A first assessment of anammox activity, which is a unique N2 emission process, was conducted in samples of coastal marine sediment from Japan with a 15 N tracer. The occurrence and diversity of bacteria possibly responsible for the anammox process were also evaluated by selective PCR-amplification of the 16S rRNA gene for known anammox bacteria. Anammox activity, detected by measuring 14 N 15 N gas production, was only found in samples collected at the intertidal sand bank located at the Yodo River estuary. In the Yodo River samples, 16S rRNA gene fragments affiliated with the known anammox bacterial lineage were also recovered, and the two major phylotypes were both "Candidatus Scalindua wagneri" relatives with 95% and 98% sequence similarity. Even from the other samples in which no recognizable anammox activity was detected, 16S rRNA gene fragments related to known anammox bacteria, but not to "Ca. S. wagneri", were detected. This is the first report of anammox-mediated N2 emission in coastal marine environments in Japan. Notably, the PCR-based analysis allowed us to discover unexpected phylogenetic diversity of anammox bacteria-related 16S rRNA gene sequences. The selective PCR primer set developed in this study could be a powerful tool to unveil the ecology of anammox bacteria in natural environments.

Although the emission of N2 via anaerobic ammonium oxidation (anammox) is a key process in the elimination of nitrogenous compounds from aquatic environments, little information is available regarding its significance and the relevant microorganisms (anammox bacteria) in eutrophic freshwater lakes. In the present study, the anammox bacteria in the sediment of a eutrophic lake in Japan, Lake Kitaura, were examined using a 15 N-tracer technique to measure their potential anammox activity. Potential anammox activity was localized to the northern region of the lake where a stable supply of both NH4 + and NO3− existed in the sediment. These results suggest the contribution of anammox bacteria to the total emission of N2 from sediment in this eutrophic lake to not be negligible. Moreover, selective PCR successfully amplified anammox bacteria-related (Brocadiales-related) 16S rRNA genes from sediment samples in which potential anammox activity was observed. The clone libraries consisted of diverse phylotypes except the genus "Scalindua"-lineages, and the lineages of genus "Brocadia" were dominantly recovered, followed by the genus "Kuenenia"-lineages. Most of them, however, were novel and phylogenetically distinguishable from known Brocadiales species. A unique population of anammox bacteria inhabits and potentially contributes to the emission of N2 from Lake Kitaura.

Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

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Distribution and seasonal fluctuation of the bacteria which inhibit the growth of a red tide marine dinoflagellate Gymnodinium mikimotoi, were surveyed in Tanabe Bay (Wakayama Pref., Japan), us ing the newly developed MPN method with an axenic culture of G. mikimotoi. G. mikimotoi's growth inhibiting bacteria (Gm-GIB) were detected at 103-104 cells/ ml before occurrences of huge red tides by G. mikimotoi at the beginning of August in 1990 and from the end of August to the beginning of Sep tember in 1991. The number of Gm-GIB fell by about two orders of magnitude at the blooming periods of G. mikimotoi, and then increased again after the blooms declined. These results suggest that the fluc tuation of Gm-GIB counts in seawater is significantly related to the development and decline process of G. mikimotoi red tide. Forty strains of Gm-GIB isolated in this study all acted as killers against this dinoflagellate rather than as suppressers on the algal growth under laboratory conditions. The precise causes of the fluctuation of Gm-GIB in seawater environments remain unknown.