Free and integrated plasmid DNA in spiroplasmas

Spiroplasma citri was found to carry an 8.0 kb plasmid that differed from previously described S. citri plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid in S. citri (R8A2, Maroc) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome. Significant quantities of integrated plasmid sequences were also observed in the corn stunt spiroplasma, S. kunkelii, while small quantities of free and/or integrated plasmid DNA could be detected in some spiroplasmas serologically and genotypically remote from S. citri. Integrated plasmid sequences were cloned into the Escherichia coli plasmid pUC13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of fragments, rather than entire plasmid DNA, inserted into specific sites in the spiroplasma chromosome. Although the biological role of the pRA1 plasmid remains unclear, the S. citri subclones containing large quantities of free plasmid exhibited slower growth rates and a tendency to lyse.