1887

Abstract

In this study we characterized a genetic locus that is predicted to encode one of the three AraC-like regulators of a homologue of MpeR of which is specific to the pathogenic species. Previous microarray studies have suggested that this gene is a member of the Fur regulon. In strain MC58, it is a pseudogene (annotated as two ORFs, NMB1879 and NMB1878) containing a frameshift mutation which we show is common to all strains tested belonging to the ST-32 hypervirulent clonal complex. Using primer extension and S1 nuclease protection assays, we mapped two promoters in the upstream intergenic region: the promoter and the NMB1880 promoter. The latter promoter drives transcription of the divergent upstream locus, which is predicted to encode a high-affinity iron uptake system. We demonstrated that both promoters are induced during iron limitation and that this regulation is also mediated by the Fur regulator. DNA-binding studies with the purified MpeR protein revealed that it binds to a region directly upstream of the NMB1880 divergent promoter, suggesting a role in its regulation. Mutants of strains lacking MpeR or overexpressing MpeR showed no significant differences in expression of the P promoter, nor did global transcriptional profiling of an MpeR knockout identify any deregulated genes, suggesting that the MpeR protein is inactive under the conditions used in these experiments. The presence of MpeR in a regulatory cascade downstream of the Fur master iron regulator implicates it as being expressed in the iron-limiting environment of the host, where it may in turn regulate a group of genes, including the divergent iron transport locus, in response to signals important for infection.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.048033-0
2011-08-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/8/2235.html?itemId=/content/journal/micro/10.1099/mic.0.048033-0&mimeType=html&fmt=ahah

References

  1. Agarwal S., Sebastian S., Szmigielski B., Rice P. A., Genco C. A.. ( 2008; ). Expression of the gonococcal global regulatory protein Fur and genes encompassing the Fur and iron regulon during in vitro and in vivo infection in women. . J Bacteriol 190:, 3129–3139. [CrossRef].[PubMed]
    [Google Scholar]
  2. Anderson M. T., Armstrong S. K.. ( 2004; ). The BfeR regulator mediates enterobactin-inducible expression of Bordetella enterobactin utilization genes. . J Bacteriol 186:, 7302–7311. [CrossRef].[PubMed]
    [Google Scholar]
  3. Banerjee-Bhatnagar N., Frasch C. E.. ( 1990; ). Expression of Neisseria meningitidis iron-regulated outer membrane proteins, including a 70-kilodalton transferrin receptor, and their potential for use as vaccines. . Infect Immun 58:, 2875–2881.[PubMed]
    [Google Scholar]
  4. Brickman T. J., Armstrong S. K.. ( 2009; ). Temporal signaling and differential expression of Bordetella iron transport systems: the role of ferrimones and positive regulators. . Biometals 22:, 33–41. [CrossRef].[PubMed]
    [Google Scholar]
  5. Busby S., Ebright R. H.. ( 1994; ). Promoter structure, promoter recognition, and transcription activation in prokaryotes. . Cell 79:, 743–746. [CrossRef].[PubMed]
    [Google Scholar]
  6. Calver G. A., Kenny C. P., Lavergne G.. ( 1976; ). Iron as a replacement for mucin in the establishment of meningococcal infection in mice. . Can J Microbiol 22:, 832–838. [CrossRef].[PubMed]
    [Google Scholar]
  7. Carson S. D., Klebba P. E., Newton S. M., Sparling P. F.. ( 1999; ). Ferric enterobactin binding and utilization by Neisseria gonorrhoeae . . J Bacteriol 181:, 2895–2901.[PubMed]
    [Google Scholar]
  8. Childers B. M., Klose K. E.. ( 2007; ). Regulation of virulence in Vibrio cholerae: the ToxR regulon. . Future Microbiol 2:, 335–344. [CrossRef].[PubMed]
    [Google Scholar]
  9. Delany I., Ieva R., Alaimo C., Rappuoli R., Scarlato V.. ( 2003; ). The iron-responsive regulator fur is transcriptionally autoregulated and not essential in Neisseria meningitidis . . J Bacteriol 185:, 6032–6041. [CrossRef] [PubMed]
    [Google Scholar]
  10. Delany I., Grifantini R., Bartolini E., Rappuoli R., Scarlato V.. ( 2006; ). Effect of Neisseria meningitidis fur mutations on global control of gene transcription. . J Bacteriol 188:, 2483–2492. [CrossRef].[PubMed]
    [Google Scholar]
  11. Ducey T. F., Carson M. B., Orvis J., Stintzi A. P., Dyer D. W.. ( 2005; ). Identification of the iron-responsive genes of Neisseria gonorrhoeae by microarray analysis in defined medium. . J Bacteriol 187:, 4865–4874. [CrossRef].[PubMed]
    [Google Scholar]
  12. Egan S. M.. ( 2002; ). Growing repertoire of AraC/XylS activators. . J Bacteriol 184:, 5529–5532. [CrossRef].[PubMed]
    [Google Scholar]
  13. Escolar G., Lozano M., Díaz-Ricart M., Rao G. H., Ordinas A., White J. G.. ( 1999a; ). Modifications in accessibility of membrane glycoproteins, binding of specific ligands and coagulation factor V during the activation of platelets in blood emerging from bleeding time wounds. . Am J Hematol 60:, 260–267. [CrossRef].[PubMed]
    [Google Scholar]
  14. Escolar L., Pérez-Martín J., de Lorenzo V.. ( 1999b; ). Opening the iron box: transcriptional metalloregulation by the Fur protein. . J Bacteriol 181:, 6223–6229.[PubMed]
    [Google Scholar]
  15. Fantappiè L., Metruccio M. M., Seib K. L., Oriente F., Cartocci E., Ferlicca F., Giuliani M. M., Scarlato V., Delany I.. ( 2009; ). The RNA chaperone Hfq is involved in stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression. . Infect Immun 77:, 1842–1853. [CrossRef].[PubMed]
    [Google Scholar]
  16. Fantappiè L., Oriente F., Muzzi A., Serruto D., Scarlato V., Delany I.. ( 2011; ). A novel Hfq-dependent sRNA that is under FNR control and is synthesized in oxygen limitation in Neisseria meningitidis . . Mol Microbiol 80:, 507–523. [CrossRef].[PubMed]
    [Google Scholar]
  17. Fetherston J. D., Bearden S. W., Perry R. D.. ( 1996; ). YbtA, an AraC-type regulator of the Yersinia pestis pesticin/yersiniabactin receptor. . Mol Microbiol 22:, 315–325. [CrossRef].[PubMed]
    [Google Scholar]
  18. Finlay B. B., Falkow S.. ( 1997; ). Common themes in microbial pathogenicity revisited. . Microbiol Mol Biol Rev 61:, 136–169.[PubMed]
    [Google Scholar]
  19. Folster J. P., Shafer W. M.. ( 2005; ). Regulation of mtrF expression in Neisseria gonorrhoeae and its role in high-level antimicrobial resistance. . J Bacteriol 187:, 3713–3720. [CrossRef].[PubMed]
    [Google Scholar]
  20. Fowler M. I., Weller R. O., Heckels J. E., Christodoulides M.. ( 2004; ). Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges. . Cell Microbiol 6:, 555–567. [CrossRef].[PubMed]
    [Google Scholar]
  21. Francis M. S., Wolf-Watz H., Forsberg A.. ( 2002; ). Regulation of type III secretion systems. . Curr Opin Microbiol 5:, 166–172. [CrossRef].[PubMed]
    [Google Scholar]
  22. Gallegos M. T., Williams P. A., Ramos J. L.. ( 1997; ). Transcriptional control of the multiple catabolic pathways encoded on the TOL plasmid pWW53 of Pseudomonas putida MT53. . J Bacteriol 179:, 5024–5029.[PubMed]
    [Google Scholar]
  23. Gao H., Zhou D., Li Y., Guo Z., Han Y., Song Y., Zhai J., Du Z., Wang X. et al. ( 2008; ). The iron-responsive Fur regulon in Yersinia pestis . . J Bacteriol 190:, 3063–3075. [CrossRef].[PubMed]
    [Google Scholar]
  24. Grifantini R., Sebastian S., Frigimelica E., Draghi M., Bartolini E., Muzzi A., Rappuoli R., Grandi G., Genco C. A.. ( 2003; ). Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B. . Proc Natl Acad Sci U S A 100:, 9542–9547. [CrossRef].[PubMed]
    [Google Scholar]
  25. Hagen T. A., Cornelissen C. N.. ( 2006; ). Neisseria gonorrhoeae requires expression of TonB and the putative transporter TdfF to replicate within cervical epithelial cells. . Mol Microbiol 62:, 1144–1157. [CrossRef].[PubMed]
    [Google Scholar]
  26. Hanahan D.. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. . J Mol Biol 166:, 557–580. [CrossRef].[PubMed]
    [Google Scholar]
  27. Heinrichs D. E., Poole K.. ( 1996; ). PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor. . J Bacteriol 178:, 2586–2592.[PubMed]
    [Google Scholar]
  28. Ieva R., Alaimo C., Delany I., Spohn G., Rappuoli R., Scarlato V.. ( 2005; ). CrgA is an inducible LysR-type regulator of Neisseria meningitidis, acting both as a repressor and as an activator of gene transcription. . J Bacteriol 187:, 3421–3430. [CrossRef].[PubMed]
    [Google Scholar]
  29. Jackson L. A., Ducey T. F., Day M. W., Zaitshik J. B., Orvis J., Dyer D. W.. ( 2010; ). Transcriptional and functional analysis of the Neisseria gonorrhoeae Fur regulon. . J Bacteriol 192:, 77–85. [CrossRef].[PubMed]
    [Google Scholar]
  30. Klee S. R., Nassif X., Kusecek B., Merker P., Beretti J. L., Achtman M., Tinsley C. R.. ( 2000; ). Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae . . Infect Immun 68:, 2082–2095. [CrossRef].[PubMed]
    [Google Scholar]
  31. Labigne-Roussel A., Courcoux P., Tompkins L.. ( 1988; ). Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni . . J Bacteriol 170:, 1704–1708.[PubMed]
    [Google Scholar]
  32. Larson J. A., Higashi D. L., Stojiljkovic I., So M.. ( 2002; ). Replication of Neisseria meningitidis within epithelial cells requires TonB-dependent acquisition of host cell iron. . Infect Immun 70:, 1461–1467. [CrossRef].[PubMed]
    [Google Scholar]
  33. Larson J. A., Howie H. L., So M.. ( 2004; ). Neisseria meningitidis accelerates ferritin degradation in host epithelial cells to yield an essential iron source. . Mol Microbiol 53:, 807–820. [CrossRef].[PubMed]
    [Google Scholar]
  34. Maiden M. C., Bygraves J. A., Feil E., Morelli G., Russell J. E., Urwin R., Zhang Q., Zhou J., Zurth K. et al. ( 1998; ). Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. . Proc Natl Acad Sci U S A 95:, 3140–3145. [CrossRef].[PubMed]
    [Google Scholar]
  35. Maxam A. M., Gilbert W.. ( 1977; ). A new method for sequencing DNA. . Proc Natl Acad Sci U S A 74:, 560–564. [CrossRef].[PubMed]
    [Google Scholar]
  36. Munson G. P., Holcomb L. G., Scott J. R.. ( 2001; ). Novel group of virulence activators within the AraC family that are not restricted to upstream binding sites. . Infect Immun 69:, 186–193. [CrossRef].[PubMed]
    [Google Scholar]
  37. Ochsner U. A., Vasil A. I., Vasil M. L.. ( 1995; ). Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. . J Bacteriol 177:, 7194–7201.[PubMed]
    [Google Scholar]
  38. Oriente F., Scarlato V., Delany I.. ( 2010; ). Expression of factor H binding protein of meningococcus responds to oxygen limitation through a dedicated FNR-regulated promoter. . J Bacteriol 192:, 691–701. [CrossRef].[PubMed]
    [Google Scholar]
  39. Perkins-Balding D., Ratliff-Griffin M., Stojiljkovic I.. ( 2004; ). Iron transport systems in Neisseria meningitidis . . Microbiol Mol Biol Rev 68:, 154–171. [CrossRef].[PubMed]
    [Google Scholar]
  40. Pradel E., Guiso N., Locht C.. ( 1998; ). Identification of AlcR, an AraC-type regulator of alcaligin siderophore synthesis in Bordetella bronchiseptica and Bordetella pertussis . . J Bacteriol 180:, 871–880.[PubMed]
    [Google Scholar]
  41. Rohde K. H., Dyer D. W.. ( 2003; ). Mechanisms of iron acquisition by the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae . . Front Biosci 8:, d1186–d1218. [CrossRef].[PubMed]
    [Google Scholar]
  42. Roncarati D., Spohn G., Tango N., Danielli A., Delany I., Scarlato V.. ( 2007; ). Expression, purification and characterization of the membrane-associated HrcA repressor protein of Helicobacter pylori . . Protein Expr Purif 51:, 267–275. [CrossRef].[PubMed]
    [Google Scholar]
  43. Rouquette C., Harmon J. B., Shafer W. M.. ( 1999; ). Induction of the mtrCDE-encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an AraC-like protein. . Mol Microbiol 33:, 651–658. [CrossRef].[PubMed]
    [Google Scholar]
  44. Rouquette-Loughlin C. E., Balthazar J. T., Hill S. A., Shafer W. M.. ( 2004; ). Modulation of the mtrCDE-encoded efflux pump gene complex of Neisseria meningitidis due to a Correia element insertion sequence. . Mol Microbiol 54:, 731–741. [CrossRef].[PubMed]
    [Google Scholar]
  45. Sambrook J., Fritsch E. F., Maniatis T.. ( 1989; ). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  46. Schleif R.. ( 2003; ). AraC protein: a love-hate relationship. . Bioessays 25:, 274–282. [CrossRef].[PubMed]
    [Google Scholar]
  47. Schryvers A. B., Gonzalez G. C.. ( 1989; ). Comparison of the abilities of different protein sources of iron to enhance Neisseria meningitidis infection in mice. . Infect Immun 57:, 2425–2429.[PubMed]
    [Google Scholar]
  48. Stojiljkovic I., Hwa V., de Saint Martin L., O’Gaora P., Nassif X., Heffron F., So M.. ( 1995; ). The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence. . Mol Microbiol 15:, 531–541. [CrossRef].[PubMed]
    [Google Scholar]
  49. Studier F. W., Moffatt B. A.. ( 1986; ). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. . J Mol Biol 189:, 113–130. [CrossRef].[PubMed]
    [Google Scholar]
  50. Turner P. C., Thomas C. E., Stojiljkovic I., Elkins C., Kizel G., Ala’Aldeen D. A., Sparling P. F.. ( 2001; ). Neisserial TonB-dependent outer-membrane proteins: detection, regulation and distribution of three putative candidates identified from the genome sequences. . Microbiology 147:, 1277–1290.[PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.048033-0
Loading
/content/journal/micro/10.1099/mic.0.048033-0
Loading

Data & Media loading...

Supplements

vol. , part 8, pp. 2235 - 2247

Microarray results from three independent experiments performed comparing RNA from cultures of strains M1239 and M2Δ grown to mid-exponential phase in GC medium and exposed for 15 min to iron-limitation. log ratios (M2Δ versus M1239) and -values for each single experiment, as well as for the merge of the three, are represented. Differentially regulated genes (log ratio greater than 1 or less than or equal to -1; -value less than 0.01) are indicated in yellow. *Indicates NMB0018 as a false positive, as this probe interacts non-specifically with the Cy5-labelled cDNA in all the previously performed microarray experiments and has been confirmed as a false positive by RT-PCR. [ Excel file, 646 kb]



EXCEL
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