INTEGRATIVE AND FREE SPIROPLASMA-CITRI ORIC PLASMIDS - EXPRESSION OF THE SPIROPLASMA-PHOENICEUM SPIRALIN IN SPIROPLASMA-CITRI

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bove, and F. Laigret, Curr. Microbiol, 29:23-29, 1994). In the present stud ies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia call replico n and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri ce lls, in which it replicated, Plasmid pBOT1 was shuttled from E. coli t o S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. How ever, the dnaA box region downstream of the dnaA gene was essential. U pon passaging of the S. citri transformants, the plasmid integrated in to the spiroplasmal host chromosome by recombination at the replicatio n origin. The integration process led to duplication of the oriC seque nces. In contrast to the integrative pBOT1, plasmid pOT1, which does n ot contain the E. coli replicon, was stably maintained as a free extra chromosomal element. Plasmid pOT1 was used as a vector to introduce in to S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pne umoniae and the spiralin gene of Spiroplasma phoeniceum. The recombina nt plasmids, pOTPG with the G fragment and pOTPS with the spiralin gen e, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.