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The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS‐1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS‐1 (WKS‐I type) or A. tamarense CU‐15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS‐1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (<0.01), whereas distance values of ITS between the catenella type and the tamarense, WKS‐1, or Thai type were 0.17, 0.18, and 0.40, respectively. Isolates of A. affine, A. insuetum, and A. pseudogonyaulax all carried unique ITS types. The ITSs of the tamarense type exhibited two distinct ITS sets, the “A gene” and the “B gene.” The two sequences occurred in a 1:1 ratio in PCR products. In contrast, the ITSs of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3′ end of ITS1 (150‐177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28‐bp region is more than 10. High diversity of this region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium.

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment‐length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra‐specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS‐1. Some restriction fragments from WKS‐1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS‐1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.

Several examples of stimulative and inhibitory effects of bacteria on microalgal growth are introduced, and the importance of bacteria in algal mass culture is investigated. Diatoms are often used as live food for planktonic larvae of sea urchin and bivalves. Monodispersed Chaetoceros ceratosporum has been cultivated by using clean, high nutrient content, deep seawater (DSW). However, the growth rate and cell yield of diatoms fluctuated, to relatively large extent, with the season that DSW was collected. When some bacterial strains isolated from DSW were added to the culture, diatom growth was often stimulated and a relatively constant cell yield was obtained. Another diatom species, C. gracilis, was also stimulated by adding some bacterial strains to cultures. The positive effect of bacteria on diatoms was observed not only for planktonic species, but also on attached species. A benthic diatom, Nitzschia sp., was stimulated by a bacterial film of Alcaligenes on the surface of the substratum. On the other hand, a strain of Flavobacterium sp. isolated from natural seawater during the decline period of an algal bloom had a strong algicidal effect on the red tide plankton, Gymnodinium mikimotoi. Recent reports demonstrate that many bacterial strains have significant algicidal effects on many species of red tide plankton. These results indicate that bacterial effects should be taken into account to obtain stable mass culture of food microalgae.

Marine bacteria that kill the noxious red tide flagellate Chattonella antiqua (Raphidophyceae) were screened and isolated from northern Hiroshima Bay, the Seto Inland Sea, Japan in 1991. Four strains (S, K, D, R) of Alteromonas spp. were selected and examined on characteristics of algicidal activities. Strains S and R showed wide algicidal range killing all cells of the 3 raphidophycean flagellates, 2 dia toms, and one dinoflagellate examined, in co-culture. Algicidal activities of the strains K and D depend on prey phytoplahkton species. Bacterial culture filtrate experiment shows that the bacterial strains K and D give lethal effects on C. antiqua by means of extracellular products, and the strains S and R not by such substances but by predation. If one or two bacterial cells were inoculated into C. antiqua cul ture, all of the host cells were killed by the 4 strains of algicidal bacteria within 7 days. All of the 4 bac terial strains could proliferate in filter-sterilized seawater, indicating their ubiquitous existence in the coastal sea. We suggest that the algicidal activity by bacteria may be a significant factor influencing the population dynamics of phytoplankton, and potentially might account for rapid termination of red tides in the coastal sea.

A novel extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent environment (depth, 22 m) in Tachibana Bay, Nagasaki Prefecture, Japan. The cells of this organism were gramnegative rods. Growth occurred at temperatures between 50 and 85°C (optimum temperature, 80°C; doubling time at optimum temperature, 90 min), at pH 5.5 and 9.0 (optimum pH, 7.0), and in the presence of 1 and 5% NaCl (optimum NaCl concentration, 3%). The new isolate was an aerobic heterotroph which utilized the following compounds as sole energy and carbon sources: yeast extract, peptone, starch, casein, Casamino Acids, a variety of sugars, some carboxylic acids, and amino acids. As determined by a sequence analysis of the 16s rRNA, the new isolate belongs to the genus Rhodothermus and represents a modern lineage of extreme thermophiles within the domain Bacteria. On the basis of the physiological and molecular properties of the new isolate, we describe a new species, Rhodothermus obamensis. The type strain of R. obamensis is strain OKD7 (= JCM 9785).Over the last 10 years, a number of new genera and species of thermophilic organisms which are capable of growth at temperatures up to 80°C have been isolated. Most of these organisms belong to the domain Archaea (31), but there are a few genera, such as the genera Themus, Thennotoga, and Aquifex, which belong to the domain Bacteria (5, 10, 11). These thermophiles have distinct physiological differences; the members of the genus Themus are strictly aerobic heterotrophs, the members of the genus Thermotoga are strictly anaerobic heterotrophs, and the members of the genus Aquifex are microaerobic chemolithoautotrophs. On the basis of 16s rRNA analysis data, the genera Thennotoga and Aquifex represent the deepest phylogenetic branches in the domain Bacteria (7, 21). Moreover, the genus Thennus also branches deeply on the phylogenetic tree inferred from 16s rRNA sequences (21). These findings and the results of a phylogenetic study of the thermophilic genus Hydrogenobacter support the hypothesis that the ancestors of bacteria were thermophilic species (1,26).The members of the genus Rhodothemus, on the other hand, are marine thermophilic bacteria that have been isolated from sites in Iceland and the Azores (2, 20). Rhodothemus marinus is an aerobic heterotrophic bacterium that has an optimum growth temperature of 65°C and has been used in studies of genetic engineering of thermostable enzymes (28). A 16s rRNA analysis of this organism placed the genus Rhodothermus close to the root of the Flexibacter-Cytophaga-Bacteroides group with affinities to the green sulfur bacteria, fibrobacteria, and spirochetes (3). This phylogenetic position of the genus Rhodothennus differs from the phylogenetic positions of other thermophilic bacteria and suggests that there are thermophilic or extremely thermophilic bacteria which are distant from the universal root of life and that there was another origin of thermophily within the Bacteria (3).In this paper, we describe the isolation and cha...

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