Prokaryotes in marine sediments taken from two neighboring semienclosed bays (the Yamada and Kamaishi bays) at the Sanriku coast in Japan were investigated by the culture-independent molecular phylogenetic approach coupled with chemical and activity analyses. These two bays were chosen in terms of their similar hydrogeological and chemical characteristics but different usage modes; the Yamada bay has been used for intensive shellfish aquaculture, while the Kamaishi bay has a commercial port and is not used for aquaculture. Substantial differences were found in the phylogenetic composition of 16S rRNA gene clone libraries constructed for the Yamada and Kamaishi sediments. In the Yamada library, phylotypes affiliated with ␦-Proteobacteria were the most abundant, and those affiliated with ␥-Proteobacteria were the second-most abundant. In contrast, the Kamaishi library was occupied by phylotypes affiliated with Planctomycetes, ␥-Proteobacteria, ␦-Proteobacteria, and Crenarchaeota. In the ␥-Proteobacteria, many Yamada phylotypes were related to freeliving and symbiotic sulfur oxidizers, whereas the Kamaishi phylotype was related to the genus Pseudomonas. These results allowed us to hypothesize that sulfate-reducing and sulfur-oxidizing bacteria have become abundant in the Yamada sediment. This hypothesis was supported by quantitative competitive PCR (qcPCR) with group-specific primers. The qcPCR also suggested that organisms closely related to Desulfotalea in the Desulfobulbaceae were the major sulfate-reducing bacteria in these sediments. In addition, potential sulfate reduction and sulfur oxidation rates in the sediment samples were determined, indicating that the sulfur cycle has become active in the Yamada sediment beneath the areas of intensive shellfish aquaculture.

Oligobrachia mashikoi, a frenulata, is a marine invertebrate living in an unusual habitat in Tsukumo Bay (20-25 m deep), Japan. It lacks a mouth and a gut, and instead possesses specialized cells called bacteriocytes, in which endosymbiotic bacterial cells are kept. The endosymbiosis involves specific interactions and obligatory metabolic exchanges between the host and endosymbiotic bacterial cells. Analyses of 16S rRNA genes from the endosymbiotic cells indicated that there are at least seven phylotypes of endosymbionts in O. mashikoi, and these phylotypes are closely related Gammaproteobacteria among which sequence homology was more than 97.6%. The analyses suggested that an adult worm predominantly accommodates one of the seven phylotypes. In situ hybridization targeting 16S rRNA demonstrated that the distribution patterns of the bacterial cells in adult worms were very similar among at least two phylotypes of the endosymbionts. The findings suggest that O. mashikoi has a strict selection mechanism for its endosymbiont.

Beard worms (Siboglinidae, Polychaeta), which lack a mouth and a digestive tract, harbor thioautotrophic or methanotrophic bacteria in special cells called bacteriocytes. These endosymbionts have been considered to be trapped at a specific larval stage from the environment. Although many species of beard worms have been discovered in various abyssal seas, Oligobrachia mashikoi inhabits Tsukumo Bay which is only 25 m deep. At least seven types of endosymbionts (endosymbiont A-G) have been distinguished in O. mashikoi. In this study, we investigated the distribution pattern of free-living cells related to the major endosymbiont (endosymbiont A) in Tsukumo Bay by quantitative PCR targeting the 16S rRNA gene. The endosymbiont A-related phylotype was detected in almost all sediment samples collected from 23 points in Tsukumo Bay, ranging in copy number of the 16S rRNA gene from 2.22×10 4 to 1.42×10 6 copies per gram of dry-sediment. Furthermore, the free-living cells made up less than 9% of the total eubacterial population, suggesting that the O. mashikoi larvae precisely select candidates for their endosymbiont from bacterial flora in the environment. This is the first report on the ecological characterization of a free-living bacterium related to the endosymbiont of the siboglinid polychaete, O. mashikoi.