Molecular Detection of Polycyclic Aromatic Hydrocarbon-degrading Bacteria in Marine Sediments

GUERRERO, LEANDRO D.; DIONISI, HEBE M.

Iguazú, Misiones, Argentina

Congreso; XL Reunión Anual Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2004

SAIB (Sociedad Argentina de Investigación Bioquímica y

Marine sediments exposed to fuel spills, industrial wastes or shipping activities tend to accumulate polycyclic aromatic hydrocarbons (PAHs) due to their low aqueous solubility, low volatility and high affinity for particulate matter. Some of these compounds are highly toxic, mutagenic, teratogenic, and carcinogenic. In this work, we use PCR to detect catabolic genes involved in PAH biodegradation in marine sediments. Surficial intertidal sediment samples were collected in Puerto Madryn city, Chubut and Ushuaia city, Tierra del Fuego. Total DNA was purified from these samples using the FastDNA® SPIN kit for Soil (Q-BIOgene) and the UltraCleanTM Soil DNA Isolation kit (MoBio Laboratories), applying the same bead beating step for cell lysis. In both methods, the majority of the obtained DNA was in the size range of 5 to 20 kb, and was found to be suitable for PCR amplification using bacterial 16S rRNA gene primers, suggesting the absence of PCR inhibition. The Q-BIOgene method, however, produced DNA yields 30 times higher than the MoBio method. PCR primers targeting the alpha subunit of initial PAH dioxygenases from marine bacteria were designed and applied to the purified DNA. Amplifications with these primers produced a band with the expected molecular weight in a contaminated sediment sample but not in less polluted sediments.