This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, X.
Right arrow Articles by Drlica, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, X.
Right arrow Articles by Drlica, K.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, January 2006, p. 362-364, Vol. 50, No. 1
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.1.362-364.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Lethal Action of Quinolones against a Temperature-Sensitive dnaB Replication Mutant of Escherichia coli

Xilin Zhao, Muhammad Malik, Nymph Chan, Alex Drlica-Wagner, Jian-Ying Wang, Xinying Li, and Karl Drlica*

Public Health Research Institute, 225 Warren St., Newark, New Jersey 07103

Received 8 September 2005/ Returned for modification 6 October 2005/ Accepted 25 October 2005


arrow
ABSTRACT
 
Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.


arrow
TEXT
 
Quinolones are broad-spectrum antibacterials that trap DNA gyrase and DNA topoisomerase IV on DNA as ternary complexes containing double-strand DNA breaks (reviewed in reference 5). The ternary complexes, which block DNA replication (8, 23), RNA synthesis (13, 24), and cell growth (7), lead to cell death by poorly understood processes. Quinolone-mediated inhibition of DNA replication, which is very rapid (19), is unlikely to account for rapid lethality because inhibition is reversible (4, 8) and because concurrent interruption of RNA or protein synthesis eliminates the lethal action of some quinolones but not their ability to block DNA replication (2, 4). A different issue is whether ongoing DNA replication is required for quinolone lethality. Such a hypothesis could be derived from studies with other topoisomerases in which collision of replication forks with ternary complexes is thought to release double-strand DNA breaks (3, 21). To address this possibility with quinolones, we blocked DNA replication by shifting a dnaB mutant of Escherichia coli to nonpermissive temperature. (DnaB is a DNA helicase [11] required for replication fork movement [1].) Subsequent treatment with quinolone killed the mutant, indicating that ongoing DNA replication is not required.

E. coli strain K12SH-28 and its dnaB-22(Ts) mutant FA22 (6) were obtained from the E. coli Stock Center (Yale University) and were grown at 28°C in M9-glucose minimal medium (14) supplemented with 0.05% Casamino Acids and 1% LB medium (14). A gyrA quinolone-resistant mutant of FA22, strain KD2672, was constructed by P1-mediated transduction (22) from strain KD2346, a spontaneous ciprofloxacin-resistant derivative of KD1366 (26) that contained GyrA amino acid substitutions Ser-83 to Leu and Asp-87 to Tyr. The dnaB-22 allele was transduced into strain DM4100 (20) using a nearby tetracycline resistance marker (malF-3089:Tn10) to produce strain KD2773. Nalidixic acid, chloramphenicol, and tetracycline were products of Sigma Chemical Co. (St. Louis, MO); ciprofloxacin was obtained from Bayer Corp. (West Haven, CT). Bactericidal activity was determined by incubation in the presence of quinolone followed by dilution and growth on drug-free agar at 28°C for 1 to 2 days. The rate of DNA synthesis was measured by incubating 100 µl bacterial culture with 0.1 µCi [3H]thymidine for 2 min followed by determination of acid-precipitable radioactivity.

When the dnaB-22 mutant was grown at 28°C and shifted to 42°C, the rate of DNA synthesis dropped by more than 95% within 10 min and was not inhibited further by subsequent nalidixic acid treatment (not shown). Under these conditions, the viable cell number remained constant for more than 2 h (Fig. 1A). If nalidixic acid was added to 60 mg/liter 15 min after the shift to 42°C and cultures were incubated for 2 h, the number of viable cells dropped by 100-fold (Fig. 1A). Under comparable conditions, the parental strain, K12SH-28, grew at 42°C and was also killed by nalidixic acid (Fig. 1A). The two strains were killed with similar kinetics, although with a long incubation time the mutant was killed slightly faster. For both strains, killing started sooner at 42°C than at 28°C. When the two strains were treated with various concentrations of nalidixic acid for a fixed time (105 min), the dnaB-22 mutant was slightly more susceptible than the wild-type strain at most drug concentrations tested (Fig. 1B). The slightly greater susceptibility observed with the mutant may reflect a defect in repair or bypass of quinolone-mediated DNA lesions, since the DnaB protein has been implicated in replication fork restart (10, 16). We established that nalidixic acid kills the dnaB-22 mutant through an interaction with DNA gyrase by treating a dnaB-22 gyrA double mutant (strain KD2672) with nalidixic acid and showing that nalidixic acid failed to kill this strain at either 28°C or 42°C (Fig. 1B).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 1. Bactericidal action of nalidixic acid with a dnaB-22 replication mutant. (A) Effect of nalidixic acid treatment time. E. coli strains K12SH-28 (wild type [wt]) and FA22 (dnaB-22) were grown at 28°C, and part of the culture was shifted to 42°C (wild type, triangles; dnaB mutant, inverted triangles). After 15 min, aliquots of K12SH-28 (circles) and FA22 (squares) at 28°C (open symbols) and 42°C (filled symbols) were treated for the indicated times with 60 mg/liter nalidixic acid, after which cells were plated on drug-free agar to determine percent survival. Similar results were obtained in a replicate experiment. (B) Effect of nalidixic acid concentration. The E. coli strains used in panel A were treated with the indicated concentrations of nalidixic acid for 105 min at 28°C (open symbols) or following a shift to 42°C (filled symbols) for strain K12SH-28 (wild type; circles), FA22 (dnaB-22; squares), and KD2672 (dnaB-22 gyrA S83L D87Y; diamonds). Similar results were obtained in a replicate experiment.

We also confirmed that DNA replication has little effect on the role of ongoing protein synthesis in nalidixic acid-mediated lethality. For such a test, dnaB-22 was transferred into a different E. coli strain, since K12SH-28 and FA22 are both hypersusceptible to chloramphenicol, an inhibitor of protein synthesis. The new dnaB-22 strain, KD2773, exhibited temperature-dependent cessation of DNA synthesis, as expected (not shown). When chloramphenicol was added to a culture of strain KD2773 after incubation at 42°C for 5 min, followed by nalidixic acid 10 min later, killing by nalidixic acid was largely inhibited (Fig. 2A). A similar effect was observed with the parental strain DM4100 (Fig. 2A). Chloramphenicol protects only partially from lethality due to ciprofloxacin (12), as seen with both the dnaB-22 mutant and the parental strain (Fig. 2B). Thus, the protective effect of chloramphenicol, which depends on quinolone structure (18), can be observed when DNA replication is blocked. For reasons that are not understood, reduction of ciprofloxacin-mediated lethality by chloramphenicol was less with the mutant (Fig. 2B).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 2. Effect of chloramphenicol on quinolone-mediated lethality with a dnaB-22 mutant. (A) Effect of chloramphenicol on survival in the presence of nalidixic acid. Cells were grown as in Fig. 1 at 28°C and shifted to 42°C for 5 min, after which zero (filled symbols) or 10 mg/liter chloramphenicol (open symbols) was added for an additional 10 min followed by the indicated concentration of nalidixic acid for 105 min. The strains were DM4100 (wild type [wt]; circles) and KD2773 (dnaB-22; squares). Similar results were obtained in a replicate experiment. (B) Effect of chloramphenicol on survival in the presence of ciprofloxacin. Cells were grown as in Fig. 1 at 28°C and shifted to 42°C for 5 min, after which zero (filled symbols) or 10 mg/liter chloramphenicol (open symbols) was added for an additional 10 min followed by the indicated concentration of ciprofloxacin for 30 min. The strains were DM4100 (wild type; circles) and KD2773 (dnaB-22; squares). Similar results were obtained in a replicate experiment.

Rapid bactericidal action of quinolones is thought to arise from release of double-strand DNA breaks from ternary complexes formed by quinolone, gyrase/topoisomerase IV, and DNA (2, 9). The experiments described above indicate that such a release of DNA breaks is not necessarily caused by collision of replication forks with the complexes because inhibition of replication, through temperature shift of a dnaB-22(Ts) replication mutant, failed to block the lethal action of nalidixic acid (Fig. 1). This conclusion is consistent with biochemical studies in which DNA breaks fail to be released by the collision of replication forks with quinolone-topoisomerase IV-DNA complexes (17). Since a 2-h shift of a dnaC-2 replication-initiation mutant to nonpermissive temperature failed to block the lethal activity of gatifloxacin (not shown), it is unlikely that the loss of dnaB-specific repair causes quinolone-mediated cell death when replication is halted. We conclude that replication fork blockage by quinolones is neither necessary nor sufficient for rapid quinolone lethality. Whether lethal chromosome fragmentation is generated by gyrase (2) or by an endonuclease (15, 25) is unknown.

Since fluoroquinolones can kill bacteria that are not undergoing DNA or protein synthesis, the possibility exists that some quinolone derivatives may be effective at killing nongrowing cells. Such a finding could have clinical importance for diseases, such as tuberculosis, that are characterized by the presence of persisting cells, since those cells may contribute to relapse and the development of antimicrobial resistance.


arrow
ACKNOWLEDGMENTS
 
We thank Marila Gennaro and Richard Pine for critical comments on the manuscript.

This work was supported by NIH grants AI35257 and AIO63431.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Public Health Research Institute, 225 Warren St., Newark, NJ 07103. Phone: (973) 854-3360. E-mail: drlica{at}phri.org. Back


arrow
REFERENCES
 
    1
  1. Caruthers, J. M., and D. B. McKay. 2002. Helicase structure and mechanism. Curr. Opin. Struct. Biol. 12:123-133.[CrossRef][Medline]
    2. 2
  2. Chen, C.-R., M. Malik, M. Snyder, and K. Drlica. 1996. DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage. J. Mol. Biol. 258:627-637.[CrossRef][Medline]
    3. 3
  3. D'Arpa, P., C. Beardmore, and L. F. Liu. 1990. Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res. 50:6919-6924.[Abstract/Free Full Text]
    4. 4
  4. Deitz, W. H., T. M. Cook, and W. A. Goss. 1966. Mechanism of action of nalidixic acid on Escherichia coli. III. Conditions required for lethality. J. Bacteriol. 91:768-773.[Abstract/Free Full Text]
    5. 5
  5. Drlica, K., and D. C. Hooper. 2003. Mechanisms of quinolone action, p. 19-40. In D. C. Hooper and E. Rubinstein (ed.), Quinolone antimicrobial agents, 3rd ed. American Society for Microbiology, Washington, D.C.
    6. 6
  6. Fangman, W., and A. Novick. 1968. Characterization of two bacterial mutants with temperature-sensitive synthesis of DNA. Genetics 60:1-17.[Free Full Text]
    7. 7
  7. Goss, W. A., W. H. Deitz, and T. M. Cook. 1964. Mechanism of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88:1112-1118.[Abstract/Free Full Text]
    8. 8
  8. Goss, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074.[Abstract/Free Full Text]
    9. 9
  9. Hiasa, H., D. Yousef, and K. Marians. 1996. DNA strand cleavage is required for replication fork arrest by a frozen topoisomerase-quinolone-DNA ternary complex. J. Biol. Chem. 271:26424-26429.[Abstract/Free Full Text]
    10. 10
  10. Jaktaji, R. P., and R. G. Lloyd. 2003. PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells. Mol. Microbiol. 47:1091-1100.[CrossRef][Medline]
    11. 11
  11. LeBowitz, J. H., and R. McMacken. 1986. The Escherichia coli dnaB replication protein is a DNA helicase. J. Biol. Chem. 261:4738-4748.[Abstract/Free Full Text]
    12. 12
  12. Lewin, C., B. Howard, and J. Smith. 1991. Protein- and RNA-synthesis independent bactericidal activity of ciprofloxacin that involves the A subunit of DNA gyrase. J. Med. Microbiol. 34:19-22.[Abstract/Free Full Text]
    13. 13
  13. Manes, S. H., G. J. Pruss, and K. Drlica. 1983. Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling. J. Bacteriol. 155:420-423.[Abstract/Free Full Text]
    14. 14
  14. Miller, J. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    15. 15
  15. Pohlhaus, J., and K. N. Kreuzer. 2005. Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo. Mol. Microbiol. 56:1416-1429.[Medline]
    16. 16
  16. Sandler, S. J., and K. J. Marians. 2000. Role of PriA in replication fork reactivation in Escherichia coli. J. Bacteriol. 182:9-13.[Free Full Text]
    17. 17
  17. Shea, M., and H. Hiasa. 1999. Interactions between DNA helicases and frozen topoisomerase IV-quinolone-DNA ternary complexes. J. Biol. Chem. 274:22747-22754.[Abstract/Free Full Text]
    18. 18
  18. Smith, J. T., and C. S. Lewin. 1988. Chemistry and mechanisms of action of the quinolone antibacterials, p. 23-82. In V. T. Andriole (ed.), The quinolones, vol. 1. Academic Press, San Diego, Calif.
    19. 19
  19. Snyder, M., and K. Drlica. 1979. DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid. J. Mol. Biol. 131:287-302.[CrossRef][Medline]
    20. 20
  20. 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.[Abstract/Free Full Text]
    21. 21
  21. Tsao, Y.-P., A. Russo, G. Nyamuswa, R. Silber, and L. F. Liu. 1993. Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system. Cancer Res. 53:5908-5914.[Abstract/Free Full Text]
    22. 22
  22. Wall, J. D., and P. D. Harriman. 1974. Phage P1 mutants with altered transducing abilities for Escherichia coli. Virology 59:532-544.[CrossRef][Medline]
    23. 23
  23. Wentzell, L., and A. Maxwell. 2000. The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex. J. Mol. Biol. 304:779-791.[CrossRef][Medline]
    24. 24
  24. Willmott, C. J. R., S. E. Critchlow, I. C. Eperon, and A. Maxwell. 1994. The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase. J. Mol. Biol. 242:351-363.[CrossRef][Medline]
    25. 25
  25. Yang, S.-W., A. B. Burgin, B. N. Huizenga, C. A. Robertson, K. C. Yao, and H. A. Nash. 1996. A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases. Proc. Natl. Acad. Sci. USA 93:11534-11539.[Abstract/Free Full Text]
    26. 26
  26. Zhao, X., C. Xu, J. Domagala, and K. Drlica. 1997. DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance. Proc. Natl. Acad. Sci. USA 94:13991-13996.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, January 2006, p. 362-364, Vol. 50, No. 1
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.1.362-364.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Liu, I-F., Annamalai, T., Sutherland, J. H., Tse-Dinh, Y.-C. (2009). Hydroxyl Radicals Are Involved in Cell Killing by the Bacterial Topoisomerase I Cleavage Complex. J. Bacteriol. 191: 5315-5319 [Abstract] [Full Text]  
  • Tse-Dinh, Y.-C. (2009). Bacterial topoisomerase I as a target for discovery of antibacterial compounds. Nucleic Acids Res 37: 731-737 [Abstract] [Full Text]  
  • Pohlhaus, J. R., Long, D. T., O'Reilly, E., Kreuzer, K. N. (2008). The {varepsilon} Subunit of DNA Polymerase III Is Involved in the Nalidixic Acid-Induced SOS Response in Escherichia coli. J. Bacteriol. 190: 5239-5247 [Abstract] [Full Text]  
  • Drlica, K., Malik, M., Kerns, R. J., Zhao, X. (2008). Quinolone-Mediated Bacterial Death. Antimicrob. Agents Chemother. 52: 385-392 [Full Text]  
  • Grossman, T. H., Bartels, D. J., Mullin, S., Gross, C. H., Parsons, J. D., Liao, Y., Grillot, A.-L., Stamos, D., Olson, E. R., Charifson, P. S., Mani, N. (2007). Dual Targeting of GyrB and ParE by a Novel Aminobenzimidazole Class of Antibacterial Compounds. Antimicrob. Agents Chemother. 51: 657-666 [Abstract] [Full Text]  
  • Reckinger, A. R., Jeong, K. S., Khodursky, A. B., Hiasa, H. (2007). RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli. Nucleic Acids Res 35: 79-86 [Abstract] [Full Text]  
  • Malik, M., Hussain, S., Drlica, K. (2007). Effect of Anaerobic Growth on Quinolone Lethality with Escherichia coli. Antimicrob. Agents Chemother. 51: 28-34 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, X.
Right arrow Articles by Drlica, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, X.
Right arrow Articles by Drlica, K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»