Specific gene targeting in Spiroplasma citri: improved vectors and production of unmarked mutations using site-specific recombination

Abstract

In Spiroplasma citri, where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, oriC 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 crr 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 crr recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the crr-disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in S. citri the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon cd TnpR/res recombination system to produce unmarked mutations in S. citri was demonstrated. In an arginine deiminase (arcA-disrupted) mutant, the tetM gene flanked by the res sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative oriC plasmid. Due to oriC 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 S. citri. It should also be relevant to other species of mollicutes.

Partial Chromosome Sequence of Spiroplasma citri Reveals Extensive Viral Invasion and Important Gene Decay

“…They encode components of phosphoenolpyruvate phosphotransferase systems (PTS), ATP binding cassette (ABC) transporters, and various other transporters. In addition to the general enzymes EI and HPr and the glucose-, fructose-, and trehalose-specific PTS permeases that were previously shown to be functional (2,3,18,25), the S. citri genome also encodes several other hypothetical PTS permeases of unknown substrates, most of which are truncated. The occurrence of PTS in spiroplasma genomes is a striking difference from the genomes of other phloemlimited bacteria such as phytoplasmas (30,36,47) and the phloem-restricted proteobacterium "Candidatus Liberibacter asiaticus" (17), which do not encode PTS.…”

The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome

“…Using random and directed mutagenesis, fructose import was identified as one of the major determinants of S. citri pathogenicity [ 23 , 24 ]. In contrast, S. citri mutants unable to import glucose through the phosphotransferase system were not affected in insect transmission nor in multiplication and symptoms induction in plants [ 25 , 26 ]. Mutants deficient in insect transmissibility were also produced by transposon mediated mutagenesis.…”

Enhancement of Targeted Homologous Recombination in Mycoplasma mycoides subsp. capri by Inclusion of Heterologous recA

“…The flanking regions were chosen to provide sufficient homologous sequences to facilitate recombination in the target gene (7)(8)(9). The insertion and the direction were verified by digestion with KpnI.…”