from the average clone library size by less than 20% of their total value (clone library sample sizes are listed in Figs. 3a and 4a). Supporting Information Table S1 Comparison of depth-integrated sulfate reduction and methane oxidation rates (mmol m-2 d-1) to concentrations fluxes of sulfate and methane (mmol m-2 d-1), respectively. Found at: doi:10.1371/journal.pone.0008738.s001 (0.04 MB DOC) Table S2 Primer sequences used in the study, their annealing temperatures, target groups, and known mismatches. Found at: doi:10.1371/journal.pone.0008738.s002 (0.07 MB DOC) Figure S1 Model fit to sulfate concentration data (red line), or sulfate reduction rate data (blue line) for Edge core. Yellow markers are the data from Figure 2c. Found at: doi:10.1371/journal.pone.0008738.s003 (9.28 MB TIF) Figure S2 Neighbor-joining tree of cDNA of full-length Deltaproteobacterial 16S rRNA sequences for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. OTUs are based on 98% similarity. Sequences from dive 3570 are in colors corresponding to those of Fig. 3a groupings. Clones given their core name (either MatB, Edge, or Out) followed by the beginning of their depth interval (0–3 cmbsf, 12–15 cmbsf, or 21–24 cmbsf), the type of cDNA (arc or bac for archaeal or bacterial 16S rRNA, mcr or dsr for mRNA), and a unique clone number. The number of sequences included in each OTU are in parentheses after the clone name, with the core and depth listed. Found at: doi:10.1371/journal.pone.0008738.s004 (10.24 MB TIF) Figure S3 Neighbor-joining tree of cDNA of full-length non- Deltaproteobacterial 16S rRNA sequences for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. OTUs are based on 98% similarity. Sequences from dive 3570 are in colors corresponding to those of Fig. 3a groupings. Found at: doi:10.1371/journal.pone.0008738.s005 (0.66 MB TIF) References 1. Hill TM, Kennett JP, Valentine DL, Yang Z, Reddy CM, et al. (2006) Climatically driven emissions of hydrocarbons from marine sediments during deglaciation. Proceedings of the National Academy of Sciences of the United States of America 103: 13570–13574. 2. Solomon EA, Kastner M, MacDonald IR, Leifer I (2009) Considerable methane fluxes to the atmosphere from hydrocarbon seep in the Gulf of Mexico. Nature Geoscience 2: 561–565. 3. Lapham LL, Chanton JP, Martens CS, Sleeper K, Woolsey JR (2008) Microbial activity in surficial sediments overlying acoustic wipeout zones at a Gulf of Mexico cold seep. Geochemistry Geophysics Geosystems 9: Q06001. doi:01029/02008GC001944. 4. Roberts HH, Carney RS (1997) Evidence of episodic fluid, gas, and sediment venting on the Northern Gulf of Mexico continental slope. Economic Geology 92: 863–879. 5. Arvidson RS, Morse JW, Joye SB (2004) The sulfur biogeochemistry of chemosynthetic cold seep communities, Gulf of Mexico, USA. Marine Chemistry 87: 97–119. 6. Joye SB, Boetius A, Orcutt BN, Montoya JP, Schulz HN, et al. (2004) The anaerobic oxidation of methane and sulfate reduction in sediments from Gulf of Mexico cold seeps. Chemical Geology 205: 219–238. 7. Orcutt B, Boetius A, Elvert M, Samarkin V, Joye SB (2005) Molecular biogeochemistry of sulfate reduction, methanogenesis and the anaerobic oxidation of methane at Gulf of Mexico cold seeps. Geochimica et Cosmochimica Acta 69: 4267–4281. 8. Orcutt BN, Boetius A, Lugo SK, MacDonald IR, Samarkin VA, et al. (2004) Life at the edge of methane ice: microbial cycling of carbon and sulfur in Gulf of Mexico gas hydrates. Chemical Geology 205: 239–251. Patterns of CH4-Seep RNA Figure S4 Comparison of Blast hits for sequence tags from V6 tag pyrosequencing and 16S rRNA sequences from RT-PCR clone libraries for 2 samples (12–15 cmbsf in Mat-B, and 12–15 cmbsf Out). Shown are 100% bar charts for A) bacteria and B) archaea. Found at: doi:10.1371/journal.pone.0008738.s006 (9.72 MB TIF) Figure S5 Neighbor-joining tree of amino acid translations of dsrAB transcripts for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. Sequences from dive 3570 are in colors corresponding to those of Fig. 3b groupings. Found at: doi:10.1371/journal.pone.0008738.s007 (5.47 MB TIF) Figure S6 Neighbor-joining tree of cDNA of full-length archaeal 16S rRNA sequences for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. OTUs are based on 98% similarity. Sequences from dive 3570 are in colors corresponding to those of Fig. 4a groupings. Found at: doi:10.1371/journal.pone.0008738.s008 (5.38 MB TIF) Figure S7 Neighbor-joining tree of amino acid translations of mcrA transcripts for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. Sequences from dive 3570 are in colors corresponding to those of Fig. 4b groupings. Found at: doi:10.1371/journal.pone.0008738.s009 (7.09 MB TIF) Acknowledgments We would like to thank Robert Woolsey and Christopher S. Martens (cruise co-chief scientists), the captain and crews of R/V Seward Johnson II and pilots of Johnson-Sea-Link submersible, as well as Richard Camilli, who directed core acquisition, Laura Lapham and Howard Mendlovitz, who helped with shipboard core-handling, Chip Peterson and Zoe Manikam, who helped with RT-PCR and cloning, Sue Huse and Hilary Morrison, who performed V6-tag pyrosequencing, and Marc Alperin, Carol Arnosti, Barbara MacGregor, and Christopher S. Martens who gave useful comments on the manuscript. 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Zhang CL, Huang Z, Cantu J, Pancost RD, Brigmon RL, et al. (2005) Lipid biomarkers and carbon isotope signatures of a microbial (Beggiatoa) mat associated with gas hydrates in the Gulf of Mexico. Applied and Environmental Microbiology 71: 2106–2112. 13. MacDonald IR, Sager WW, Peccini MB (2003) Gas hydrate and chemosynthetic biota in mounded bathymetry at mid-slope hydrocarbon seeps: Northern Gulf of Mexico. Marine Geology 198: 133–158. 14. MacDonald IR, Bender LC, Vardaro M, Bernard B, Brooks JM (2005) Thermal and visual time-series at a seafloor gas hydrate deposit on the Gulf of Mexico slope. Earth and Planetary Science Letters 233: 45– 59. 15. Mills HJ, Martinez RJ, Story S, Sobecky PA (2004) Identification of members of the metabolically active microbial populations associated with Beggiatoa species mat communities from Gulf of Mexico cold-seep sediments. Applied and Environmental Microbiology 70: 5447–5458. PLoS ONE | www.plosone.org 11 January 2010 | Volume 5 | Issue 1 | e8738