Population structure of Alexandrium (Dinophyceae) cyst formation-promoting bacteria in Hiroshima Bay, Japan

Abstract

A total of 31 bacterial isolates that have potential Alexandrium cyst formation-promoting activity (Alex-CFPB) were isolated from Hiroshima Bay (Japan), which is characterized by seasonal blooms of the toxic dinoflagellate Alexandrium tamarense. The population structure of Alex-CFPB was analyzed by means of restriction fragment length polymorphism analysis of the 16S rRNA genes (16S rDNA). Fourteen ribotypes, A to N, were observed among the 31 isolates of Alex-CFPB by using four restriction enzymes, MboI, HhaI, RsaI and BstUI. Among them, seven isolates, which were obtained from the seawater samples taken during the peak and termination periods of the A. tamarense bloom in 1998, belonged to ribotype A. This result suggests that bacterial strains of ribotype A may be dominant in the Alex-CFPB assemblages during these periods. The partial 16S rDNA-based phylogenetic tree of 10 ribotypes studied showed that nine of them fell into the Rhodobacter group of the alpha subclass of the Proteobacteria: Eight of nine ribotypes of the Rhodobacter group fell into the lineage of the Roseobacter subgroup, and one fell into the Rhodobacter subgroup. The non-Rhodobacter group type fell into the Marinobacterium-Neptunomonas-Pseudomonas group of the gamma-Proteobacteria: Isolates of Alex-CFPB ribotypes A and C do not have clear growth-promoting activities but have strong cyst formation-promoting activities (CFPAs) under our laboratory conditions. These results show that the Alex-CFPB assemblage may consist of various bacteria that belong mainly to the Roseobacter group and have strong CFPAs. These results suggest that not only the Alexandrium cyst formation-inhibiting bacteria (Alex-CFIB) reported previously but also Alex-CFPB, especially bacteria of ribotype A, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment.

FIG. 1.

Amplified products of the 16S rDNA of various Alex-CFPB (A) and digestion of the PCR products with RsaI (B), BstUI (C), MboI (D), and HhaI (E). Lane M, 100-bp ladder markers; lane 1, CFPB-A9; lane 2, CFPB-B1; lane 3, CFPB-C1; lane 4, CFPB-D1; lane 5, CFPB-E1; lane 6, CFPB-F1; lane 7, CFPB-G1; lane 8, CFPB-H1; lane 9, CFPB-I1; lane 10, CFPB-J1; lane 11, CFPB-K1; lane 12, CFPB-L1; lane 13, CFPB-M1; lane 14, CFPB-N1.

FIG. 2.

Molecular phylogenetic tree inferred from the partial 16S rDNA for Alex-CFPB in the Rhodobacter group of the α subclass of the Proteobacteria by the NJ method. Numbers are percentages of 1,000 bootstrap repetitions. Bootstrap values greater than 50% are indicated. The tree is based on positions 50 to 410 (E. coli numbering) of the 16S rDNA described by González et al. (20, 21). Black squares indicate isolates that were found in association with phycospheres. The names of algal species associated with bacteria are shown in parentheses. Black circles indicate isolates that have abilities to transform organic and/or inorganic sulfur compounds.

FIG. 3.

Molecular phylogenetic tree inferred from the partial 16S rDNA for CFPB-D1 in the Pseudomonas group of the γ subclass of the Proteobacteria by the NJ method. Numbers are percentages of 1,000 bootstrap repetitions. Bootstrap values greater than 50% are indicated. The tree is based on positions 50 to 410 (E. coli numbering) of the 16S rDNA described by González et al. (20, 21).