| The purposes of this study were to evaluate variation among spiroplasma species/lines by molecular typing using rep-PCR, to evaluate a transposome system for mutagenesis of Spiroplasma citri BR3-3X, and to enrich for natural non-adherent variants of S. citri BR3-3X by passaging on tissue cultured cells of the vector, Circulifer tenellus (CT-1), to which the bacterium is known to adhere. Rep-PCRs employing BOX and ERIC primers generated DNA sequences from Spiroplasma citri lines/strains (BR3-3X, BR3-T, BR3-G, R8A2, and ASP-1), S. melliferum TS-2, S. floricola 23-6, S. kunkelii CR2-3X and S. phoenicium. The DNA patterns were relatively simple and distinguished among species but not lines/strains. Mutagenesis of S. citri BR3-3X using a Transposome(TM) (Epicentre RTM) system mediated by electroporation resulted in transformation of spiroplasmas at an efficiency of 28.8 CFUs/ng transposome. PCR using primers for the dihydrofolate reductase (DHFR) marker gene confirmed the transposon's presence in the genome of selected transformants, and Southern-blot hybridization showed its insertion to be random, mostly single and chromosome-located. The insertions were stable as indicated by the consistent presence of the gene and sustainability of transformants sub-cultured to high passages. All transformed spiroplasmas retained the ability to adhere to the Circulifer tenellus (CT-1) cell line, but grew more slowly in LD8 broth. Enrichment for naturally occurring, non-adherent Spiroplasma citri BR3-3X variants was done by selection following sequential passages on the CT-1 cells. The number of unbound spiroplasmas declined with each passage until the 8th to 10th passage, after which it stabilized at less than 0.5% of the number of cells initially applied. The leveling of the population suggests that spiroplasmas remaining in the suspension by the 8th--10th passage were non-adherent. |
