Archaeal rDNA can be retrieved using eukariotic specific primers

COVACEVICH F.; CUMINO A. C.; SILVA R. I; NEGRI R.; SALERNO G. L.

La Plata, Argentina

Workshop; SAMIGE; 2006

Sociedad Argentina Microbiología General

The Archaea are a group of prokaryotes that were recognized in 1977 as an independent monophyletic domain of life. Although many archaea colonize extreme environments, archaeans are not restricted to extreme environments. New research is showing that archaeans are also quite abundant in the plankton of the open sea. The aim of this work was to recovery picoeukariotic organisms from the environmental samples of the Argentinean Sea. Picoplankton were collected from surface well-mixed water collected in September, October and November 2003 at the fixed EPEA Station (38º28’S-57º41’W at 20 km south of Mar del Plata, Argentine). Water samples were prefiltered through a 5 µm pore size membrane and then filtered through a 0.2 µm pore size. Filters were transferred into a cryovial tube containing a DNA lysis buffer, immediately frozen in liquid nitrogen until analysis in the laboratory. Nucleic acid (DNA) extraction was performed, using the phenol-chloroform method and DNA integrity was checked by agarose gel electrophoresis. Extracted DNA was used as the template in a PCR reaction in which eukaryotic 18S ribosomal DNA (rDNA)-specific primers for (Diez et al., 2001). An aliquot of the PCR product was electrophoresed in an agarose gel and quantified. Two bands were obtained from the samples extracted in September and October. Higher bands were about 1700 kb and lower bands were about 1500 kb. Only one band of about 1700 kb was obtained from sample extracted in November. Bands were separately excised and DNA from de agarose gel was extracted. The PCR product was used to construct libraries with a pGemTeasy cloning kit. The presence of rDNA inserts was confirmed by PCR amplification with the same amplification conditions. DNA products of expected sizes were digested with restriction enzyme HaeIII. The restriction fragment length polymorphism (RFLP) products were visualized after electrophoresis in low-melting-point agarose gel. Clones with different RFLP patterns were sequenced (Macrogen, Corea). Nucleotide sequences were subjected to a BLAST search to determine the phylogenetic affiliation. Sequence alignments were generated with the CLUSTALX software and dendograms were compiled by using the neighbor-joining method of CLUSTALX and the maximum parsimony algorithm of the PHYLIP package. Sequences of higher bands corresponded to eukariotic phytoplanktonic organisms. However, sequences of lower DNA bands corresponded to archaeal organisms. Phylogenetic analysis showed that sequeces of lower bands corresponded to eight different archaea. All of them were grouped with the Marine Group II of Euryarchaota organisms, which is characterized as a methanogen-halophile lineage of the Euryarchaeota. The organisms of this group have been shown to fix nitrogen and have been reported, in general, in the deep sea. This study rely in the importance of recovering rDNA archaeal sequences from surface water samples from the Argentinean Sea by using specific primers of eukariotic organisms. References Diez, B., Pedros-Alio, C., Massana, R. 2001. Appl. Environ. Microbiol. 67:2932–2941. Acknowledgments This project was supported by PICT 2002 N° 12233