Phylogenetic Diversity of Dissimilatory Sulfite Reductase Genes from Deep-sea Cold Seep Sediment Tatsuhiro Fukuba, 1 Mari Ogawa, 2 Teruo Fujii, 1 and Takeshi Naganuma 2, * 1 Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8508, Japan 2 School of Biosphere Sciences, Hiroshima University, Higashi-hiroshima 739-8528, Japan Abstract: The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) a-subunit genes from a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for amplification by polymerase chain reaction (PCR) of DSR a-subunit gene. Two sizes of PCR products, 1.4 kb (expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of 1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs, i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About 400 bp of the 5¢ ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the 5¢-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the d-Proteobacterial lineage with the DSR genes from e-Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from 1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron donors including methane. Key words: sulfate reduction, sulfate-reducing prokaryotes, methane seep, Sagami Sea Knoll. INTRODUCTION Sulfate-reducing prokaryotes (SRPs) are obligatory or fac- ultative anaerobes capable of anaerobic respiration using sulfate (SO 2À 4 ) as a terminal electron acceptor (Brock et al., 1997). SRPs utilize diverse forms of electron donors (hy- drogen sources) such as fatty acids. Certain species are capable of chemolithoautotrophic growth only with carbon dioxide (CO 2 ) as the sole carbon source, molecular hy- drogen (H 2 ) as the electron donor, and sulfate as the ter- minal electron acceptor (Brysch et al., 1987; Schauder et al., 1989). Sulfur (S) is not assimilated during these sulfate- reducing processes. Dissimilatory sulfate-reducing metab- olism is commonly known among the domains of Bacteria and Archaea, but not in Eukarya. Sulfate reducers are regarded as major players in global sulfur cycling (Jørgensen, 1982). Their roles in producing hydrogen sulfide (H 2 S) and decomposing organic matter are essential processes in dark and reducing habitats such as marine sediments, deep-sea hot vents, and cold seeps. Received June 20 2002; accepted November 13 2002. *Corresponding author: telephone +81-824-24-7986; fax +81-824-22-7059; e-mail: takn@hiroshima-u.ac.jp Mar. Biotechnol. 5, 458–468, 2003 DOI: 10.1007/s10126-002-0091-0 Ó 2003 Springer-Verlag New York Inc.