Large-scale transposon mutagenesis of Mycoplasma pulmonis

• Published in: Molecular microbiology Vol. 69; no. 1; pp. 67 - 76
• Main Authors: French, Christopher T, Lao, Ping, Loraine, Ann E, Matthews, Brian T, Yu, Huilan, Dybvig, Kevin
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.07.2008; Blackwell Publishing Ltd; Blackwell Science
• Subjects: Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacillus subtilis - radiation effects; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; DNA repair; DNA Transposable Elements; Fundamental and applied biological sciences. Psychology; Gene Library; Genes; Genetics; Microbiology; Miscellaneous; Molecular Sequence Data; Mutagenesis; Mutagenesis, Insertional; Mutation; Mycoplasma genitalium - genetics; Mycoplasma genitalium - metabolism; Mycoplasma genitalium - radiation effects; Mycoplasma pulmonis; Mycoplasma pulmonis - genetics; Mycoplasma pulmonis - metabolism; Mycoplasma pulmonis - radiation effects; Open Reading Frames; Phenotype; Mollicutes; Mycoplasmatales; Microbiology; Mutagenesis; Mycoplasma pulmonis; Transposon; Bacteria; Transposable element; Mycoplasmataceae
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2008.06262.x

To obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn4001T was determined for 1700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared with those reported for Mycoplasma genitalium and Bacillus subtilis. Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA, uvrA, uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N-acetylglucosamine was identified.