1887

Abstract

The quorum-sensing regulators B and C (QseBC) two-component system were previously shown to regulate biofilm growth of the oral pathogen and to be essential for virulence. In this study, we use RT-PCR to show that an open reading frame, , residing upstream of and encoding a hypothetical protein is co-expressed with . In addition, using a series of transcriptional fusion constructs and 5′-rapid amplification of cDNA Ends (RACE), the promoter that drives expression of the operon and the transcriptional start site was mapped to the 372 bp intergenic region upstream from . No internal promoters drive expression independently from . However, expression is attenuated by approximately ninefold by a putative attenuator stem–loop (Δ = −77.0 KJ/mol) that resides in the 137 bp intergenic region between and . The QseB response regulator activates expression of the operon and transcription from the promoter is drastically reduced in Δ and Δ mutants of . In addition, transcriptional activity of the promoter is significantly reduced in a mutant expressing an in-frame deletion of that lacks the sensor domain of QseC, suggesting that a periplasmic signal is required for QseB activation. Finally, a non-polar in-frame deletion in had little effect on biofilm depth but caused a significant increase in surface coverage relative to wild-type. Complementation of the mutant with a plasmid-borne copy of reduced surface coverage back to wild-type levels. Interestingly, deletion of the sensor domain of QseC or of the entire open reading frame resulted in significant reductions in biofilm depth, biomass and surface coverage, indicating that the sensor domain is essential for optimal biofilm formation by . Thus, although and are co-expressed, they regulate biofilm growth by distinct mechanisms.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.066183-0
2013-06-01
2019-12-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/6/989.html?itemId=/content/journal/micro/10.1099/mic.0.066183-0&mimeType=html&fmt=ahah

References

  1. Amarasinghe J. J. , Scannapieco F. A. , Haase E. M. . ( 2009; ). Transcriptional and translational analysis of biofilm determinants of Aggregatibacter actinomycetemcomitans in response to environmental perturbation. . Infect Immun 77:, 2896–2907. [CrossRef] [PubMed]
    [Google Scholar]
  2. Bearson B. L. , Bearson S. M. . ( 2008; ). The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium. . Microb Pathog 44:, 271–278. [CrossRef] [PubMed]
    [Google Scholar]
  3. Brogan J. M. , Lally E. T. , Demuth D. R. . ( 1996; ). Construction of pYGK, an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector. . Gene 169:, 141–142. [CrossRef] [PubMed]
    [Google Scholar]
  4. Clarke M. B. , Sperandio V. . ( 2005; ). Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli. . Mol Microbiol 57:, 1734–1749. [CrossRef] [PubMed]
    [Google Scholar]
  5. Clarke M. B. , Hughes D. T. , Zhu C. , Boedeker E. C. , Sperandio V. . ( 2006; ). The QseC sensor kinase: a bacterial adrenergic receptor. . Proc Natl Acad Sci U S A 103:, 10420–10425. [CrossRef] [PubMed]
    [Google Scholar]
  6. Demuth D. R. , Novak E. A. , Shao H. . (2011);. Alternative autoinducer-2 quorum sensing response circuits: impact on microbial community development. . In Oral Microbial Communities: Genomic Inquiry and Interspecies Communication, pp. 263–282. Edited by Kolenbrander P. E. . . Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  7. Fine D. H. , Kaplan J. B. , Furgang D. , Karched M. , Velliyagounder K. , Yue G. . ( 2010; ). Mapping the epithelial-cell-binding domain of the Aggregatibacter actinomycetemcomitans autotransporter adhesin Aae. . Microbiology 156:, 3412–3420. [CrossRef] [PubMed]
    [Google Scholar]
  8. Fong K. P. , Chung W. O. , Lamont R. J. , Demuth D. R. . ( 2001; ). Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS. . Infect Immun 69:, 7625–7634. [CrossRef] [PubMed]
    [Google Scholar]
  9. Fong K. P. , Gao L. , Demuth D. R. . ( 2003; ). luxS and arcB control aerobic growth of Actinobacillus actinomycetemcomitans under iron limitation. . Infect Immun 71:, 298–308. [CrossRef] [PubMed]
    [Google Scholar]
  10. Ginalski K. , Kinch L. , Rychlewski L. , Grishin N. V. . ( 2004; ). BOF: a novel family of bacterial OB-fold proteins. . FEBS Lett 567:, 297–301. [CrossRef] [PubMed]
    [Google Scholar]
  11. Hadjifrangiskou M. , Kostakioti M. , Chen S. L. , Henderson J. P. , Greene S. E. , Hultgren S. J. . ( 2011; ). A central metabolic circuit controlled by QseC in pathogenic Escherichia coli. . Mol Microbiol 80:, 1516–1529. [CrossRef] [PubMed]
    [Google Scholar]
  12. Haffajee A. D. , Socransky S. S. . ( 1994; ). Microbial etiological agents of destructive periodontal diseases. . Periodontol 2000 5:, 78–111. [CrossRef] [PubMed]
    [Google Scholar]
  13. Henderson B. , Nair S. P. , Ward J. M. , Wilson M. . ( 2003; ). Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans. . Annu Rev Microbiol 57:, 29–55. [CrossRef] [PubMed]
    [Google Scholar]
  14. Hughes D. T. , Clarke M. B. , Yamamoto K. , Rasko D. A. , Sperandio V. . ( 2009; ). The QseC adrenergic signaling cascade in enterohemorrhagic E. coli (EHEC). . PLoS Pathog 5:, e1000553. [CrossRef] [PubMed]
    [Google Scholar]
  15. Hyvärinen K. , Mäntylä P. , Buhlin K. , Paju S. , Nieminen M. S. , Sinisalo J. , Pussinen P. J. . ( 2012; ). A common periodontal pathogen has an adverse association with both acute and stable coronary artery disease. . Atherosclerosis 223:, 478–484. [CrossRef] [PubMed]
    [Google Scholar]
  16. James D. , Shao H. , Lamont R. J. , Demuth D. R. . ( 2006; ). The Actinobacillus actinomycetemcomitans ribose binding protein RbsB interacts with cognate and heterologous autoinducer 2 signals. . Infect Immun 74:, 4021–4029. [CrossRef] [PubMed]
    [Google Scholar]
  17. Jinadasa R. N. , Bloom S. E. , Weiss R. S. , Duhamel G. E. . ( 2011; ). Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages. . Microbiology 157:, 1851–1875. [CrossRef] [PubMed]
    [Google Scholar]
  18. Juárez-Rodríguez M. D. , Torres-Escobar A. , Demuth D. R. . ( 2013; ). Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans. . Plasmid 69:, 211–222. [CrossRef] [PubMed]
    [Google Scholar]
  19. Kachlany S. C. . ( 2010; ). Aggregatibacter actinomycetemcomitans leukotoxin: from threat to therapy. . J Dent Res 89:, 561–570. [CrossRef] [PubMed]
    [Google Scholar]
  20. Khajanchi B. K. , Kozlova E. V. , Sha J. , Popov V. L. , Chopra A. K. . ( 2012; ). The two-component QseBC signalling system regulates in vitro and in vivo virulence of Aeromonas hydrophila. . Microbiology 158:, 259–271.[CrossRef]
    [Google Scholar]
  21. Kostakioti M. , Hadjifrangiskou M. , Pinkner J. S. , Hultgren S. J. . ( 2009; ). QseC-mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli. . Mol Microbiol 73:, 1020–1031. [CrossRef] [PubMed]
    [Google Scholar]
  22. Lee J. , Hiibel S. R. , Reardon K. F. , Wood T. K. . ( 2010; ). Identification of stress-related proteins in Escherichia coli using the pollutant cis-dichloroethylene. . J Appl Microbiol 108:, 2088–2102.[PubMed]
    [Google Scholar]
  23. Li J. , Attila C. , Wang L. , Wood T. K. , Valdes J. J. , Bentley W. E. . ( 2007; ). Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: effects on small RNA and biofilm architecture. . J Bacteriol 189:, 6011–6020. [CrossRef] [PubMed]
    [Google Scholar]
  24. Merighi M. , Septer A. N. , Carroll-Portillo A. M. , Bhatiya A. , Porwollik S. , McClelland M. , Gunn J. S. . ( 2009; ). Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium. . BMC Microbiol 9:, 42. [CrossRef] [PubMed]
    [Google Scholar]
  25. Miller J. H. . ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  26. Moreira C. G. , Weinshenker D. , Sperandio V. . ( 2010; ). QseC mediates Salmonella enterica serovar typhimurium virulence in vitro and in vivo. . Infect Immun 78:, 914–926. [CrossRef] [PubMed]
    [Google Scholar]
  27. Moreira C. G. , Herrera C. M. , Needham B. D. , Parker C. T. , Libby S. J. , Fang F. C. , Trent M. S. , Sperandio V. . ( 2013; ). Virulence and stress-related periplasmic protein (VisP) in bacterial/host associations. . Proc Natl Acad Sci U S A 110:, 1470–1475. [CrossRef] [PubMed]
    [Google Scholar]
  28. Murzin A. G. . ( 1993; ). OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. . EMBO J 12:, 861–867.[PubMed]
    [Google Scholar]
  29. Njoroge J. , Sperandio V. . ( 2012; ). Enterohemorrhagic Escherichia coli virulence regulation by two bacterial adrenergic kinases, QseC and QseE. . Infect Immun 80:, 688–703. [CrossRef] [PubMed]
    [Google Scholar]
  30. Nørskov-Lauritsen N. , Kilian M. . ( 2006; ). Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. . Int J Syst Evol Microbiol 56:, 2135–2146. [CrossRef] [PubMed]
    [Google Scholar]
  31. Novak E. A. , Shao H. , Daep C. A. , Demuth D. R. . ( 2010; ). Autoinducer-2 and QseC control biofilm formation and in vivo virulence of Aggregatibacter actinomycetemcomitans. . Infect Immun 78:, 2919–2926. [CrossRef] [PubMed]
    [Google Scholar]
  32. Paturel L. , Casalta J. P. , Habib G. , Nezri M. , Raoult D. . ( 2004; ). Actinobacillus actinomycetemcomitans endocarditis. . Clin Microbiol Infect 10:, 98–118. [CrossRef] [PubMed]
    [Google Scholar]
  33. Perez B. A. , Planet P. J. , Kachlany S. C. , Tomich M. , Fine D. H. , Figurski D. H. . ( 2006; ). Genetic analysis of the requirement for flp-2, tadV, and rcpB in Actinobacillus actinomycetemcomitans biofilm formation. . J Bacteriol 188:, 6361–6375. [CrossRef] [PubMed]
    [Google Scholar]
  34. Pilonieta M. C. , Erickson K. D. , Ernst R. K. , Detweiler C. S. . ( 2009; ). A protein important for antimicrobial peptide resistance, YdeI/OmdA, is in the periplasm and interacts with OmpD/NmpC. . J Bacteriol 191:, 7243–7252. [CrossRef] [PubMed]
    [Google Scholar]
  35. Rahamat-Langendoen J. C. , van Vonderen M. G. , Engström L. J. , Manson W. L. , van Winkelhoff A. J. , Mooi-Kokenberg E. A. . ( 2011; ). Brain abscess associated with Aggregatibacter actinomycetemcomitans: case report and review of literature. . J Clin Periodontol 38:, 702–706. [CrossRef] [PubMed]
    [Google Scholar]
  36. Rasko D. A. , Moreira C. G. , Li R. , Reading N. C. , Ritchie J. M. , Waldor M. K. , Williams N. , Taussig R. , Wei S. . & other authors ( 2008; ). Targeting QseC signaling and virulence for antibiotic development. . Science 321:, 1078–1080. [CrossRef] [PubMed]
    [Google Scholar]
  37. Reading N. C. , Torres A. G. , Kendall M. M. , Hughes D. T. , Yamamoto K. , Sperandio V. . ( 2007; ). A novel two-component signaling system that activates transcription of an enterohemorrhagic Escherichia coli effector involved in remodeling of host actin. . J Bacteriol 189:, 2468–2476. [CrossRef] [PubMed]
    [Google Scholar]
  38. Saito T. , Ishihara K. M. , Ryu M. , Okuda K. , Sakurai K. . ( 2010; ). Fimbriae-associated genes are biofilm-forming factors in Aggregatibacter actinomycetemcomitans strains. . Bull Tokyo Dent Coll 51:, 145–150. [CrossRef] [PubMed]
    [Google Scholar]
  39. Sambrook J. , Russell D. W. . ( 2001; ). Molecular Cloning: a Laboratory Manual, , 3rd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory Press;.
    [Google Scholar]
  40. Shao H. , Lamont R. J. , Demuth D. R. . ( 2007a; ). Autoinducer 2 is required for biofilm growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans. . Infect Immun 75:, 4211–4218. [CrossRef] [PubMed]
    [Google Scholar]
  41. Shao H. , James D. , Lamont R. J. , Demuth D. R. . ( 2007b; ). Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2. . J Bacteriol 189:, 5559–5565. [CrossRef] [PubMed]
    [Google Scholar]
  42. Sperandio V. , Torres A. G. , Kaper J. B. . ( 2002; ). Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. . Mol Microbiol 43:, 809–821. [CrossRef] [PubMed]
    [Google Scholar]
  43. Steele K. H. , O’Connor L. H. , Burpo N. , Kohler K. , Johnston J. W. . ( 2012; ). Characterization of a ferrous iron-responsive two-component system in nontypeable Haemophilus influenzae. . J Bacteriol 194:, 6162–6173. [CrossRef] [PubMed]
    [Google Scholar]
  44. Torres-Escobar A. , Juárez-Rodríguez M. D. , Curtiss R. III . ( 2010; ). Biogenesis of Yersinia pestis PsaA in recombinant attenuated Salmonella Typhimurium vaccine (RASV) strain. . FEMS Microbiol Lett 302:, 106–113. [CrossRef] [PubMed]
    [Google Scholar]
  45. Torres-Escobar A. , Juárez-Rodríguez M. D. , Lamont R. J. , Demuth D. R. . ( 2013; ). Transcriptional regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK operons and their role in biofilm formation. . J Bacteriol 195:, 56–65. [CrossRef] [PubMed]
    [Google Scholar]
  46. Wang C. Y. , Wang H. C. , Li J. M. , Wang J. Y. , Yang K. C. , Ho Y. K. , Lin P. Y. , Lee L. N. , Yu C. J. . & other authors ( 2010; ). Invasive infections of Aggregatibacter (Actinobacillus) actinomycetemcomitans. . J Microbiol Immunol Infect 43:, 491–497. [CrossRef] [PubMed]
    [Google Scholar]
  47. Wang X. , Wang Q. , Yang M. , Xiao J. , Liu Q. , Wu H. , Zhang Y. . ( 2011; ). QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda. . Fish Shellfish Immunol 30:, 944–953. [CrossRef] [PubMed]
    [Google Scholar]
  48. Yue G. , Kaplan J. B. , Furgang D. , Mansfield K. G. , Fine D. H. . ( 2007; ). A second Aggregatibacter actinomycetemcomitans autotransporter adhesin exhibits specificity for buccal epithelial cells in humans and Old World primates. . Infect Immun 75:, 4440–4448. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.066183-0
Loading
/content/journal/micro/10.1099/mic.0.066183-0
Loading

Data & Media loading...

Supplements

Supplementary material 

PDF
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