METAGENOMIC ANALYSIS OF AROMATIC RING-HYDROXYLATING DIOXYGENASES FROM COLD MARINE ENVIRONMENTS

LOVISO, CLAUDIA; GUIBERT, LILIAN; MARCOS, MAGALÍ; DI MARZIO, WALTER; LOZADA, MARIANA; DIONISI, HEBE

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímicay Biología Molecular (SAIB 2010); 2010

Sociedad Argentina de Investigación en Bioquímicay Biología Molecular

Pollution, low temperatures and high UV-B radiation levels make intertidal sediments of Ushuaia Bay an interesting target for the construction of a metagenomic library, to study the microbial community from this extreme environment and its biodegradation potential. We performed molecular ecological analyses (catabolic gene library and qPCR) in the chosen sample, an important step for a targeted screening of the metagenomic library. PCR fragments of ring-hydroxylating dioxygenase genes were cloned into plasmids and sequenced. The library showed three different gene groups previously detected at this site (B, C and nahAc). The two most abundant genes in the library, C and nahAc, were estimated by qPCR to be at 105 and 104copies/μg DNA, respectively. In addition, phnA1 genes from Cycloclasticus spp. were found to be at 104 copies/μg DNA, and bacterial 16S rRNA genes were 4 orders of magnitude more abundant in the sediments. The purification of metagenomic DNA from the sediment was optimized to meet the requirements for the construction of a metagenomic fosmid library, where high yields, purity and an appropriate molecular weight is needed. DNA with an approximately size of 40 Kb was cloned into the fosmid vector Copy Control pCC2FOS (Epicentre). Currently, the screening for clones carrying PAH biodegradation pathway genes is being performed by both functional and molecular analyses.