Community Proteogenomics of a Cold-methane Seep Sediment at Nyegga, Mid-Norwegian Margin

Anaerobic oxidation of methane (AOM) is limited to anoxic environments and differs in its rates from a few pmol cm-3day-1 in subsurface SMTZ (sulfate-methane transition zone) of deep margins, to a few μmol cm-3 day-1 in surface sediments above gas hydrates [1]. This process is catalyzed by consortia of anaerobic methane oxidizing archaea (ANME) in association with sulfate-reducing bacteria. The Nyegga area is located on the Mid-Norwegian continental slope at the northern flank of the Storegga Slide at 700-800 mbsl. Hundreds of pockmarks are widespread on the seabed in Nyegga and sub-zero temperatures (-0.7 °C), and pingo-structures within the pockmarks are indicators of active fluid flow locations. Preliminary microbial and geochemical profiling of a 22 cm push-core within the G11 pockmark gave strong indications of an ANME-1 dominated community at 14-16 cmbsf. In light of these findings we submitted extracted DNA to 454-pyrosequencing. Sequencing data (829,527 reads) was assembled using the Newbler v2.3, resulting in 13,151 contigs (357,530 reads) over 500 bp with the longest contig being 24,521 bp. MEGAN taxonomic analysis supported the high abundance of Euryarchaea (70%) with 66% of the assembled metagenome belonging to ANME-1. In order to obtain functional information of the ANME-1 community, protein extraction protocols from sediment samples was established. Extracted proteins was separated on a large (18cm) 1D-SDS-PAGE and subsequently cut in 30 gel slices. Peptides extracted after In-gel tryptic digest was injected into an Ultimate 3000 nanoLC system connected to a linear quadropole ion trap-orbitrap (LTQ-Orbitrap XL) mass spectrometer equipped with a nanoelectrospray ion source. A custom database of open reading frames (ORFs) from the metagenome including known contaminants such as trypsin and human keratin was search against using Mascot 2.2. IRMa tool box [2] was used in peptide validation and peptides whose score >= 25.0 (i.e avg identity, p<0.05) and rank <= 1 was marked as significant. Validated protein ORFs were subjected to a local Blastp search against NCBInr with an E-value cut-off 0.001. A total of 370 proteins met the criteria with 254 of the proteins belonging to ANME-1. The protein dataset contained enzymes involved in C1-metabolism in the reverse metanogenesis pathway of ANME-1 as well as the enzymes sulfite reductase and APS-reductase (of presumably bacterial origin). Hence, the key enzymes involved in anaerobic methane oxidation were expressed in the environment. Results will be further discussed. [1] Knittel, K., and Boetius, A. (2009) Anaerobic Oxidation of Methane: Progress with an Unknown Process. Annu. Rev. Microbiol. 63, 311-334. [2] Dupierris, V., Masselon, C., Court, M., Kieffer-Jaquinod, S., and Bruley, C. (2009) A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25, 1980-1981.