Spiroplasma plasmids

Abstract

Extrachromosomal DNA, constituting plasmids or replicative forms of viruses, has been detected in a variety of spiroplasmas, particularly in Spiroplasma citri. Only a few of the S. citri plasmids were characterized by restriction enzyme mapping, and essentially nothing is known on functions encoded by the plasmids. Our studies revealed in S. citri (R8A2) an 8.0-kbp plasmid that differed from previously described 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) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome, as revealed by Southern hybridization blots of digested spiroplasmal DNA with nick-translated pRA1 or its recombinant as probes. Significant quantities of integrated plasmid sequences were also observed in S. kunkelii and in Spiroplasma sp. P40. Small quantities of free and/or integrated plasmid DNA were detected in some spiroplasmas serologically and genotypically remote from S. citri. Chromosome-integrated pRA1 sequences were cloned into the Escherichia coli plasmids pUC13 and M13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of small repetitive sequences inserted into specific sites on the spiroplasma chromosome. Despite the large number of the inserts they do not appear to affect significantly gene expression in the spiroplasma. Due to the abundance of free and integrated pRA1 in S. citri, nick-translated pRA1 was effective as a DNA probe in detecting small numbers of S. citri in infected periwinkle plants and leafhoppers.