1887

Abstract

In , where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, plasmids. To improve the probability of selecting rare recombination events without fastidious, extensive passaging of the transformants, a new targeting vector was constructed, which was used to inactivate the gene encoding the IIA component of the glucose phosphotransferase system (PTS) permease. Selection of recombinants was based on a two-step strategy using two distinct selection markers, one of which could only be expressed once recombination had occurred through one single crossover at the target gene. According to this strategy, spiroplasmal transformants were screened and multiplied in the presence of gentamicin before the recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the -disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon TnpR/res recombination system to produce unmarked mutations in was demonstrated. In an arginine deiminase (-disrupted) mutant, the gene flanked by the sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative plasmid. Due to incompatibility, plasmid loss occurred spontaneously when selection pressure was removed. This approach will be helpful for constructing unmarked mutations and generating multiple mutants with the same selection marker in . It should also be relevant to other species of mollicutes.

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2005-08-01
2020-09-21
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