1887

Abstract

The peptide wrwycr inhibits Holliday junction resolution and is a potent antimicrobial. To study the physiological effects of wrwycr treatment on cells, we partially screened the Keio collection of knockout mutants for those with increased sensitivity to wrwycr. Strains lacking part of the ferric-enterobactin (iron-bound siderophore) uptake and utilization system, parts of the enterobactin synthesis pathway, TolC (an outer-membrane channel protein) or Fur (an iron-responsive regulator) were hypersensitive to wrwycr. We provide evidence that the Δ mutant was hypersensitive to wrwycr due to its reduced ability to efflux wrwycr from the cell rather than due to its export of newly synthesized enterobactin. Deleting , which encodes a small RNA involved in iron regulation, mostly relieved the wrwycr hypersensitivity of the and ferric-enterobactin uptake mutants, indicating that the altered regulation of a RyhB-controlled gene was at least partly responsible for the hypersensitivity of these strains. Chelatable iron in the cell, measured by electron paramagnetic resonance spectroscopy, increased dramatically following wrwycr treatment, as did expression of Fur-repressed genes and, to some extent, mutation frequency. These incongruous results suggest that while wrwycr treatment caused accumulation of chelatable iron in the cell, iron was not available to bind to Fur. This is corroborated by the observed induction of the system, which assembles iron–sulfur clusters in low-iron conditions. Disruption of iron metabolism by wrwycr, in addition to its effects on DNA repair, may make it a particularly effective antimicrobial in the context of the low-iron environment of a mammalian host.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.054361-0
2012-02-01
2020-12-04
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/2/547.html?itemId=/content/journal/micro/10.1099/mic.0.054361-0&mimeType=html&fmt=ahah

References

  1. Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K. A., Tomita M., Wanner B. L., Mori H.. ( 2006;). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol2: [CrossRef][PubMed]
    [Google Scholar]
  2. Bagg A., Neilands J. B.. ( 1987;). Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli . Biochemistry26:5471–5477 [CrossRef][PubMed]
    [Google Scholar]
  3. Bleuel C., Grosse C., Taudte N., Scherer J., Wesenberg D., Krauss G. J., Nies D. H., Grass G.. ( 2005;). TolC is involved in enterobactin efflux across the outer membrane of Escherichia coli . J Bacteriol187:6701–6707 [CrossRef][PubMed]
    [Google Scholar]
  4. Boal A. K., Yavin E., Lukianova O. A., O’Shea V. L., David S. S., Barton J. K.. ( 2005;). DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters. Biochemistry44:8397–8407 [CrossRef][PubMed]
    [Google Scholar]
  5. Boldt J. L., Pinilla C., Segall A. M.. ( 2004;). Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution. J Biol Chem279:3472–3483 [CrossRef][PubMed]
    [Google Scholar]
  6. Brickman T. J., McIntosh M. A.. ( 1992;). Overexpression and purification of ferric enterobactin esterase from Escherichia coli. Demonstration of enzymatic hydrolysis of enterobactin and its iron complex. J Biol Chem267:12350–12355[PubMed]
    [Google Scholar]
  7. Buss K., Müller R., Dahm C., Gaitatzis N., Skrzypczak-Pietraszek E., Lohmann S., Gassen M., Leistner E.. ( 2001;). Clustering of isochorismate synthase genes menF and entC and channeling of isochorismate in Escherichia coli . Biochim Biophys Acta1522:151–157[PubMed][CrossRef]
    [Google Scholar]
  8. Campoy S., Jara M., Busquets N., de Rozas A. M., Badiola I., Barbé J.. ( 2002;). Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants. Microbiology148:1039–1048[PubMed]
    [Google Scholar]
  9. Cherepanov P. P., Wackernagel W.. ( 1995;). Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene158:9–14 [CrossRef][PubMed]
    [Google Scholar]
  10. Cunningham R. P., Asahara H., Bank J. F., Scholes C. P., Salerno J. C., Surerus K., Münck E., McCracken J., Peisach J., Emptage M. H.. ( 1989;). Endonuclease III is an iron–sulfur protein. Biochemistry28:4450–4455 [CrossRef][PubMed]
    [Google Scholar]
  11. Daruwala R., Kwon O., Meganathan R., Hudspeth M. E.. ( 1996;). A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene. FEMS Microbiol Lett140:159–163 [CrossRef][PubMed]
    [Google Scholar]
  12. Davis R. W., Botstein D., Roth J. R.. ( 1980;). Advanced Bacterial Genetics: a Manual for Genetic Engineering Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  13. Fenton H.. ( 1894;). Oxidation of tartaric acid in presence of iron. J Chem Soc Trans65:899 [CrossRef]
    [Google Scholar]
  14. Forbes J. R., Gros P.. ( 2001;). Divalent-metal transport by NRAMP proteins at the interface of host–pathogen interactions. Trends Microbiol9:397–403 [CrossRef][PubMed]
    [Google Scholar]
  15. Furrer J. L., Sanders D. N., Hook-Barnard I. G., McIntosh M. A.. ( 2002;). Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter. Mol Microbiol44:1225–1234 [CrossRef][PubMed]
    [Google Scholar]
  16. Goetz D. H., Holmes M. A., Borregaard N., Bluhm M. E., Raymond K. N., Strong R. K.. ( 2002;). The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell10:1033–1043 [CrossRef][PubMed]
    [Google Scholar]
  17. Gu M., Imlay J. A.. ( 2011;). The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide. Mol Microbiol79:1136–1150 [CrossRef][PubMed]
    [Google Scholar]
  18. Guerinot M. L.. ( 1994;). Microbial iron transport. Annu Rev Microbiol48:743–772 [CrossRef][PubMed]
    [Google Scholar]
  19. Gunderson C. W., Segall A. M.. ( 2006;). DNA repair, a novel antibacterial target: Holliday junction-trapping peptides induce DNA damage and chromosome segregation defects. Mol Microbiol59:1129–1148 [CrossRef][PubMed]
    [Google Scholar]
  20. Gunderson C. W., Boldt J. L., Authement R. N., Segall A. M.. ( 2009;). Peptide wrwycr inhibits the excision of several prophages and traps Holliday junctions inside bacteria. J Bacteriol191:2169–2176 [CrossRef][PubMed]
    [Google Scholar]
  21. Haber F., Weiss J.. ( 1932;). Über die Katalyse des Hydroperoxydes. Naturwiss20:948–950 [CrossRef]
    [Google Scholar]
  22. Haber F., Weiss J.. ( 1934;). The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Lond A Math Phys Sci147:332–351 [CrossRef]
    [Google Scholar]
  23. Haber F., Willstätter R.. ( 1931;). Unpaarigkeit und Radikalketten im Reaktionsmechanismus organischer und enzymatischer Vorgänge. Chem Ber64:2844–2856[CrossRef]
    [Google Scholar]
  24. Halliwell B., Gutteridge J. M.. ( 1984;). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J219:1–14[PubMed]
    [Google Scholar]
  25. Hantke K.. ( 1990;). Dihydroxybenzoylserine – a siderophore for E. coli . FEMS Microbiol Lett55:5–8[PubMed]
    [Google Scholar]
  26. Hubbard J. A., Lewandowska K. B., Hughes M. N., Poole R. K.. ( 1986;). Effects of iron-limitation of Escherichia coli on growth, the respiratory chains and gallium uptake. Arch Microbiol146:80–86 [CrossRef][PubMed]
    [Google Scholar]
  27. Imlay J. A., Chin S. M., Linn S.. ( 1988;). Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro . Science240:640–642 [CrossRef][PubMed]
    [Google Scholar]
  28. Jonnalagadda U. M.. ( 2009;).The effects of antibacterial peptides on bacterial and membrane integrity and their mechanism of cellular entry
  29. Kepple K. V., Boldt J. L., Segall A. M.. ( 2005;). Holliday junction-binding peptides inhibit distinct junction-processing enzymes. Proc Natl Acad Sci U S A102:6867–6872 [CrossRef][PubMed]
    [Google Scholar]
  30. Kohanski M. A., Dwyer D. J., Hayete B., Lawrence C. A., Collins J. J.. ( 2007;). A common mechanism of cellular death induced by bactericidal antibiotics. Cell130:797–810 [CrossRef][PubMed]
    [Google Scholar]
  31. Koronakis V.. ( 2003;). TolC – the bacterial exit duct for proteins and drugs. FEBS Lett555:66–71 [CrossRef][PubMed]
    [Google Scholar]
  32. Koronakis V., Eswaran J., Hughes C.. ( 2004;). Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu Rev Biochem73:467–489 [CrossRef][PubMed]
    [Google Scholar]
  33. Li Y., Schellhorn H. E.. ( 2007;). Rapid kinetic microassay for catalase activity. J Biomol Tech18:185–187[PubMed]
    [Google Scholar]
  34. Lino M., Kus J. V., Tran S. L., Naqvi Z., Binnington B., Goodman S. D., Segall A. M., Foster D. B.. ( 2011;). A novel antimicrobial peptide significantly enhances acid-induced killing of Shiga toxin-producing Escherichia coli O157 and non-O157 serotypes. Microbiology157:1768–1775 [CrossRef][PubMed]
    [Google Scholar]
  35. Lukianova O. A., David S. S.. ( 2005;). A role for iron–sulfur clusters in DNA repair. Curr Opin Chem Biol9:145–151 [CrossRef][PubMed]
    [Google Scholar]
  36. Massé E., Gottesman S.. ( 2002;). A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli . Proc Natl Acad Sci U S A99:4620–4625 [CrossRef][PubMed]
    [Google Scholar]
  37. Massé E., Escorcia F. E., Gottesman S.. ( 2003;). Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli . Genes Dev17:2374–2383 [CrossRef][PubMed]
    [Google Scholar]
  38. Massé E., Vanderpool C. K., Gottesman S.. ( 2005;). Effect of RyhB small RNA on global iron use in Escherichia coli . J Bacteriol187:6962–6971 [CrossRef][PubMed]
    [Google Scholar]
  39. Miller J. H.. ( 1972;). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  40. Nairz M., Theurl I., Ludwiczek S., Theurl M., Mair S. M., Fritsche G., Weiss G.. ( 2007;). The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium . Cell Microbiol9:2126–2140 [CrossRef][PubMed]
    [Google Scholar]
  41. Neidhardt F. C., Bloch P. L., Smith D. F.. ( 1974;). Culture medium for enterobacteria. J Bacteriol119:736–747[PubMed]
    [Google Scholar]
  42. Neilands J. B.. ( 1981;). Iron absorption and transport in microorganisms. Annu Rev Nutr1:27–46 [CrossRef][PubMed]
    [Google Scholar]
  43. Outten F. W., Djaman O., Storz G.. ( 2004;). A suf operon requirement for Fe–S cluster assembly during iron starvation in Escherichia coli . Mol Microbiol52:861–872 [CrossRef][PubMed]
    [Google Scholar]
  44. Prévost K., Salvail H., Desnoyers G., Jacques J. F., Phaneuf E., Massé E.. ( 2007;). The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis. Mol Microbiol64:1260–1273 [CrossRef][PubMed]
    [Google Scholar]
  45. Raymond K. N., Dertz E. A., Kim S. S.. ( 2003;). Enterobactin: an archetype for microbial iron transport. Proc Natl Acad Sci U S A100:3584–3588 [CrossRef][PubMed]
    [Google Scholar]
  46. Ren B., Duan X., Ding H.. ( 2009;). Redox control of the DNA damage-inducible protein DinG helicase activity via its iron-sulfur cluster. J Biol Chem284:4829–4835 [CrossRef][PubMed]
    [Google Scholar]
  47. Schwyn B., Neilands J. B.. ( 1987;). Universal chemical assay for the detection and determination of siderophores. Anal Biochem160:47–56 [CrossRef][PubMed]
    [Google Scholar]
  48. Su L. Y., Willner D. L., Segall A. M.. ( 2010;). An antimicrobial peptide that targets DNA repair intermediates in vitro inhibits Salmonella growth within murine macrophages. Antimicrob Agents Chemother54:1888–1899 [CrossRef][PubMed]
    [Google Scholar]
  49. Vassinova N., Kozyrev D.. ( 2000;). A method for direct cloning of Fur-regulated genes: identification of seven new Fur-regulated loci in Escherichia coli . Microbiology146:3171–3182[PubMed]
    [Google Scholar]
  50. Woodmansee A. N., Imlay J. A.. ( 2002;). Quantitation of intracellular free iron by electron paramagnetic resonance spectroscopy. Methods Enzymol349:3–9 [CrossRef][PubMed]
    [Google Scholar]
  51. Yeo W. S., Lee J. H., Lee K. C., Roe J. H.. ( 2006;). IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe–S assembly proteins. Mol Microbiol61:206–218 [CrossRef][PubMed]
    [Google Scholar]
  52. Zhang Z., Gosset G., Barabote R., Gonzalez C. S., Cuevas W. A., Saier M. H. Jr. ( 2005;). Functional interactions between the carbon and iron utilization regulators, Crp and Fur, in Escherichia coli . J Bacteriol187:980–990 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.054361-0
Loading
/content/journal/micro/10.1099/mic.0.054361-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error