Patterns of CH4-Seep RNA Figure 6. Correlation of geochemical and microbial stratification. Comparisons of geochemical fluxes and molecular microbiological data for three cores within a bacterial mat (Mat-B), at its edge (Edge), and less than a meter outside the mat (Outside). Plotted are percent of bacterial clones from putative sulfate reducing bacterial rRNA transcripts; percent of archaeal clones from putative methane oxidizer/methanogen ANME groups; presence or absence of dsrAB and mcrA mRNA clones (100%) or absence (0%), slightly offset from each other for visibility; the absolute value of the sulfate flux, since all were negative values; and methane flux multiplied by 10. Sulfate-related data is shown in red, and methane-related data is shown in blue. A) data for surface sediments (0–3 cmbsf), B) data for deeper sediments (12–15 cmbsf). Methane fluxes could not be accurately calculated for B) because methane concentrations were above atmospheric saturation and were not reliable. doi:10.1371/journal.pone.0008738.g006 that they do not cause a detectable decrease in sulfate concentrations with depth. Transcripts of dsrAB retrieved from deep samples would have had to persist in inactive cells for at least 161 years at 5 cm depth below the point where a sulfate flux is no longer measureable, assuming the maximum sedimentation rate for MC118 (31 cm/kyr [67]). Since such persistence of mRNA is extremely unlikely, we conclude that living sulfate-reducing populations are active at these depths. Sulfate reducers undergoing fermentation express dsrAB constitutively [34], so the presence of dsrAB may not necessarily indicate the occurrence of sulfate reduction. Alternately, sulfate could be recycled by reoxidation of sulfide coupled to iron reduction, described as ‘‘cryptic sulfate formation’’ in a study of Black Sea sediments [49]; however, this process would require the presence of a large bioavailable amount of a suitable electron acceptor such as iron to drive the sulfide reoxidation. Another possibility for deep sulfate regeneration is the occasional redistribution of nitrate (an electron donor for sulfide oxidation) to deep sediments through Beggiatoa spp., although we saw no direct evidence of the presence of Beggiatoa spp. deeper in the cores. Since ANME archaeal mcrA mRNA transcripts occur deeper in sediment cores where methane oxidation rates are very low, a small ANME population might survive on these low methane oxidation rates. Alternatively, since the edge core is net methanogenic (according to the d13C profile of methane), ANME populations could switch to methanogenesis, as has been suggested previously [7]. Unlike surficial sediments, no large-scale changes were observed in the active microbial communities at 12–15 cmbsf between mat, edge, and outside cores (Fig. 6B). This deep sulfur cycling could therefore be the result of recent shifts in the location of vent conduits and the microbial population has yet to equilibrate with their new chemical environment. Seafloor observatories are needed to determine over what timescales these vent fluid shifts occur, and assess their relative importance in gene expression levels of the microbial community. Trends in Bacterial and Archaeal Diversity Among the many possible controls that affect bacterial and archaeal diversity, we consider the roles of electron acceptor and carbon substrate availability in determining relative bacterial and archaeal diversity. Bacterial diversity was highest at 1.5 cmbsf outside the mat, suggesting the combined effects of pelagically- derived organic matter and energetically advantageous electron acceptors such as oxygen and nitrate. By comparison, all bacterial samples under the Beggiatoa spp. mat show decreased diversity, possibly a consequence of strongly reducing conditions in sulfate- reducing and methane-cycling sediments. The archaeal diversity trends do not show this strong contrast between sediments PLoS ONE | www.plosone.org 7 January 2010 | Volume 5 | Issue 1 | e8738