Large-scale transposon mutagenesis of Mycoplasma pulmonis

Abstract

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.

Fig. 1

Map illustrating the density of transposon insertion sites in the M. pulmonis genome. Each tick mark represents a transposon insertion. The genome was divided into 480 blocks of 2-kb each, with the number of transposon insertion sites in each block color coded as indicated. oriC refers to the chromosomal origin of replication. HS1 and HS2 refer to hot spots for transposon insertion.

Fig. 2

Number of mutated ORFs of size greater than 2 kb, greater than 1 kb, greater than 500 bp or of any size plotted as a function of the number of transposition insertion sites mapped.

Fig. 3

Repair of UV-damaged DNA. The surviving fraction of CFU was plotted as a function of applied UV exposure. Non-irradiated samples of each mutant were assessed as a control. The data shown are from a single experiment and are representative of 4 individual experiments.

Fig. 4

Transport of GlcNAc. Incorporation of radiolabeled GlcNAc at 18 hours. Error bars indicate standard deviations.