1887

Abstract

Mycoplasmas are thought to control gene expression through simple mechanisms. The switching mechanisms needed to regulate transcription during significant environmental shifts do not seem to be required for these host-adapted organisms. , a swine respiratory pathogen, undergoes differential gene expression, but as for all mycoplasmas, the mechanisms involved are still unknown. Since mycoplasmas contain only a single sigma factor and few regulator-type proteins, it is likely that other mechanisms control gene regulation, possibly involving intergenic (IG) regions. To study this further, we investigated whether IG regions are transcribed in , and measured transcription levels across five specific regions. Microarrays were constructed with probes covering 343 IG regions of the genome, and RNA isolated from laboratory-grown cells was used to interrogate the arrays. Transcriptional signals were identified in 321 (93.6 %) of the IG regions. Five large (>500 bp) IG regions were chosen for further analysis by qRT-PCR by designing primer sets whose products reside in flanking ORFs, bridge flanking ORFs and the IG region, or reside solely within the IG region. The results indicate that no single transcriptional start site can account for transcriptional activity within IG regions. Transcription can end abruptly at the end of an ORF, but this does not seem to occur at high frequency. Rather, transcription continues past the end of the ORF, with RNA polymerase gradually releasing the template. Transcription can also be initiated within IG regions in the absence of accepted promoter-like sequences.

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2010-08-01
2019-12-05
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References

  1. Adams, C., Pitzer, J. E. & Minion, F. C. ( 2005; ). In vivo expression analysis of the P97 and P102 paralog families of Mycoplasma hyopneumoniae. Infect Immun 73, 7784–7787.[CrossRef]
    [Google Scholar]
  2. Benders, G. A., Powell, B. C. & Hutchison, C. A., III ( 2005; ). Transcriptional analysis of the conserved ftsZ gene cluster in Mycoplasma genitalium and Mycoplasma pneumoniae. J Bacteriol 187, 4542–4551.[CrossRef]
    [Google Scholar]
  3. Burnett, T. A., Dinkla, K., Rohde, M., Chhatwal, G. S., Uphoff, C., Srivastava, M., Cordwell, S. J., Geary, S., Liao, X. & other authors ( 2006; ). P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryotic cells. Mol Microbiol 60, 669–686.[CrossRef]
    [Google Scholar]
  4. Casella, G. & Berger, R. L. ( 2002; ). Statistical Inference, 2nd edn. Pacific Grove, CA: Duxbury.
  5. Chang, L.-J., Chen, W.-H., Minion, F. C. & Shiuan, D. ( 2008; ). Mycoplasmas regulate the expression of heat shock protein genes through CIRCE–HrcA interaction. Biochem Biophys Res Commun 367, 213–218.[CrossRef]
    [Google Scholar]
  6. Farnham, P. J. & Platt, T. ( 1981; ). Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro. Nucleic Acids Res 9, 563–577.[CrossRef]
    [Google Scholar]
  7. Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A., Fleischmann, R. D., Bult, C. J., Kerlavage, A. R., Sutton, G. & other authors ( 1995; ). The minimal gene complement of Mycoplasma genitalium. Science 270, 397–403.[CrossRef]
    [Google Scholar]
  8. Friis, N. F. ( 1975; ). Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare, a survey. Nord Vet Med 27, 337–339.
    [Google Scholar]
  9. Gallup, J. M. & Ackermann, M. R. ( 2006; ). Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system ‘Focusfield2–6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions. Biol Proced Online 8, 87–152.[CrossRef]
    [Google Scholar]
  10. Glass, J. I., Lefkowitz, E. J., Glass, J. S., Heiner, C. R., Chen, E. Y. & Cassell, G. H. ( 2000; ). The complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature 407, 757–762.[CrossRef]
    [Google Scholar]
  11. Gottesman, S. ( 2005; ). Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet 21, 399–404.[CrossRef]
    [Google Scholar]
  12. Güell, M., van Noort, V., Yus, E., Chen, W. H., Leigh-Bell, J., Michalodimitrakis, K., Yamada, T., Arumugam, M., Doerks, T. & other authors ( 2009; ). Transcriptome complexity in a genome-reduced bacterium. Science 326, 1268–1271.[CrossRef]
    [Google Scholar]
  13. Hutchison, C. A., III & Montague, M. G. ( 2002; ). Mycoplasmas and the minimal genome concept. In Molecular Biology and Pathogenicity of Mycoplasmas, pp. 221–253. Edited by S. Razin & R. Herrmann. New York: Kluwer Academic/Plenum Publishers.
  14. Hyman, H. C., Gafny, R., Glaser, G. & Razin, S. ( 1988; ). Promoter of the Mycoplasma pneumoniae rRNA operon. J Bacteriol 170, 3262–3268.
    [Google Scholar]
  15. Janis, C., Lartigue, C., Frey, J., Wrâoblewski, H., Thiaucourt, F., Blanchard, A. & Sirand-Pugnet, P. ( 2005; ). Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum. Appl Environ Microbiol 71, 2888–2893.[CrossRef]
    [Google Scholar]
  16. Knudtson, K. L. & Minion, F. C. ( 1993; ). Construction of Tn4001 lac derivatives to be used as promoter probe vectors in mycoplasmas. Gene 137, 217–222.[CrossRef]
    [Google Scholar]
  17. Knudtson, K. L. & Minion, F. C. ( 1994; ). Use of lac gene fusions in the analysis of Acholeplasma upstream gene regulatory sequences. J Bacteriol 176, 2763–2766.
    [Google Scholar]
  18. Lluch-Senar, M., Vallmitjana, M., Querol, E. & Pinol, J. ( 2007; ). A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium. Microbiology 153, 2743–2752.[CrossRef]
    [Google Scholar]
  19. Madsen, M. L., Nettleton, D., Thacker, E. L. & Minion, F. C. ( 2006a; ). Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays. Microbiology 152, 937–944.[CrossRef]
    [Google Scholar]
  20. Madsen, M. L., Nettleton, D., Thacker, E. L., Edwards, R. & Minion, F. C. ( 2006b; ). Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays. Infect Immun 74, 160–166.[CrossRef]
    [Google Scholar]
  21. Madsen, M. L., Puttamreddy, S., Thacker, E. L., Carruthers, M. D. & Minion, F. C. ( 2008; ). Transcriptome changes in Mycoplasma hyopneumoniae during infection. Infect Immun 76, 658–663.[CrossRef]
    [Google Scholar]
  22. Mare, C. J. & Switzer, W. P. ( 1965; ). New species: Mycoplasma hyopneumoniae, a causitive agent of virus pig pneumonia. Vet Med Small Anim Clin 60, 841–846.
    [Google Scholar]
  23. Minion, F. C., Lefkowitz, E. L., Madsen, M. L., Cleary, B. J., Swartzell, S. M. & Mahairas, G. G. ( 2004; ). The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 186, 7123–7133.[CrossRef]
    [Google Scholar]
  24. Musatovova, O., Dhandayuthapani, S. & Baseman, J. B. ( 2006; ). Transcriptional heat shock response in the smallest known self-replicating cell, Mycoplasma genitalium. J Bacteriol 188, 2845–2855.[CrossRef]
    [Google Scholar]
  25. Muto, A. & Ushida, C. ( 2002; ). Transcription and translation. In Molecular Biology and Pathogenicity of Mycoplasmas, pp. 323–345. Edited by S. Razin & R. Herrmann. New York: Plenum Press.
  26. Neimark, H. C. ( 1986; ). Origins and evolution of wall-less prokaryotes. In The Bacterial L-Forms, pp. 21–42. Edited by S. Madoff. New York: Marcel Dekker.
  27. Oneal, M. J., Schafer, E. R., Madsen, M. L. & Minion, F. C. ( 2008; ). Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine. Microbiology 154, 2581–2588.[CrossRef]
    [Google Scholar]
  28. Pointon, A. M., Byrtr, D. & Heap, P. ( 1985; ). Effect of enzootic pneumonia of pigs on growth performance. Aust Vet J 62, 13–18.
    [Google Scholar]
  29. Pollack, J. D., Williams, M. V. & McElhaney, R. N. ( 1997; ). The comparative metabolism of the mollicutes (Mycoplasmas): the utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells. Crit Rev Microbiol 23, 269–354.[CrossRef]
    [Google Scholar]
  30. Razin, S. & Herrmann, R. (editors) ( 2002; ). Molecular Biology and Pathogenicity of Mycoplasmas. New York: Kluwer Academic/Plenum Publishers.
  31. Razin, S., Yogev, D. & Naot, Y. ( 1998; ). Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62, 1094–1156.
    [Google Scholar]
  32. Ross, R. F. ( 1992; ). Mycoplasmal disease. In Diseases of Swine, pp. 537–551. Edited by A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire & D. J. Taylor. Ames: Iowa State University Press.
  33. Rozen, S. & Skaletsky, H. J. ( 2000; ). Primer3 on the WWW for general users and for biologist programmers. Source code available at http://fokker.wi.mit.edu/primer3/. In Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp. 365–386. Edited by S. Misener & S. A. Krawetz. Totowa, NJ: Humana Press.
  34. Schafer, E. R., Oneal, M. J., Madsen, M. L. & Minion, F. C. ( 2007; ). Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to hydrogen peroxide. Microbiology 153, 3785–3790.[CrossRef]
    [Google Scholar]
  35. Szymanski, M., Erdmann, V. A. & Barciszewski, J. ( 2003; ). Noncoding regulatory RNAs database. Nucleic Acids Res 31, 429–431.[CrossRef]
    [Google Scholar]
  36. Taschke, C. & Herrmann, R. ( 1988; ). Analysis of transcription and processing signals in the 5′ regions of the two Mycoplasma capricolum rRNA operons. Mol Gen Genet 212, 522–530.[CrossRef]
    [Google Scholar]
  37. Taschke, C., Klinkert, M.-Q., Wolters, J. & Herrmann, R. ( 1986; ). Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes. Mol Gen Genet 205, 428–433.[CrossRef]
    [Google Scholar]
  38. Washio, T., Sasayama, J. & Tomita, M. ( 1998; ). Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination. Nucleic Acids Res 26, 5456–5463.[CrossRef]
    [Google Scholar]
  39. Waters, L. S. & Storz, G. ( 2009; ). Regulatory RNAs in bacteria. Cell 136, 615–628.[CrossRef]
    [Google Scholar]
  40. Weiner, J., III, Herrmann, R. & Browning, G. F. ( 2000; ). Transcription in Mycoplasma pneumoniae. Nucleic Acids Res 28, 4488–4496.[CrossRef]
    [Google Scholar]
  41. Weiner, J., III, Zimmerman, C.-U., Gohlmann, H. W. H. & Herrmann, R. ( 2003; ). Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures. Nucleic Acids Res 31, 6306–6320.[CrossRef]
    [Google Scholar]
  42. Wilton, J., Stewart, K. K., Minion, F. C., Young, P., Collins, A., Walker, M. F. & Djordjevic, S. P. ( 2004; ). Domains in the carboxy-terminal of the cilium adhesin of Mycoplasma hyopneumoniae bind fibronectin and heparin. In 15th Congress of the International Organization for Mycoplasmology, p. 152. Athens, GA: International Organization for Mycoplasmology.
  43. Wilton, J., Jenkins, C., Cordwell, S. J., Falconer, L., Minion, F. C., Oneal, D. C., Djordjevic, M. A., Connolly, A., Barchia, I. & other authors ( 2009; ). Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae. Mol Microbiol 71, 566–582.[CrossRef]
    [Google Scholar]
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Histogram of IG region size [ PDF] (511 kb) The 343 IG regions of 50 bp or more [ PDF] (16 kb) (including Figs S2-S4, and Tables S2-S5) [ PDF] (38 kb)

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Histogram of IG region size [ PDF] (511 kb) The 343 IG regions of 50 bp or more [ PDF] (16 kb) (including Figs S2-S4, and Tables S2-S5) [ PDF] (38 kb)

PDF

Histogram of IG region size [ PDF] (511 kb) The 343 IG regions of 50 bp or more [ PDF] (16 kb) (including Figs S2-S4, and Tables S2-S5) [ PDF] (38 kb)

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