1887

Abstract

The mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTn minitransposon and containing a promoterless reporter gene was constructed to analyse transcription . Recovered transposon insertions were in monocopy, with 16 % expressing enough -galactosidase (-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The -Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in .

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2007-08-01
2024-11-03
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References

  1. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
    [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
    [Google Scholar]
  3. Chiner E., Signes-Costa J., Andreu A. L., Andreu L. 2003; [ Mycoplasma pneumoniae pneumonia: and uncommon cause of adult respiratory distress syndrome]. An Med Interna 20:597–598 (in Spanish
    [Google Scholar]
  4. Dybvig K., French C. T., Voelker L. L. 2000; Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis . J Bacteriol 182:4343–4347
    [Google Scholar]
  5. Ermolaeva M. D., Khalak H. G., White O., Smith H. O., Salzberg S. L. 2000; Prediction of transcription terminators in bacterial genomes. J Mol Biol 301:27–33
    [Google Scholar]
  6. 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
    [Google Scholar]
  7. Glass J. I., Assad-Garcia N., Alperovich N., Yooseph S., Lewis M. R., Maruf M., Hutchison C. A. III, Smith H. O., Venter J. C. 2006; Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A 103:425–430
    [Google Scholar]
  8. Halbedel S., Stulke J. 2006; Probing in vivo promoter activities in Mycoplasma pneumoniae : a system for generation of single-copy reporter constructs. Appl Environ Microbiol 72:1696–1699
    [Google Scholar]
  9. Henry I., Sharp P. M. 2007; Predicting gene expression level from codon usage bias. Mol Biol Evol 24:10–12
    [Google Scholar]
  10. Hutchison C. A., Peterson S. N., Gill S. R., Cline R. T., White O., Fraser C. M., Smith H. O., Venter J. C. 1999; Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286:2165–2169
    [Google Scholar]
  11. Iverson-Cabral S. L., Astete S. G., Cohen C. R., Rocha E. P., Totten P. A. 2006; Intrastrain heterogeneity of the mgpB gene in Mycoplasma genitalium is extensive in vitro and in vivo and suggests that variation is generated via recombination with repetitive chromosomal sequences. Infect Immun 74:3715–3726
    [Google Scholar]
  12. Jaffe J. D., Stange-Thomann N., Smith C., DeCaprio D., Fisher S., Butler J., Calvo S., Elkins T., FitzGerald M. G. other authors 2004; The complete genome and proteome of Mycoplasma mobile . Genome Res 14:1447–1461
    [Google Scholar]
  13. Jensen J. S. 2004; Mycoplasma genitalium : the aetiological agent of urethritis and other sexually transmitted diseases. J Eur Acad Dermatol Venereol 18:1–11
    [Google Scholar]
  14. Knudtson K. L., Minion F. C. 1993; Construction of Tn 4001lac derivatives to be used as promoter probe vectors in mycoplasmas. Gene 137:217–222
    [Google Scholar]
  15. Lloyd A. L., Marshall B. J., Mee B. J. 2005; Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and genescan analysis. J Microbiol Methods 60:291–298
    [Google Scholar]
  16. Madsen M. L., Nettleton D., Thacker E. L., Edwards R., Minion F. C. 2006a; Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays. Infect Immun 74:160–166
    [Google Scholar]
  17. Madsen M. L., Nettleton D., Thacker E. L., Minion F. C. 2006b; Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays. Microbiology 152:937–944
    [Google Scholar]
  18. Margolin W. 2005; FtsZ and the division of prokaryotic cells and organelles. Nat Rev Mol Cell Biol 6:862–871
    [Google Scholar]
  19. McInerney J. O. 1998; GCUA: general codon usage analysis. Bioinformatics 14:372–373
    [Google Scholar]
  20. Miller F. D., Hershberger C. L. 1984; A quantitative beta-galactosidase alpha-complementation assay for fusion proteins containing human insulin B-chain peptides. Gene 29:247–250
    [Google Scholar]
  21. Morfeldt E., Taylor D., von Gabain A., Arvidson S. 1995; Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J 14:4569–4577
    [Google Scholar]
  22. Musatovova O., Dhandayuthapani S., Baseman J. B. 2003; Transcriptional starts for cytadherence-related operons of Mycoplasma genitalium . FEMS Microbiol Lett 229:73–81
    [Google Scholar]
  23. Musatovova O., Dhandayuthapani S., Baseman J. B. 2006a; Transcriptional heat shock response in the smallest known self-replicating cell, Mycoplasma genitalium . J Bacteriol 188:2845–2855
    [Google Scholar]
  24. Musatovova O., Herrera C., Baseman J. B. 2006b; Proximal region of the gene encoding cytadherence-related protein permits molecular typing of Mycoplasma genitalium clinical strains by PCR-restriction fragment length polymorphism. J Clin Microbiol 44:598–603
    [Google Scholar]
  25. Osada Y., Saito R., Tomita M. 1999; Analysis of base-pairing potentials between 16S rRNA and 5′ UTR for translation initiation in various prokaryotes. Bioinformatics 15:578–581
    [Google Scholar]
  26. Osawa M., Erickson H. P. 2005; Probing the domain structure of FtsZ by random truncation and insertion of GFP. Microbiology 151:4033–4043
    [Google Scholar]
  27. Peterson S. N., Bailey C. C., Jensen J. S., Borre M. B., King E. S., Bott K. F., Hutchison C. A III. 1995; Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation. Proc Natl Acad Sci U S A 92:11829–11833
    [Google Scholar]
  28. Pich O. Q., Burgos R., Ferrer-Navarro M., Querol E., Pinol J. 2006a; Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence. Mol Microbiol 60:1509–1519
    [Google Scholar]
  29. Pich O. Q., Burgos R., Planell R., Querol E., Pinol J. 2006b; Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium : application to studies of the minimal gene complement. Microbiology 152:519–527
    [Google Scholar]
  30. Reddy S. P., Rasmussen W. G., Baseman J. B. 1996; Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium . FEMS Immunol Med Microbiol 15:199–211
    [Google Scholar]
  31. Robertson J. A. R., Stemke G. W. 1995; Measurement of mollicute growth by ATP-dependent luminometry. In Molecular and Diagnostic Procedures in Mycoplasmology pp 65–71 Edited by Razin S., Tully J. G. San Diego, CA: Academic Press;
    [Google Scholar]
  32. Rocha E. P., Danchin A., Viari A. 1999; Translation in Bacillus subtilis : roles and trends of initiation and termination, insights from a genome analysis. Nucleic Acids Res 27:3567–3576
    [Google Scholar]
  33. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. 1983; New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene 25:71–82
    [Google Scholar]
  34. Sharp P. M., Bailes E., Grocock R. J., Peden J. F., Sockett R. E. 2005; Variation in the strength of selected codon usage bias among bacteria. Nucleic Acids Res 33:1141–1153
    [Google Scholar]
  35. Tully J. G., Rose D. L., Whitcomb R. F., Wenzel R. P. 1979; Enhanced isolation of Mycoplasma pneumoniae from throat washings with a newly-modified culture medium. J Infect Dis 139:478–482
    [Google Scholar]
  36. Wagner E. G., Flardh K. 2002; Antisense RNAs everywhere?. Trends Genet 18:223–226
    [Google Scholar]
  37. Waldo R. H. III., Krause D. C. 2006; Synthesis, stability, and function of cytadhesin P1 and accessory protein B/C complex of Mycoplasma pneumoniae . J Bacteriol 188:569–575
    [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
    [Google Scholar]
  39. Weiner J. III., Herrmann R., Browning G. F. 2000; Transcription in Mycoplasma pneumoniae . Nucleic Acids Res 28:4488–4496
    [Google Scholar]
  40. Weiner J. III., Zimmerman C. U., Gohlmann H. W., Herrmann R. 2003; Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures. Nucleic Acids Res 31:6306–6320
    [Google Scholar]
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