Apr 5, 1991 - (H. Westerhoff, M. O'Dea, A. Maxwell, and M. Gellert, Cell Biophys. 12:157-181, 1988), the ..... J., and J. Messing. 1982. The pUC plasmids, an.
JOURNAL
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BACTERIOLOGY, June 1991, p. 3914-3917
Vol. 173, No. 12
0021-9193/91/123914-04$02.00/0 Copyright © 1991, American Society for Microbiology
Bacterial DNA Supercoiling and [ATP]/[ADP] Ratio: Changes Associated with Salt Shock LI-SHAN HSIEH,1 JOSETTE ROUVIERE-YANIV,2 AND KARL DRLICAl*
Public Health Research Institute, 455 First Avenue, New York, New York 10016,1 and Laboratoire de Physiologie Bacterienne, Institut de Biologie Physico-Chimique, 75005 Paris, France2 Received 17 December 1990/Accepted
5
April 1991
When Escherichia coli K-12 was shifted from a medium lacking salt to one containing 0.5 M NaCl, both the
[ATP]/[ADP] ratio and negative supercoiling of plasmid DNA increased within a few minutes. After about 10 min both declined, eventually reaching a level slightly above that observed with cells growing exponentially in the absence of salt. Since in vitro the [ATP]/[ADP] ratio influences the level of supercoiling generated by gyrase (H. Westerhoff, M. O'Dea, A. Maxwell, and M. Gellert, Cell Biophys. 12:157-181, 1988), the physiological response of supercoiling to salt shock is most easily explained by the sensitivity of gyrase to changes in the intracellular [ATPJ/[ADP] ratio. This raises the possibility that the [ATP]/[ADP] ratio is an important factor in the control of supercoiling.
When circular DNA is extracted from bacterial cells, it is under negative superhelical tension because of a deficit of duplex turns (for reviews, see references 2 and 5). Superhelical tension is altered by treatments that perturb the activities of gyrase and topoisomerase I, indicating that these two enzymes are involved in the control of DNA supercoiling. Gyrase is thought to be responsible for introducing supercoils (6), and topoisomerase I is thought to be responsible for preventing supercoiling from reaching unacceptably high levels (3, 16). Supercoiling appears to be controlled in part by homeostatic regulation of the genes encoding gyrase and topoisomerase I: treatments that lower supercoiling increase gyrase expression (14) and lower topoisomerase I expression (22); a treatment that raises supercoiling increases topoisomerase I expression (23). Superimposed on this general scheme is the finding that processes such as transcription can alter supercoiling (for a review, see reference 15). When gyrase and topoisomerase I fail to correct the topological perturbations caused by transcription, local variations in supercoiling can arise (17). Cellular energetics may also play a role in the control of supercoiling. In vitro the ratio of [ATP] to [ADP] strongly influences the level of supercoiling attained in the presence of gyrase, regardless of whether gyrase is introducing or removing negative supercoils (25). While examining the effect of anaerobiosis on DNA supercoiling in Escherichia coli, we found that lowering of oxygen tension caused both supercoiling and the [ATP]/[ADP] ratio to transiently drop (9). Eventually both reached levels higher than those observed under aerobic conditions. These two correlations between [ATP]/[ADP] ratio and supercoiling, plus the clear effect of the [ATP]/[ADP] ratio on gyrase in vitro (25), led to the suggestion that this adenine nucleotide ratio might be an important factor in the regulation of supercoiling. To further test the idea that cellular energetics plays a role in DNA supercoiling, we have examined the effect of salt shock on [ATP]/[ADP] ratio and supercoiling. Plasmids extracted from E. coli cells grown in the presence of high concentrations of NaCl exhibit higher levels of negative supercoiling than those extracted from cells grown in the *
Corresponding author. 3914
absence of salt (7). Below we report that within a few minutes after addition of NaCl to bacterial cultures both the [ATP]/[ADP] ratio and plasmid supercoiling increase. Subsequently, both drop to a steady-state level that is elevated relative to values obtained from cells growing in low-salt medium. We first confirmed that a high concentration of NaCl in the growth medium causes plasmid supercoiling to be more negative. Strain JTT1 (18) containing plasmid pUC9 (24) was grown to mid-exponential phase in LB medium containing 0 or 0.5 M NaCl. Plasmid DNA was extracted, and plasmid topoisomers were then separated by gel electrophoresis in the presence of chloroquine under conditions in which all of the topoisomers were negatively supercoiled. As shown in Fig. 1A, plasmid supercoiling was about 1.5 bands or 10% more negative when DNA was obtained from cells growing in 0.5 M NaCl. Next, the effect of NaCl on the ratio of [ATP] to [ADP] was measured by using a luciferase method (1). In cells grown to mid-exponential phase in LB medium containing 0.5 M NaCl, the [ATP]/[ADP] ratio was 4.3, as compared with 3.6 when cells were grown in the absence of salt. To measure the effect of salt shock, we grew strain JTT1 containing pUC9 to early log phase without NaCl. NaCl was then added to 0.5 M, and at various times aliquots were removed. Plasmid DNA was extracted, and the DNA topoisomers were separated by gel electrophoresis. Figure 1B shows densitometric tracings of plasmid topoisomer distributions: the addition of NaCl caused supercoiling to transiently increase by about 4 bands (25%). The [ATP]/[ADP] ratio showed a transient five- to sixfold increase following salt shock (Fig. 1C). Relaxation lags substantially behind the drop in [ATP]/[ADP] ratio. This may be due to the high ratio of [ATP] to [ADP] being saturating with respect to gyrase so that relaxation occurs only after a substantial drop in this ratio. We have not ruled out secondary effects due to other factors. If gyrase is the source of the increased supercoiling associated with salt shock, pretreatment with coumermycin, a competitive inhibitor of the interaction of gyrase and ATP (20), should interfere with the ability of NaCl to increase supercoiling. The relaxing effect of several concentrations of coumermycin on DNA supercoiling in the absence of salt is
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VOL. 173, 1991
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-2 .2 C cm ci
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FIG. 1. Effect of NaCl on plasmid supercoiling and [ATP]/[ADP] ratio. (A) Plasmid supercoiling during exponential growth. E. coli JTT1 containing pUC9 was grown at 37°C in LB medium lacking salt (lane a) or containing 0.5 M NaCl (lane b). At the mid-exponential phase of growth, plasmid DNA was extracted by an alkaline-sodium dodecyl sulfate miniprep method (11, 21), omitting the polyethylene glycol step. Plasmid topoisomers were then separated by electrophoresis in 0.8% agarose containing 22.5 jig of chloroquine per ml in Tris-phosphate buffer (12). Plasmid DNA was detected by fluorescence following staining with ethidium bromide (1 jig/ml) and illumination with UV light. The direction of migration is from top to bottom; under the conditions of electrophoresis, more-negatively supercoiled DNA migrated more rapidly. (B) Plasmid supercoiling following a shift to 0.5 M NaCl. E. coli JTT1 containing pUC9 was grown in LB medium lacking NaCl to approximately 3 x 108 cells per ml. NaCl was added to 0.5 M by dilution from 5 M NaCl, and at various times aliquots were removed for plasmid isolation and supercoil analysis as described for panel A. Densitometric tracings of gels similar to that in Fig. 1A are shown. Times of incubation in NaCl were 0 min (a), 5 min (b), 10 min (c), 15 min (d), 20 min (e), and 40 min (f). Results for samples taken at 60 and 80 min were identical to those obtained at 40 min. The arrow indicates the direction of migration. (C) Plasmid supercoiling and [ATP]/[ADP] ratio following a shift to 0.5 M NaCl. E. coli cells were grown in LB medium and treated with NaCl as described for panel B. At various times aliquots were removed for determination of the [ATP]/[ADP] ratio (open circles). Changes in plasmid supercoiling (closed circles) are expressed as the change in linking number determined by visual inspection of the data shown in panel B.
shown in Fig. 2A. Increased supercoiling due to salt shock (Fig. 2B, lanes a and b) was largely prevented (Fig. 2B, lane d) by a concentration of coumermycin that had no detectable effect on supercoiling in the absence of salt. The relaxation phase was also sensitive to coumermycin; at a low concentration the drug accelerated the DNA relaxation following the initial increase in supercoiling (compare Fig. 2C and D). These results are unlikely to be due to salt affecting drug uptake, since salt had no effect on the ability of coumermycin to inhibit growth on agar plates (data not shown). None of the coumermycin treatments affected the [ATP]/[ADP] ratio (data not shown). Since topoisomerase I also influences supercoiling, it was
D
FIG. 2. Effect of coumermycin on plasmid supercoiling. (A) Effect of coumermycin in the absence of NaCl. E. coli JTT1, growing exponentially in LB lacking NaCl, was treated with water (lane a), dimethyl sulfoxide (lane b; dimethyl sulfoxide was the solvent for coumermycin Al), or dimethyl sulfoxide plus coumermycin A1 at 0.4 p.g/ml (lane c), 1.0 ,ug/ml (lane d), 4.0 ,ug/ml (lane e), 10 ,ug/ml (lane f), or 20 ,ug/ml (lane g) at a cell density of approximately 2 x 108/ml. Twenty minutes later cells were harvested and plasmids were isolated for supercoiling comparison as described for Fig. 1A, using 18 jig of chloroquine per ml during electrophoresis. (B) Effect of coumermycin on supercoiling after addition of NaCl. Strain JTT1, transformed with pUC9 and growing in the absence of NaCl, was treated with coumermycin as described for panel A, and 20 min later NaCl was added to 0.5 M for an additional 5 min. Plasmid DNA was extracted, and the topoisomers were displayed by gel electrophoresis as described for Fig. 1A. One sample (lane a) received only dimethyl sulfoxide and remained in medium lacking NaCl. Another (lane b) received only dimethyl sulfoxide, but the NaCl concentration was raised to 0.5 M. The other samples received dimethyl sulfoxide plus coumermycin A1 at 0.4 ,ug/ml (lane c), 1.0 ,ug/ml (lane d), 4.0 ,ug/ml (lane e), 10 ,ug/ml (lane f), or 20 ,ug/ml (lane g) and 20 min later received NaCl. (C) Time course in the absence of coumermycin. Strain JTT1 was grown in LB medium lacking NaCl and then exposed to 0.5 M NaCl for various times. Plasmid DNA was isolated, and the topoisomers were separated by gel electrophoresis as described for Fig. 1A. The times of exposure to NaCl were 0 min (lane a), 5 min (lane b), 10 min (lane e), 15 min (lane d), 20 min (lane e), 40 min (lane f), and 60 min (lane g). (D) Time course following pretreatment with 0.4 ,ug of coumermycin per ml. Strain JTl1 transformed with pUC9 was treated with coumermycin at 0.4 ,ug/ml for 20 min, after which NaCl was added to 0.5 M for the same times as in panel C. Plasmid DNA was isolated and treated as for panel A. Similar results were obtained by treatment with 1.0 ,ug of coumermycin per ml.
of interest to examine the effect of NaCl on supercoiling in a topA mutant. Strain RS2, which contains the topA10 mutation in the same genetic background as JTT1 and has only 1% of the wild-type topoisomerase I relaxing activity (18), was transformed with pUC9 and grown in LB medium
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J. BACTERIOL.
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FIG. 3. Effect of mutation of HU on plasmid supercoiling. (A) Plasmid supercoiling in HU mutants. Mutations in hupA and hupB (10) were transduced into E. coli JTT1. Plasmid pUC9 was then introduced by transformation. Each strain was grown in LB medium to the mid-log phase, plasmid DNA was extracted, and plasmid topoisomers were separated by gel electrophoresis as described for Fig. 1A. Lanes a, strain JRY878 (hupB::KmD; b, strain JRY880 (hupA::Cmr); c, strain JRY892 (hupA::Cmr hupB::Kmr); d, strain JTT1 (wild type). (B) Plasmid supercoiling following a shift to 0.5 M NaCl. E. coli KD629 (hupA::Cmr hupB::Kmr) was grown and treated with NaCl for various times as described for Fig. 2C. Cells were harvested, plasmid was isolated, and plasmid topoisomers were separated by electrophoresis as described for Fig. 1A. The times of exposure to NaCl were 0 min (lane a), 5 min (lane b), 10 min (lane c), 15 min (lane d), 20 min (lane e), 40 min (lane f), 60 min (lane g), and 80 min (lane h).
lacking NaCi. NaCl was added to 0.5 M, and plasmid DNA extracted at various times for supercoil comparison. Supercoiling increased by the same amount seen with the wild-type strain, but little relaxation followed up to 40 min after the shift (data not shown). Thus, topoisomerase I has little influence on initiating the initial response of supercoiling to salt shock. However, it does appear to play a role in the recovery phase, which emphasizes that the [ATP]/[ADP] ratio is not the only factor influencing supercoiling following salt shock. Another protein that could influence supercoiling is the histonelike protein HU, through its ability to wrap DNA (for a review, see reference 4). The availability of mutations in the two genes encoding HU allowed us to examine whether the absence of HU affects the change in supercoiling introduced by salt shock. The loss of HU lowers supercoiling of extracted DNA by about 12% (Fig. 3A; compare lanes c and d), a value similar to that observed with Salmonella typhimurium (8). A slight reduction in supercoiling is also seen when only hupA is mutant (Fig. 3A, lane b); mutation of hupB has no noticeable effect on supercoiling (19; Fig. 3A, lane a). The absence of both genes encoding HU proteins appears to have little effect on the ability of salt shock to raise supercoiling, since mutant and wild-type responses were similar (compare Fig. 2C and 3B). Our data with extracted DNA do not allow us to distinguish between effects of salt on free superhelical tension and was
on constrained supercoils. Others, however, have used cruciform formation to assess intracellular superhelical tension, and they argue that the entire linking change associated with salt treatment is expressed as a change in tension (13). Thus, it is very likely that our measurements reflect changes in intracellular superhelical tension. If the intracellular superhelix density at low salt concentrations is 0.025 (see reference 13 and references therein), the addition of four superhelical turns to pUC9 because of salt shock would increase superhelix density by about 60%. This value is similar to that reported for a salt shift that followed extensive treatment with chloramphenicol (13); apparently protein synthesis is not involved in the salt-induced increase in supercoiling, a result consistent with the rapid response of supercoiling to salt shock that we observed. There is now evidence that two types of environmental shift, salt shock and anaerobic shock (9), rapidly alter the [ATP]/[ADP] ratio and DNA supercoiling. These correlations lead us to propose that changes in the [ATP]/[ADP] ratio affect gyrase in vivo as they do in vitro, thereby contributing to the control of supercoiling. Since the most striking effects of salt shock are transient, time-course studies should be useful for examining relationships among environmental change, cellular energetics, and DNA supercoiling. We thank Sam Kayman and Ellen Murphy for critical comments on the manuscript and Anna Almeida for technical assistance in constructing the HU mutants. This work was supported by grant PMB 8718115 from the National Science Foundation, grant URA 1139 from the Centre de la Recherche Scientifique, and the Association de la Recherche sur le Cancer.
REFERENCES 1. Chapman, A., L. Fall, and D. Atkinson. 1971. Adenylate energy charge in Escherichia coli during growth and starvation. J. Bacteriol. 108:1072-1086. 2. Cozzarelli, N. R. 1980. DNA gyrase and the supercoiling of DNA. Science 207:953-960. 3. DiNardo, S., K. A. Voelkel, R. Sternglanz, A. E. Reynolds, and A. Wright. 1982. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell 31:43-51. 4. Drlica, K., and J. Rouviere-Yaniv. 1987. Histonelike proteins of bacteria. Microbiol. Rev. 51:301-319. 5. GelHert, M. 1981. DNA topoisomerases. Annu. Rev. Biochem. 50:879-910. 6. Gellert, M., M. H. O'Dea, T. Itoh, and J. Tomizawa. 1976. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Natl. Acad. Sci. USA 73:44744478. 7. Higgins, C. F., C. J. Dorman, D. A. Stirling, L. Waddell, I. R. Booth, G. May, and E. Bremer. 1988. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell 52:569-584. 8. Hillyard, D. R., M. Edlund, K. Hughes, M. Marsh, and N. P. Higgins. 1990. Subunit-specific phenotypes of Salmonella typhimurium HU mutants. J. Bacteriol. 172:5402-5407. 9. Hsieh, L., R. M. Burger, and K. Drlica. Bacterial DNA supercoiling and [ATP]/[ADP]: changes associated with a transition to anaerobic growth. J. Mol. Biol., in press. 10. Huisman, O., M. Faelen, D. Girard, A. Jaffe, A. Toussaint, and J. Rouviere-Yaniv. 1989. Multiple defects in Escherichia coli mutants lacking HU protein. J. Bacteriol. 171:3704-3712. 11. Ish-Horwicz, D., and J. Burke. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9:2989-2998. 12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
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VOL. 173, 1991 13. McClellan, J., P. Boublikova, E. Palecek, and D. Lilley. 1990. Superhelical torsion in cellular DNA responds directly to environmental and genetic factors. Proc. Nati. Acad. Sci. USA 87:8373-8377. 14. Menzel, R., and M. Geliert. 1983. Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell 34:105-113. 15. Pruss, G., and K. Drlica. 1989. DNA supercoiling and prokaryotic transcription. Cell 56:521-523. 16. Pruss, G. J., S. H. Manes, and K. Drlica. 1982. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell 31:35-42. 17. Rahmouni, A. R., and R. D. Wells. 1989. Stabilization of Z DNA in vivo by localized supercoiling. Science 246:358-363. 18. Sternglanz, R., S. DiNardo, K. A. Voelkel, Y. Nishimura, Y. Hirota, A. K. Becherer, L. Zumstein, and J. C. Wang. 1981. Mutations in the gene coding for Escherichia coli DNA topoisomerase I affecting transcription and transposition. Proc. Natl. Acad. Sci. USA 78:2747-2751. 19. Storts, D., and A. Markovitz. 1988. Construction and character-
20. 21. 22.
23. 24.
25.
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ization of mutations in hupB, the gene encoding HU-P (HU-1) in Escherichia coli K-12. J. Bacteriol. 170:1541-1547. Sugino, A., N. Higgins, P. Brown, C. Peebles, and N. Cozzarelli. 1978. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc. Natl. Acad. Sci. USA 75:4838-4842. Treisman, R. 1985. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell 42:889-902. Tse-Dinh, Y. 1985. Regulation of the Escherichia coli DNA topoisomerase I gene by DNA supercoiling. Nucleic Acids Res. 13:4751-4763. Tse-Dinh, Y., and R. Beran. 1988. Multiple promoters for transcription of the E. coli DNA topoisomerase I gene and their regulation by DNA supercoiling. J. Mol. Biol. 202:735-742. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268. Westerhoff, H., M. O'Dea, A. Maxwell, and M. Gellert. 1988. DNA supercoiling by DNA gyrase. A static head analysis. Cell Biophys. 12:157-181.
