Identification of gene clusters involved in alkane biodegradation from polluted subantarctic sediments by two complementary metagenomic approaches

GUIBERT, L.; LOVISO, C.L.; JANSSON, J.; DIONISI, H.; LOZADA, M.

Rosario

Congreso; IX CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL (SAMIGE 2013); 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Alkanes occur in nature as chemo-attractants or protecting agents produced by many living organisms, and also as a major constituent of crude oil. In this study, we analyzed alkane biodegradation genes from indigenous microorganisms from a chronically-polluted Subantarctic site (Ushuaia Bay) within their genomic context, by two complementary metagenomic approaches. In the first approach, a metagenomic fosmid library, composed of 46,000 clones, was constructed from intertidal sediments using CopyControl™ HTP Fosmid Library Production Kit (Epicentre Biotechnologies). Three fosmid clones containing alkane monooxygenase (alkB) genes were detected by a combination of molecular and functional screening methods. The fosmids were completely sequenced by 454-pyrosequencing and assembled (INDEAR, Rosario). The average insert size was 38 Kbp. Percent amino acid identity values of the three identified alkB genes with sequences from public databases ranged from 50 to 70%. Two of these genes were phylogenetically related to alkB genes described in Bacteroidetes. Although these two genes clustered together, they shared only 47% identity and 73% similarity at the amino acid level. Other genes related to alkane metabolism, as well as a putative transposase, were also found in the corresponding genomic fragments. The alkB gene from the third clone was phylogenetically related to alkane monooxygenase genes described in Alphaproteobacteria, as well as Gammaproteobacteria including obligate alkane degraders. The genomic context of this alkB gene was markedly different from the organization of the other two clones, resembling an archetypical operon-like structure (alkBGHJLST). Putative mobile genetic elements were also found in this genome fragment. For the second approach, the database generated by whole genome shotgun sequencing (WGS) of six sediment samples from Ushuaia Bay was analyzed (2 sites, 3 replicates each). A total of 1.97 x 106 sequences annotated from assembled data in these metagenomes were screened using BLASTP, with the alkB gene sequences from the fosmids as query. Fifty-one sequences were retrieved, three of which were full-length alkB genes. These genes clustered with the two Bacteroidetes-related sequences from the fosmid clones, although with amino acid identity values ranging from 33% to 47%. The corresponding contigs ranged between 1388-4043 bp, noticeably shorter than the ones obtained with the metagenomic library approach. In conclusion, complementary metagenomic approaches allowed us to characterize novel alkane biodegradation genes from Subantarctic marine sediments. However, only the metagenomic library approach was able to retrieve neighboring genomic information.