1887

Abstract

has been proposed as a suitable model for an in-depth understanding of the biology of a free-living organism. This paper reports that the expression of the aminoglycoside resistance gene (′)-(″), the only selectable marker hitherto available for genetic studies, correlates with a growth impairment of the resistant strains. In light of this finding, a construction based on the tetracycline resistance gene was developed; it can be used efficiently in and confers multiple advantages when compared to (′)-(″). The use of significantly improves transformation efficiency and generates visible colonies faster. Finally, the improvements in the pMTn construction made it possible to obtain insertions in genes which have not been previously considered to be dispensable under laboratory growth conditions.

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2006-02-01
2020-01-24
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References

  1. Bergemann A. D, Whitley J. C, Finch L. R. 1989; Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194. J Bacteriol171:593–595
    [Google Scholar]
  2. Boehr D. D, Daigle D. M, Wright G. D. 2004; Domain-domain interactions in the aminoglycoside antibiotic resistance enzyme AAC(6′)-APH(2″). Biochemistry43:9846–9855[CrossRef]
    [Google Scholar]
  3. Byrne M. E, Rouch D. A, Skurray R. A. 1989; Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001. Gene81:361–367[CrossRef]
    [Google Scholar]
  4. Cao J, Kapke P. A, Minion F. C. 1994; Transformation of Mycoplasma gallisepticum with Tn 916 , Tn 4001 , and integrative plasmid vectors. J Bacteriol176:4459–4462
    [Google Scholar]
  5. Cordova C. M, Lartigue C, Sirand-Pugnet P, Renaudin J, Cunha R. A, Blanchard A. 2002; Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids. J Bacteriol184:5426–5435[CrossRef]
    [Google Scholar]
  6. Culebras E, Martinez J. L. 1999; Aminoglycoside resistance mediated by the bifunctional enzyme 6′- N -aminoglycoside acetyltransferase-2″- O -aminoglycoside phosphotransferase. Front Biosci4:D1–D8[CrossRef]
    [Google Scholar]
  7. Daigle D. M, McKay G. A, Thompson P. R, Wright G. D. 1999; Aminoglycoside antibiotic phosphotransferases are also serine protein kinases. Chem Biol6:11–18[CrossRef]
    [Google Scholar]
  8. Dhandayuthapani S, Rasmussen W. G, Baseman J. B. 1999; Disruption of gene mg218 of Mycoplasma genitalium through homologous recombination leads to an adherence-deficient phenotype. Proc Natl Acad Sci U S A96:5227–5232[CrossRef]
    [Google Scholar]
  9. Dhandayuthapani S, Blaylock M. W, Bebear C. M, Rasmussen W. G, Baseman J. B. 2001; Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium . J Bacteriol183:5645–5650[CrossRef]
    [Google Scholar]
  10. Dirksen L. B, Krebes K. A, Krause D. C. 1994; Phosphorylation of cytadherence-accessory proteins in Mycoplasma pneumoniae . J Bacteriol176:7499–7505
    [Google Scholar]
  11. Dybvig K, Voelker L. L. 1996; Molecular biology of mycoplasmas. Annu Rev Microbiol50:25–57[CrossRef]
    [Google Scholar]
  12. Dybvig K, French C. T, Voelker L. L. 2000; Construction and use of derivatives of transposon Tn 4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis . J Bacteriol182:4343–4347[CrossRef]
    [Google Scholar]
  13. Franke A. E, Clewell D. B. 1981; Evidence for a chromosome-borne resistance transposon (Tn 916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J Bacteriol145:494–502
    [Google Scholar]
  14. Fraser C. M, Gocayne J. D, White O.26 other authors 1995; The minimal gene complement of Mycoplasma genitalium . Science270:397–403[CrossRef]
    [Google Scholar]
  15. Gawron-Burke C, Clewell D. B. 1984; Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn 916 in Escherichia coli : strategy for targeting and cloning of genes from gram-positive bacteria. J Bacteriol159:214–221
    [Google Scholar]
  16. Hedreyda C. T, Lee K. K, Krause D. C. 1993; Transformation of Mycoplasma pneumoniae with Tn 4001 by electroporation. Plasmid30:170–175[CrossRef]
    [Google Scholar]
  17. 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 . Science286:2165–2169[CrossRef]
    [Google Scholar]
  18. King K. W, Dybvig K. 1992; Nucleotide sequence of Mycoplasma mycoides subspecies mycoides plasmid pKMK1. Plasmid28:86–91[CrossRef]
    [Google Scholar]
  19. Lartigue C, Blanchard A, Renaudin J, Thiaucourt F, Sirand-Pugnet P. 2003; Host specificity of mollicutes oriC plasmids: functional analysis of replication origin. Nucleic Acids Res31:6610–6618[CrossRef]
    [Google Scholar]
  20. Lyon B. R, May J. W, Skurray R. A. 1984; Tn 4001 : a gentamicin and kanamycin resistance transposon in Staphylococcus aureus . Mol Gen Genet193:554–556[CrossRef]
    [Google Scholar]
  21. Musatovova O, Dhandayuthapani S, Baseman J. B. 2003; Transcriptional starts for cytadherence-related operons of Mycoplasma genitalium . FEMS Microbiol Lett229:73–81[CrossRef]
    [Google Scholar]
  22. Peterson S. N, Bailey C. C, Jensen J. S, Borre M. B, King E. S, Bott K. F, Hutchison C. A 3rd. 1995; Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation. Proc Natl Acad Sci U S A92:11829–11833[CrossRef]
    [Google Scholar]
  23. Platt M. W, Rottem S, Milner Y, Barile M. F, Peterkofsky A, Reizer J. 1988; Protein phosphorylation in Mycoplasma gallisepticum . Eur J Biochem176:61–67[CrossRef]
    [Google Scholar]
  24. 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 Microbiol23:269–354[CrossRef]
    [Google Scholar]
  25. Pour-El I, Adams C, Minion F. C. 2002; Construction of mini-Tn 4001tet and its use in Mycoplasma gallisepticum . Plasmid47:129–137[CrossRef]
    [Google Scholar]
  26. Reddy S. P, Rasmussen W. G, Baseman J. B. 1996; Isolation and characterization of transposon Tn 4001 -generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium . FEMS Immunol Med Microbiol15:199–211[CrossRef]
    [Google Scholar]
  27. Roberts R. J. 2004; Identifying protein function – a call for community action. PLoS Biol2:E42[CrossRef]
    [Google Scholar]
  28. Sabelnikov A. G, Greenberg B, Lacks S. A. 1995; An extended −10 promoter alone directs transcription of the Dpn II operon of Streptococcus pneumoniae . J Mol Biol250:144–155[CrossRef]
    [Google Scholar]
  29. Sambrook J, Russell D. W. 2001; Molecular Cloning: a Laboratory Manual, 3rd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  30. Sharples G. J, Benson F. E, Illing G. T, Lloyd R. G. 1990; Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination. Mol Gen Genet221:219–226
    [Google Scholar]
  31. 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 Dis139:478–482[CrossRef]
    [Google Scholar]
  32. Weiner J., 3rd, Herrmann R, Browning G. F. 2000; Transcription in Mycoplasma pneumoniae . Nucleic Acids Res28:4488–4496[CrossRef]
    [Google Scholar]
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