PCR-based survey of methane-cycling archaea in methane-soaked subsurface sediments of Guaymas Basin, Gulf of California

Abstract

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, rapid deposition of organic-rich sediments, steep geothermal gradients, and abundant methane of mixed thermogenic and microbial origin. Subsurface sediment samples were selected from eight drilling sites to explore the diversity, depth range, and in-situ temperature range of methane-cycling archaea in the Guaymas Basin subsurface, using PCR amplification with general (mcrIRD) and ANME-1 specific primers targeting the alpha (α) subunit of methyl coenzyme M reductase, encoded by themcrAgene. Diverse ANME-1 lineages were detected in seven drilling sites, preferentially around the methane-sulfate interface, and in several cases showed preferences for specific sites and types of sediment. Phylogenetically, most ANME-1 sequences from the Guaymas Basin subsurface were related to similar sequences from marine mud volcanoes, seep sites, and the shallow marine subsurface. Methanogenic lineages were found in three sites. The most frequently recovered phylotypes, closely affiliated with the hyperthermophilicMethanocaldococcaceae, were found at the hydrothermally influenced Ringvent site. The coolest drilling site, in the northern axial trough of Guaymas Basin, yielded the greatest diversity of methanogen lineages. Our survey indicates extensive microbial methane cycling within subsurface sediments of Guaymas Basin, and calls attention to deep sources of thermogenic methane where potential microbial processing remains to be shown.ImportanceThe Guaymas Basin has received significant attention from microbiologists and biogeochemists due to the abundance of methane in its sediments. This study has identified new ANME-1 and methanogenic lineages and phylogenetic clusters present in subsurface Guaymas Basin sediments. Our work allows for a greater understanding of 1) the diversity of methane-cycling archaea and 2) methane-cycling in the Guaymas Basin.

The DNA event horizon in the Guaymas Basin subsurface biosphere: technical advances and re-defined limits in bulk extractions of nucleic acids from deep marine sediments

“…75 mbsf at Ringvent sites U1547B and U1548B, and 170 mbsf at the Northwestern sites U1545B and U1546B (Table 2). PCR amplification of mcrA genes with the ANME1-targeted primers gave positive results generally near and within methane-sulfate interfaces (Hinkle et al, 2023). Amplification of mcrA gene for methanogens worked only for few samples, consistent with the apparent rarity of methanogens in the Guaymas Basin subsurface (Bojanova et al, 2023).…”

“…PCR allows for the amplification of targeted gene sequences within dilute DNA extracts, increasing the sensitivity of detection. DNA extracts from all drilling sites were used for PCR amplification of bacterial & archaeal 16S rRNA gene segments with primer combinations of 515F-Y (Parada et al 2015) and 926R (Quince et al 2011), and partial archaeal 16S rRNA genes using primer combination 25F and 806R (Mara et al, 2023), and for mcrA genes (Hinkle et al, 2023) using primer combinations mcrIRD and mcrANME1 (Lever and Teske 2015). In contrast to the metagenomic extractions in Table 1, these DNA extractions for PCR were performed in single samples rather than in triplicate, and the sample size (0.5 g) was based on wet weight, not volume.…”

“…Home brew methods that have been developed are as simple as performing serial dilutions to reduce inhibitory compounds, or more complex as with newly formulated reagents and solutions. The ability to overcome these challenges has led to a surge in high quality data from previously inaccessible sample materials (Bojanova et al, 2023, Mara et al, 2023.…”