Submitted to: International Organization for Mycoplasmology
Publication Type: Abstract only
Publication Acceptance Date: 3/2/2004
Publication Date: 7/11/2004
Citation: Dally, E.L., Barros, T., Zhao, Y., Jomantiene, R., Davis, R.E. 2004. Physical and genetic map of the spiroplasma kunkelii cr2-3x chromosome. International Organization for Mycoplasmology. p. 131-132. Interpretive Summary:
Technical Abstract: Spiroplasma kunkelii is the characteristically helical, wall-less bacterium that causes corn stunt disease. Its ability to parasitize both plant and insect hosts, as well as its phylogenetic position in class Mollicutes, draws intense interest in the genetic make-up of this microbe. The genomic complement of S. kunkelii consists of a single chromosome and mobile genetic elements (plasmids and spiroplasmavirus). To aid our concurrent project to sequence the entire genome of S. kunkelii strain CR2-3X, we have constructed a physical and genetic map of this strain's chromosome using restriction enzyme analyses, pulsed-field gel electrophoresis (PFGE), and DNA hybridizations. Single digests of the chromosome were performed with restriction enzymes I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI. The number of sites in the chromosome for a given enzyme ranged from 1 to 20. The sizes of the resulting fragments for each enzyme were determined by PFGE and totaled to determine the size of the chromosome. The size of the S. kunkelii CR2-3X chromosome was estimated at 1.55 Mbp. The order of the restriction fragments on the chromosome was determined by analyses of reciprocal double digests, and adjacent fragments were identified on two-dimensional PFGE gels. The distribution of the enzyme recognition sites indicated localized regions of higher G+C content compared to the rest of the chromosome. A partial genetic map was obtained by Southern hybridization analyses, using homologous probes, of DNA fragments separated in PFGE gels. We determined the locations of twenty-five S. kunkelii genes, including those encoding 16SrRNA, spiralin, DnaA, GyrA, SMC, ParE, RpoB, FolA, DHFR, SRP54, SecA, and SecY. The positions of dnaA and gyrA on the map indicated the probable location of the chromosomal origin of replication. Information on the organization of the S. kunkelii chromosome gained in this study will contribute to an understanding of spiroplasma evolution.