A metagenomic study of methanotrophic microorganisms in Coal Oil Point seep sediments

Abstract

Background: Methane oxidizing prokaryotes in marine sediments are believed to function as a methane filter reducing the oceanic contribution to the global methane emission. In the anoxic parts of the sediments, oxidation of methane is accomplished by anaerobic methanotrophic archaea (ANME) living in syntrophy with sulphate reducing bacteria. This anaerobic oxidation of methane is assumed to be a coupling of reversed methanogenesis and dissimilatory sulphate reduction. Where oxygen is available aerobic methanotrophs take part in methane oxidation. In this study, we used metagenomics to characterize the taxonomic and metabolic potential for methane oxidation at the Tonya seep in the Coal Oil Point area, California. Two metagenomes from different sediment depth horizons (0-4 cm and 10-15 cm below sea floor) were sequenced by 454 technology. The metagenomes were analysed to characterize the distribution of aerobic and anaerobic methanotrophic taxa at the two sediment depths. To gain insight into the metabolic potential the metagenomes were searched for marker genes associated with methane oxidation.

Results: Blast searches followed by taxonomic binning in MEGAN revealed aerobic methanotrophs of the genus Methylococcus to be overrepresented in the 0-4 cm metagenome compared to the 10-15 cm metagenome. In the 10-15 cm metagenome, ANME of the ANME-1 clade, were identified as the most abundant methanotrophic taxon with 8.6% of the reads. Searches for particulate methane monooxygenase (pmoA) and methyl-coenzyme M reductase (mcrA), marker genes for aerobic and anaerobic oxidation of methane respectively, identified pmoA in the 0-4 cm metagenome as Methylococcaceae related. The mcrA reads from the 10-15 cm horizon were all classified as originating from the ANME-1 clade.

Conclusions: Most of the taxa detected were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were mainly due to abundance differences. The results suggests that the Tonya Seep sediment is a robust methane filter, where taxa presently dominating this process could be replaced by less abundant methanotrophic taxa in case of changed environmental conditions.

Figure 1

Flowchart showing the workflow for taxonomic binning, marker gene annotation and pathway mapping. Abbreviations used in the figure: ncbiP-nr (NCBIs non-redundant Protein Database), mcrA (methyl-coenzyme M reductase), pmoA (particulate methane monooxygenase), dsrAB (dissimilatory sulphite reductase), KAAS (KEGG Automatic Annotation Server) and KEGG (Kyoto Encyclopedia of Genes and Genomes).

Figure 2

Rarefaction curves created in MEGAN. Rarefaction analysis was performed at the most resolved taxonomic level of the NCBI taxonomy in MEGAN for each metagenome. The curves for all taxa include Bacteria, Archaea, Eukaryota, Viruses, unclassified and other sequences.

Figure 3

Normalized MEGAN tree at the domain level. Comparative tree view of the two metagenomes from the root to the domain level. The 0-4 cm metagenome is presented in red and the 10-15 cm metagenome in blue. The numbers in brackets give the percentage of total reads assigned to each node for the two metagenomes. The size of the individual nodes is scaled logarithmically to indicate number of reads assigned.

Figure 4

Potential methanotrophic genera detected. Shown is the proportion of reads assigned to methanotrophic genera at the genus level in MEGAN for each metagenome. In the left section known aerobic methanotrophic genera are presented. In the middle section known taxa involved in anaerobic methane oxidation are presented. In the right section known genera of sulphate reducing bacteria are presented. The archaeal sulphate reducing genus Archaeoglobus is also included in this section. The 0-4 cm metagenome is presented in red. The 10-15 cm metagenome is presented in blue. Numbers are given as log(10) percentage of total reads in each metagenome.

Figure 5

Anaerobic oxidation of methane/methanogenesis pathway. The figure is based on the KEGG-map for methane metabolism and includes the enzymes involved in methanogenesis and reverse methanogenesis. Colours are used to indicate from which metagenome the enzymes were identified by KAAS annotation.

Figure 6

Taxonomic distribution of marker genes for methane oxidation. Shown is the number of reads matching marker genes associated with oxidation of methane and the taxonomic distribution of these reads in each metagenome. Reads matching the marker genes for anaerobic oxidation of methane (mcrA), aerobic oxidation of methane (pmoA) and sulphate reduction (dsrAB) are presented in the left, middle and right section respectively. The 0-4 cm metagenome is presented in red and the 10-15 cm metagenome in blue.