Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition

Abstract Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi . This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ ( rrmJ ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.