Screening of a Metagenomic Library from Polluted Subantarctic Sediments for the Identification of Gene Clusters Involved in Hydrocarbon Biodegradation

LOVISO, CLAUDIA .L.; GUIBERT, L.M.; SARANGO, S.; LOZADA, MARIANA; DIONISI, HEBE

Denver, Colorado

Congreso; ASM2013 113th general meeting; 2013

American Society for Microbiology

Hydrocarbon pollution can be highly persistent in cold marine environments, where it represents a significant threat to the ecosystem health. In previous studies, we identified a high diversity and abundance of novel hydrocarbon biodegradation genes in intertidal sediments of a Subantarctic coastal environment chronically-exposed to refined petroleum products, using both PCR clone libraries and qPCR. The goal of this study was to identify gene clusters involved in hydrocarbon biodegradation in this environment, using a metagenomic approach. A composite sample was obtained from intertidal sediments of UshuaiaBay, which is located within the Beagle Channel in South Patagonia. A metagenomic fosmid library was constructed from this sample using the CopyControl™ HTP Fosmid Library Production Kit (Epicentre Biotechnologies). A total of 46,000 clones, representing approximately 1.8 Gb of genetic information from this environment, were screened using both molecular and functional approaches. Four clones were detected by PCR using two broad specificity primer sets. Two clones contained alkane monooxygenase genes that shared 45% identity at the amino acid level in the amplified fragment. The closest matches in a BLAST analysis included sequences from uncultured microorganisms from cold environments (42-45% identity at the protein level). Two clones sharing 41% identity at the amino acid level contained aromatic ring-hydroxylating dioxygenase (ARHD) genes. Close matches from the databases encoded two different large subunit ARHD-like proteins identified in the genome of the pyrene-degrading bacterium Cycloclasticus sp. strain P1 (65 and 62% identity at the amino acid level). One of these clones presented 98.9-100% identity at the amino acid level with a gene fragment that was highly abundant in PCR clone libraries constructed from intertidal sediments from Central and South Patagonia. In addition, one clone was detected based on indigo production from indol and five clones showed catechol dioxygenase activity in the functional screening. Clones of interest are currently being sequenced by 454-pyrosequencing, in order to analyze gene organization. The information obtained in this study will increase our knowledge regarding aerobic hydrocarbon biodegradation pathways from cold marine environments.  Credits This research was supported with grants from CONICET and ANPCyT (Argentina).