Spiroplasmas and the transfer of genetic material by transformation and transfection.

Two plasmids, pMH1 with 7 kbp and pM41 with 8 kbp were purified from Spiroplasma citri strains MH and M4 respectively. On the basis of guanine + cytosine content and restriction enzyme mapping, the two plasmids are different. The linearized pMH1 plasmid was introduced into Escherichia coli plasmid vector pBR328 and could be cloned in E. coli. Using radioactive probes specific for each plasmid, we found that pM41 was present in three additional S. citri strains and in three other spiroplasmas not belonging to the S. citri species. pMH1 was found as a free 7-kbp plasmid only in the S. citri strain MH. However, the pMH1 probe hybridized strongly with high molecular weight DNA of several S. citri strains and strains of spiroplasmas other than S. citri. The major membrane protein of S. citri, spiralin, is strongly antigenic and rabbit antibodies against whole S. citri cells strongly react with spiralin. Thus, the enzyme-linked immunosorbent assay (ELISA) has been used to screen E. coli clones that were transformed with HindIII-generated S. citri DNA fragments inserted into the HindIII site of pBR328. One E. coli transformant strongly reacted in ELISA with S. citri polyclonal antiserum. The same transformant also gave a positive reaction with monospecific antiserum against spiralin. These results demonstrate that a gene from S. citri, the spiralin gene, could be expressed in a bacterium. The isometric virus SV4, infecting honeybee spiroplasmas of Group I-2, was shown to possess circular single-stranded DNA of molecular weight 1.7 X 10(6) Da. Transfection of spiroplasma G1 with purified DNA of SV4 was achieved. These experiments open the way to the introduction of foreign genes into spiroplasmas.