N-179. DNA Repair Capacity of the Great Salt Plains Cyanobacterium Aphanothece (SP24)

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

Cyanobacteria at the Great Salt Plains (GSP) are constantly exposed to the severe conditions of high salinity, extreme temperatures and direct UV radiation that cause damage to the organisms’ DNA and must be repaired for survival. Various studies have shown that the photoreactivation gene, phrA , is the main factor responsible for repairing DNA damage in cyanobacteria. In the study reported here, our initial focus has been to investigate whether phrA is a major DNA repair factor in the cyanobacterium Aphanothece from the GSP. DNA survival curves have been used to estimate repair capacity by exposing the organisms to various doses of UV radiation and determining the percent survival under dark and light incubation conditions. Our results showed that Aphanothece survived higher doses of UV only when incubated in light after UV exposure, indicating the importance of light-induced DNA repair in this organism. This conclusion is being confirmed by mutating the Aphanothece phrA gene that we have identified by developing degenerate primers for the phrA gene and obtaining the PCR product. Our working hypothesis is that the phrA gene of GSP-isolated Aphanothece is more efficient in DNA repair than similar genes present in cyanobacteria from non-extreme environments (i.e., Synechocystis ). To test this hypothesis we performed comparative survival curve analysis, which showed that Aphanothece survives higher doses of UV irradiation than Synechocystis . Based on these findings further investigations are being carried out to show that the phrA gene of Aphanothece is indeed more efficient in DNA repair than the Synechosystis gene. First, a phrA deficient Synechocystis mutant is being complemented with the phrA gene of Aphanothece and UV survival capacity tested. Second, the mRNA expressed by the two cyanobacteria after UV exposure is being quantified using RT-PCR. Third, the PhrA protein is being quantified using Western Blot analysis and PhrA antibodies following UV exposure to various doses. These studies will provide insight into the DNA repair capacity of these photosynthetic organisms that have evolved to survive the harsh conditions of the GSP.