the seafloor at methane seeps and hydrate sites in complex, patchy patterns [11,12,13]. Beggiatoa spp. mats are often used as visual locators of active hydrocarbon seeps and seep-related microbial communities [6,14,15], but it is not clear how tightly coupled the presence of mat is to underlying seepage. Are the edges of a mat associated with diminished seepage rates that gradually transition to no seep influence in sediments some distance away from the mat? Or is the transition from seep-influenced to non-seep-influenced sediments and associated microbial communities abrupt, indi- cating a focused subsurface flow? Finally, since microbial mats cover only a fraction of the seafloor even at active seep sites, what can be inferred about the patchiness and distribution of seepage- associated microbial processes, such as methanogenesis, sulfate reduction, sulfate-dependent methane oxidation? We explored the relationship between geochemical activity measurements, and genetic analysis of the active microbial community with depth at different locations across a large (,10 meter diameter, Fig. 1a) Beggiatoa spp. mat at a hydrocarbon seep in the Gulf of Mexico (Mississippi Canyon 118) (Fig. 1b). This habitat transect gives insights into the ecophysiology, activity, habitat preference, and diversity of these mostly uncultured microbial communities [16]. Establishing microbial activity with analysis of nucleic acids in the environment is difficult since DNA from inactive cells may be stable in cold anoxic sediments [17]. Therefore we used two forms of RNA obtained directly from bulk sediment to identify active microbial populations. In order to link sulfate reduction and methane oxidation/production as closely as possible to the corresponding gene expression pattern of the microbial commu- nity, messenger RNA (mRNA) of the genes for dissimilatory sulfite reductase (dsrAB) for sulfate reduction [18]and methyl coenzyme Patterns of CH4-Seep RNA M reductase (mcrA) for methanogenesis and possibly also anaerobic methane oxidation [19], were reverse-transcribed and sequenced. Active bacteria and archaea have higher cellular rRNA concen- trations relative to inactive bacteria and archaea [20], and rRNA content is positively correlated with independent measurements of cellular activity such as cellular protein levels [21] bromodeoxy- uridine (BrdU) uptake [22], 3H-adenine incorporation [23], oxygen consumption [24], and chlorophyll a content and 14C- fixation rates [25]. Accordingly, RT-PCR-based studies have shown a significantly different population in marine sediments than were derived from PCR-based methods [22,26,27,28]. For these reasons, RNA is more likely than DNA to reflect the active population, but preservation mechanisms may also exist for RNA in anoxic sediments or inactive cells, for instance low levels of 16S rRNA transcripts can persist in inactive methanogens at least a few hundred days [29]. A closer link to metabolic activity can be found in certain types of mRNA [30]. Transcription of mcrA is closely linked to metabolism in both Methanococcus vanielii, where mcrA has a maximum half-life of 15 minutes [31], and Methanosarcina acetivorans, where mutants can nonetheless arise that are capable of constitutive expression [32]. The expression of dsrAB genes is also coupled to sulfate-reducing activity in sediments [33] and in pure cultures of Desulfobacterium autotrophicum [34], although small amounts of constitutive expression during fermentation or thiosulfate reduction were also detected [34]. Since the small sizes of cDNA clone libraries often miss much of the microbial diversity present in the environment [35], we also checked selected samples using amplicon tag sequencing, where the V6 hypervariable regions of 16S rRNA genes undergo high throughput amplicon pyrosequencing to improve upon the sampling depth of clone libraries by at least two orders of magnitude and fully explore the microbial diversity [35]. Figure 1. Locations and photos of sampling area. A) View of Beggiatoa spp. mat used for sampling. The mat covers the entire visible seafloor area of the photo; the white circle in the upper lefthand corner is the reflection of a light from the Johnson-Sea-Link submersible, B) Overview map of Mississippi Canyon block 118 (MC118) off the coast of Louisiana, and C) Mosaic image of seafloor a few tens of meters away from the large microbial mat in A; approximate width of the whole strip is 1.83 to 2.44 m given a submersible altitude of 3 to 4 m above the seafloor. doi:10.1371/journal.pone.0008738.g001 PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8738