Howe, W. E., and D. W. Mount. 1975. Production of cells without deoxyribonucleic acid during thymidine starvation of lexA- cultures of Escherichia coli K-12. J. Bacteriol. 124:1113-1121. 13. Inouye, M. 1969. Unlinking of cell division from deoxyribonucleic acid replication in a temperature-sensitive deoxyribonucleic acid synthesis mutant of Escherichia coli. J. Bacteriol. 99:842-850.
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14. Inouye, M. 1971. Pleiotropic effect of the recA gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid replication. J. Bacteriol. 106:539-542. 15. Inouye, M., and A. B. Pardee. 1970. Changes in membrane proteins and their relation to deoxyribonucleic acid synthesis and cell division of Escherichia coli. J. Biol. Chem. 245:5813-5819. 16. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:329-348. 17. James, R. 1975. Identification of an outer membrane protein of Escherichia coli, with a role in the coordination of deoxyribonucleic acid replication and cell elongation. J. Bacteriol. 124:918-929. 18. Jay, E., R. Roychoudhury, and R. Wu. 1976. Nucleotide sequence with elements of an unusual two-fold rotational symmetry in the region oforigin ofreplication of SV40 DNA. Biochem. Biophys. Res. Commun. 69:678-686. 19. Jones, N. C., and W. D. Donachie. 1973. Chromosome replication, transcription and control of cell division in Escherichia coli. Nature (London) New Biol. 243:100-103. 20. Kirby, E. P., F. Jacob, and D. A. Goldthwait. 1967. Prophage induction and filament formation in a mutant strain of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 58:1903-1910. 21. Kohiyama, M., R. Kollek, W. Goebel, and A. Kepes. 1977. Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid. J. Bacteriol. 129:658-667. 22. Leibowitz, P. J., and M. Schaechter. 1975. The attachment of the bacterial chromosome to the cell membrane. Int. Rev. Cytol. 41:1-28. 23. McEntee, K., J. E. Hesse, and W. Epstein. 1976. Identification and radiochemical purification of the recA protein of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 73:3979-3983. 24. MacKay, V., and S. Linn. 1976. Selective inhibition of the DNase activity of the recBC enzyme by the DNA binding protein from Escherichia coli. J. Biol. Chem. 251:3716-3719. 25. Mount, D. W., A. C. Walker, and C. Kosel. 1973.
J. BACTERIOL. Suppression of kx mutations affecting deoxyribonucleic acid repair in Escherichia coli K-12 by closely linked thermosensitive mutations. J. Bacteriol. 116:950-956. 26. Pardee, A. B., P. C. Wu, and D. R. Zusman. 1973. Bacterial division and the cell envelope, p. 357412. In L. Leive (ed.), Bacterial membranes and walls. Marcel Dekker, New York. 27. Sargent, M. G. 1975. Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis. J. Bacteriol. 123:1218-1234. 28. Satta, G., P. Canepari, R. Fontana, and L. Callegari. 1974. Envelope protein alteration in a conditional mutant of Klebsiellapneumoniae with pH dependent morphology and temperature dependent division. Ann. Microbiol. (Paris) 125B:259-273. 29. Sedgwick, S. G. 1975. UV-inducible protein associated with error-prone repair in E. coli B. Nature (London) 255:349-350. 30. Shannon, K. P., and R. J. Rowbury. 1972. Alteration of the ratio of cell division independent of the rate of DNA synthesis in a mutant of Salmonella typhimurium. Mol. Gen. Genet. 115:122-125. 31. Siccardi, A. G., B. M. Shapiro, Y. Hirota, and F. Jacob. 1971. On the process of cellular division in Escherichia coli. IV. Altered protein composition and turnover of the membranes of thermosensitive mutants defective in chromosomal replication. J. Mol. Biol. 56:475-490. 32. Spratt, B. G. 1975. Distinct penicillin binding proteins involved in the division, elongation and shape of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 72:2999-3003. 33. Spratt, B. G., and R. J. Rowbury. 1970. A mutant in the initiation of DNA synthesis in Salmonella typhimurium. J. Gen. Microbiol. 64:127-138. 34. Witkin, E. AL 1967. The radiation sensitivity of Escherichia coli B: a hypothesis relating filament formation and prophage induction. Proc. Natl. Acad. Sci. U.S.A. 57:1275-1279. 35. Zaritsky, A., and R. H. Pritchard. 1973. Changes in cell size and shape associated with changes in the replication time of the chromosome of Escherichia coli. J. Bacteriol. 114:824-837.
