Incidence of Ultraviolet Radiation (UVR) on Colobutane Pirimidine Dimers (CPDs) accumulation and bacterioplankton composition in a high altitude argenitne andean wetland

MARÍA V. FERNANDEZ ZENOFF; MARÍA R. FLORES; MARÍA C. ESTEVEZ; MARÍA E. FARÍAS

Cairns, Australia

Congreso; 12th International Symposium on Microbial Ecology; 2008

ISME

Incidence of Ultraviolet Radiation (UVR) on Colobutane Pirimidine Dimers (CPDs) accumulation and bacterioplankton composition in a high altitude argenitne andean wetland V. Fernández Zenoff, R. Flores, C. Estévez and M. E. Farías Bacterioplankton are the most abundant planktonic organism in freshwater lakes, and are centrally involved in biogeochemical cycling (5). They constitute an essential component of microbial food webs and play significant roles in global carbon and mineral cycles. Exposure of bacterioplankton to surface solar radiation results in the formation of cyclobutane pyrimidine dimers (2,8). These DNA lesions obstruct RNA and DNA polymerases and consequently inhibit gene transcription and DNA replication (11) which eventually can lead to decreased growth rates (4) and changes in bacterioplankton community composition.The aim of this study is evaluated CPDs accumulation and changes in bacterioplankton community composition by denaturing gradient gel electrophoresis (DGGE) in connection with natural UVR in Laguna Vilama. DESCRIPTION OF SAMPLING SITE. Laguna Vilama is located in the plateau of Jujuy Province, Argentina, at 4,600 m above sea level. The location is a very isolated site with no access roads. Rainfall is scarce, the lake is shallow and present high metalcontent. It is included in the List of Wetlands of International Importance (RAMSAR). SAMPLING AND IN SITU EXPERIMENTS. Surface water from L. Vilama was sampling in duplicated during 5 h centered on local noon during summer 2007. Samples (50 ml) were vacuum filtered trough 0.22 polycarbonate Millipore Isopore Membrane Filter(GTTP 04700), by duplicate. The filters were immediately frozen in liquid nitrogen. DNA biodosimeters (calf thymus DNA) were incubated in situ to determinate the DNA effective dose (3). The amount of CPDs in the biodosimeters were determined as described above for water samples. UV radiation was quantified with a radiometer (09811-56, Cole Parmer Instrument Company) for 312 nm with half bandwidth: 300 to 325 nm. DNA EXTRACTION AND CUANTIFICATION. For quantification of DNA damage and amplification of 16S rDNA sequence, DNA was extracted following the CTAB method (6). After extraction, the DNA was quantified fluorometrically using Hoechst 33258 dye. CPDS DETERMINATION. The amount of CPDs was determined using the method of Boelen et al. (1) employing the H3 antibody (Affitech, Oslo) directed mainly to thymine dimers. PCR AMPLIFICATION, DGGE AND SEQUENCING. The variable V3 region of 16S was enzymatically amplified in the PCR (9). After electrophoresis the gel was stained and distinguishable bands were excised from the gel; the eluted DNA was used as template for re-amplified and these products were sequenced in Macrogen Korea and aligned with the reference 16S rRNA gene sequence using the Basic Local Alignment Search Tool (BLAST) analysis.