N-149. DNA Repair Potential of the Halomonas spp. from the Great Salt Plain

D. G. Rudrappa, R. V. Miller;
Oklahoma State Univ., Stillwater, OK.

Great Salt Plains (GSP) National Wildlife Reserve located in northcentral Oklahoma is a ecologically diverse, extreme, hypersaline environment. Evaporated mineral salt crusts form the surface layer of these plains. Microorganisms living in the GSP are continuously exposed to desiccating conditions, high surface temperatures, freezing winters, high salinity and direct Ultra Violet (UV) radiation. Survival under these conditions may lead to selection for unique survival strategies. DNA repair mechanisms associated with UV light or chemically-induced DNA damage have been shown to be important in protecting microorganisms from desiccation. Because very little is known about the DNA repair capacity of microorganisms from hypersaline terrestrial environments, we decided to study the DNA repair potential of Halomonas spp. isolates that are the dominant Gram-negative bacterial group in the GSP. Survival after exposure to UV light was used as a tool to assess the DNA repair capacity. The two most common Halomonas spp. isolated from the GSP (H.salina and H.venusta) were tested and compared with Escherichia coli and Pseudomonas aeruginosa for DNA repair capacity. Survival of Halomonas spp. from GSP was on a par with E.coli but was greater than P. aeruginosa. These results led us to isolate and characterize the DNA repair gene (recA) from these Halomonas spp. Primers for PCR amplification were designed using a highly conserved region from the alignment of seven previously characterized recA gene sequences. A 200bp product was successfully amplified, gel purified, cloned into the pDrive cloning vector, and sequenced. This PCR product has high sequence similarity with the recA gene sequences of several Gram-negative bacteria. Further amplification to obtain the complete gene sequence is under way. This clone will be used in physiological study to determine enzymatic activity by gene replacement and to assess the genetic diversity of this group of organisms from GSP.