Diversity of aromatic hydrocarbon-degrading genes in Sub-Antarctic coastal marine sediments

MARCOS, M. S.; LOZADA, M.; DIONISI, H. M.

Rosario

Congreso; V Congreso Argentino de Microbiología General; 2008

Sociedad Argentina de Microbiología General (SAMIGE)

Functional characterization of aromatic hydrocarbon-degrading bacterial populations can be achieved by studying genes encoding for aromatic ring-hydroxylating dioxygenases (ARHDs), enzymes responsible for the first step of their biodegradation pathways. We amplified ARHD gene fragments from total DNA extracted from intertidal sediments of Patagonia, using a degenerate primer set designed to specifically target these catabolic genes in Gram-positive bacteria. Two gene libraries were constructed by cloning the amplification products obtained from two sediment samples of Ushuaia Bay (Tierra del Fuego), retrieved at the same site but two years apart. Clones carrying amplification products produced due to mispriming events were detected and excluded from further analysis using the TBLASTX program of BLAST. Bioinformatic tools were used to describe and compare both clone libraries. The DOTUR software identified 15 different groups of genes, which were called gene types I to XV. In order to infer the possible function of these gene fragments, their sequences were compared to four different databases: NCBI, COG (Clusters of Orthologous Groups: a phylogenetic classification of proteins), KEGG (Kyoto Encyclopedia of Genes and Genomes: a metabolic pathway database) and GO (Gene Ontology: an ontology-based gene products database). Deduced amino acid sequences of group XIII showed 85% identity (94% similarity) at the amino acid level with the dioxygenase IpbAa from Pseudomonas putida RE204 (AAC03436), a cumene-degrading bacteria. The remaining sequences showed less than 78% amino acid identity to ARHDs published in the NCBI database. An alignment of the deduced amino acid sequences from clones with ARHD sequences obtained from the NCBI database, showed conserved key amino acid residues associated to the active site of these enzymes. On the other hand, searches performed in COG, KEGG and GO databases resulted in all sequences matching ring-hydroxylating dioxygenases, and E-values were lower than 10-4, 10-17 and 10-16, respectively, indicating a very low probability of matching these genes by chance. Gene libraries were compared by calculating diversity and similarity indexes. Both libraries were equally diverse (Shannon?s diversity index between 1.86 and 1.89), showed low dominance indexes (Simpson?s dominance index between 0.14 and 0.21) and were moderately similar (Chao?s Jaccard abundance-based similarity index: 0.59). Most ARHD gene fragments analyzed in this study were highly divergent from those previously described, showing that there is still much uncertainty about aromatic hydrocarbon catabolic pathways in marine environments. Further research aiming to quantify the abundance of these functional genes will be performed, in order to assess the ecological role of these populations in the degradation of these harmful pollutants in marine sediments.