Submitted to: Canadian Journal of Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/27/2006
Publication Date: 4/27/2006
Citation: Dally, E.L., Barros, T.S., Zhao, Y., Shaoping, L., Roe, B.A., Davis, R.E. 2006. Physical and genetic map of the spiroplasma kunkelii cr2-3x chromosome. Canadian Journal of Microbiology. 52:857-867. Interpretive Summary: Corn stunt disease is a major limiting factor in corn production in the Americas. The disease is caused by an unusual, very small bacterium that has a helical shape and is known as Spiroplasma kunkelii. In order to gain a better understanding of the spiroplasma's genetic properties, we determined the size of its DNA, designed tools for detecting specific spiroplasma genes, and determined the relative positions of 27 genes on the spiroplasma's DNA (the chromosome). The results of this study will be of interest to scientists and students who study bacteria that are capable of causing diseases in plants, insects, and animals, and to plant quarantine agencies and companies involved in detecting such bacteria.
Technical Abstract: Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analyses, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analyses was used to construct a physical and genetic map of the S. kunkelii strain CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal restriction enzyme double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; and adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kbp. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.