Mycoplasmas, the smallest free-living microorganisms, are widely distributed in nature and commonly produce disease in plants, insects, and animals, including man. Although the economic impact of pathogenesis and effective methods of control are unavailable. The development of mycoplasma genetics has been limited by a lack of genetic tools such as selectable markers (e.g. auxotrophic mutants), methods to transfer DNA (e.g. conjugation, transformation, and transduction), and vectors suitable for cloning. The goal of this proposal is to develop mycoplasma genetic tools, with the long-range goal of using these tools to study the basic biology and disease pathogenesis of these organisms. We have recently established that plasmid pAM120, containing the streptococcal transposon Tn916, can be transformed into Acholeplasma laidlawii, Mycoplasma pulmonis, and Mycoplasma hyorhinis using a polyethylene glycol-mediated procedure. Transformed cells can be selected by their expression of the tetracycline resistance determinant, tetM, located on Tn916, Tetracycline-resistant transformants contain Tn916 inserted into the mycoplasmal chromosome, with different transformants containing Tn916 inserted at different sites; Tn916 apparently behaves as a transposable element in these mycoplasmas. Thus, Tn916 is a potentially important new tool that can be used to study mycoplasma genetics. In addition, we have recently established that the streptococcal replicon in plasmid pVA868 functions in A. laidlawii. Thus, replicons of Gram-positive origin may be useful for the development of mycoplasma vectors, To further develop mycoplasma genetic tools, we propose to: (1) isolate and characterize mutants of M. pulmonis, thereby demonstrating the utility of Tn916 as an insertions mutagen of this organism; (ii) develop a mycoplasma cloning vector by combining broad-host-range Gram-positive replicons with a selectable marker (the tetM gene); and (ii) determine whether the conjugative properties of Tn916 can be used of establish gene transfer techniques in mycoplasmas.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI025640-02
Application #
3454536
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1989-03-01
Project End
1994-02-28
Budget Start
1990-03-01
Budget End
1991-02-28
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
King, K W; Dybvig, K (1994) Transformation of Mycoplasma capricolum and examination of DNA restriction modification in M. capricolum and Mycoplasma mycoides subsp. mycoides. Plasmid 31:308-11
King, K W; Dybvig, K (1994) Mycoplasmal cloning vectors derived from plasmid pKMK1. Plasmid 31:49-59
King, K W; Woodard, A; Dybvig, K (1994) Cloning and characterization of the recA genes from Mycoplasma pulmonis and M. mycoides subsp. mycoides. Gene 139:111-5
Panangala, V S; Stringfellow, J S; Dybvig, K et al. (1993) Mycoplasma corogypsi sp. nov., a new species from the footpad abscess of a black vulture, Coragyps atratus. Int J Syst Bacteriol 43:585-90
Schoeb, T R; Dybvig, K; Davidson, M K et al. (1993) Cultivation of cilia-associated respiratory bacillus in artificial medium and determination of the 16S rRNA gene sequence. J Clin Microbiol 31:2751-7
Dybvig, K; Woodard, A (1992) Cloning and DNA sequence of a mycoplasmal recA gene. J Bacteriol 174:778-84
Dybvig, K; Woodard, A (1992) Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment. Plasmid 28:262-6
Bhugra, B; Dybvig, K (1992) High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching. Mol Microbiol 6:1149-54
King, K W; Dybvig, K (1992) Nucleotide sequence of Mycoplasma mycoides subspecies Mycoides plasmid pKMK1. Plasmid 28:86-91
Blanchard, A; Crabb, D M; Dybvig, K et al. (1992) Rapid detection of tetM in Mycoplasma hominis and Ureaplasma urealyticum by PCR: tetM confers resistance to tetracycline but not necessarily to doxycycline. FEMS Microbiol Lett 74:277-81

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