Usio Simidu

Phylogenetic relationships of marine bacteria, mainly members of the family Vibrionaceae, determined on the basis of 16S rRNA sequences.

The phylogenetic relationships of 50 reference strains, mostly marine bacteria which require Na+ for growth, were determined on the basis of 600 16S rRNA nucleotides by using reverse transcriptase sequencing. Strains belonging to 10 genera were included (four genera of the family Vibrionaceae, the genus Aeromonas of the family Aeromonadaceae, and the genera Alteromonas, Marinomonas, Shewanella, Pseudomonas, and Deleya). The sequences were aligned, the similarity values and evolutionary distance values were determined, and a phylogenetic tree was constructed by using the neighbor-joining method. On the basis of our results, the family Vibrionaceae was separated into at least seven groups (genera and families). Vibrio marinus clearly was on a line of descent that was remote from other vibrios. As determined by the similarity and evolutionary distance values, V. marinus is more distantly related to the family Vibrionaceae than the members of the Aeromonadaceae are. Also, Vibrio cholerae strains formed a separate group with Vibrio mimicus at the genus level. Of 30 species of the Vibrionaceae, 17 formed a large phylogenetic cluster. The genus Listonella was found to be a heterogeneous group, and the species were distributed in various subgroups of the Vibrionaceae. The separation of the family Aeromonadaceae from the family Vibrionaceae and the separation of the genera Marinomonas and Shewanella from the genus Alteromonas were confirmed in this phylogenetic study. However, a marine Pseudomonas species, Pseudomonas nautica, was clearly separated from two terrestrial Pseudomonas species. Each group that was separated by the phylogenetic analysis had characteristic 16S rRNA sequence patterns that were common only to species in that group. Therefore, the characteristic sequences described in this paper may be useful for identification purposes.

Taxonomy of four marine bacterial strains that produce tetrodotoxin

Four strains of tetrodotoxin-producing bacteria isolated from a red alga and from pufferfish were characterized. Two of these strains are members of the genus Listonella MacDonell and Colwell. The phenotypic characteristics, guanine-plus-cytosine contents, and base sequences of the 16S rRNAs of these organisms indicated that they are members of Listonella pelagia (Vibrio pelagius) biovar II. The other two strains are members of the genus Alteromonas Baumann et al. and the genus Shewanella MacDonell and Colwell. These two strains are mutually distinct and distinct from the previously described Alteromonas and Shewanella species and therefore are placed in new species. The names Shewanella alga and Alteromonas tetraodonis are proposed for these organisms; the type strains are strains OK-1 and GFC, respectively.

Erythrobacter longus gen. nov., sp. nov., an Aerobic Bacterium Which Contains Bacteriochlorophyll a

Four orange-pigmented and seven pink-pigmented strains of bacteria which contained bacteriochlorophyll a were isolated from high-tidal seaweeds, such as Enteromorpha linza (L.) J. Ag. and Porphyra sp. All of the isolates were gram negative. The orange-pigmented bacteria were rods with parallel sides and rounded ends, and the pink-pigmented bacteria were ovoids and short rods. All were motile by means of subpolar flagella. None of the strains produced growth anaerobically in the light. No growth occurred with an atmosphere containing H2 and CO2. All of these bacteria grew aerobically and utilized glucose, pyruvate, acetate, butyrate, and glutamate as sole organic carbon sources. The best growth occurred on complex media formulated for heterotrophic marine bacteria. Biotin was required. Oxidase and catalase were present. Small amounts of acid were produced from a wide range of carbohydrates under microaerobic conditions. Gelatin was hydrolyzed. The strains which we investigated fell into the following three clusters: cluster A, all of the orange strains; cluster B, three pink strains; and cluster C, four pink strains. The strains of clusters B and C required thiamine and nicotinic acid and were susceptible to streptomycin. Tween 80 was hydrolyzed and phosphatase activity was produced by the strains of clusters A and B. Pantothenate was required only by the strains of cluster C. The guanine-plus-cytosine contents of the deoxyribonucleic acids of these organisms ranged from 60 to 64 mol%. These organisms are recognized here as members of a new genus, Erythrobacter. Although these organisms did not grow phototrophically, the presence of bacteriochlorophyll a indicated that they are most closely related to the Rhodospirillaceae. The type species is Erythrobacter longus, the type strain of which is an orange strain, OCh101 (= IFO 14126).

A tissue culture assay for tetrodotoxin, saxitoxin and related toxins

In the presence of ouabain, veratridine enhances sodium influx in the mouse neuroblastoma cell line Neuro-2A (ATCC, CCL131), causing cellular swelling and subsequent death. Tetrodotoxin (puffer fish toxin) or saxitoxin (paralytic shellfish poison), both of which block the sodium channel of excitable membranes, antagonize this effect, enabling cell growth to continue. This phenomenon was used as the basis of a new assay for these toxins. It is also possible to estimate the quantity of TTX from the relationship between TTX concentration and percentage of living cells. This new method is simple, inexpensive, and sensitive, and may replace the conventional mouse bioassay.

Tetrodotoxin-producing bacteria from the blue-ringed octopus Octopus maculosus

Several live specimens of the blue-ringed octopus Octopus maculosus were collected from the Philippines in November 1985, and from Japan in February 1986, and the distribution of toxicity, along with toxin composition, in the posterior salivary gland and other soft parts were examined. Tetrodotoxin (TTX: 1400 mouse units g-1) was detected in the posterior salivary gland of a Japanese specimen, while not only the salivary gland but other soft parts were toxic in the Philippine specimens. The Philippine specimens contained TTX and anhydrotetrodotoxin, the Japanese specimen TTX, 4-epitetrodotoxin, and an unknown toxin. The posterior salivary gland, intestine and other parts were excised from the Philippine specimens and examined for bacterial flora. Twenty-two dominant strains were isolated and cultured in a 2xORI medium (Ocean Research Institute, Simidu and Tsukamoto 1985) at 20°C for 20 to 48 h. Cells were harvested by centrifugation, and disrupted by ultrasonication. The toxins were partially purified from the cell lyzate by ultrafiltration and Bio-Gel P-2 column-chromatography. Instrumental analyses disclosed that 16 of the 22 strains produced TTX and/or related substances. Six strains which clearly exhibited TTX productivity were identified as Alteromonas (2 strains), Bacillus (2), Pseudomonas (1) and Vibrio (1), based on biochemical and biological characteristics. Of these, one strain each of Bacillus and Pseudomonas produced TTX at a level detectable by the mouse assay.

Microbiological studies of Tokyo Bay

The generic composition of the heterotrophic bacterial population of Tokyo Bay, which is now highly polluted and eutrophic, was compared with that of the adjacent, less polluted regions of Sagami Bay and Suruga Bay. Members of Vibrionaceae predominated in the bacterial flora of seawater and zooplankton samples from Sagami Bay, Suruga Bay, and the mouth of Tokyo Bay. However,Vibrio spp. formed only a small proportion of the bacterial population of the water and sediment samples from the inner Tokyo Bay; there the Gram-negative, nonmotile, nonpigmented bacteria, which were tentatively identified asAcinetobacter, were predominant. The result of experiments, in which seawater samples from Tokyo Bay were incubated under various experimental conditions, indicated that two significant factors apparently control the growth ofVibrio spp. in seawater; (1) a direct antagonism betweenVibrios and phytoplankton undergoing rapid growth, and (2) a limiting organic nutrient forvibrios.

Bacterial attachment to phytoplankton in sea water

Abstract Bacterial attachment to phytoplankton cells was investigated in natural sea water and with a mixed culture of Flavobacterium T-8D and marine diatom, Skeletonema costatum (Grev.) Cleve. The attached bacteria on incubated natural phytoplankton cells were considerably different from those in the surrounding sea water. The attachment rate of T-8D to the alga depended on the algal growth phase and light, but not on the bacterial concentration. The attachment of this strain to glass surfaces depended on the bacterial concentration. The results indicate that characteristic bacterial groups will attach to phytoplankton cells depending on the physiological condition of the algae, but not on the concentration of bacteria or dissolved organic matter.

A new method to detect viable bacteria in natural seawater using 16SrRNA oligonucleotide probe

Fluorescent oligonucleotide probes were used to detectVibrio cholerae directly under epifluorescence microscope. The probe which is complementary to the specific 16SrRNA sequence ofVibrio cholerae was labelled with Texas-Red, whereas the universal probe was labelled with Fluorescein. These probes allowed the distinctionVibrio cholerae from other eubacteria under the same microscopic field. In order to detect and enumerate specific bacteria in natural seawater, this method was combined with the direct viable count (DVC) technique. The combined method increased intracellular rRNA levels in the sample, and made it possible to detect the target bacteria with the specific gene probe. The applicability of this new method was confirmed both for the laboratory mixed culture system and natural seawater.

Fluctuation of the communities of heterotrophic bacteria during the decomposition process of phytoplankton

Abstract The fluctuations of the number and the communities of heterotrophic bacteria were investigated during the decomposition processes of two marine phytoplanktons, Chlorella sp. and Skeletonema costatum (Grev.) Cleve, under laboratory conditions. The concentration of particulate organic carbon (POC) decreased rapidly during the first 5 days. The number of the heterotrophic bacteria increased rapidly on the first day, the maximum value of plate counts was obtained on the 5th day. During the decomposition of phytoplankton, the communities of bacteria attached to particulate organic matter (POM) were quite different from that of free-living bacteria in the surrounding sea water. The dominant communities of heterotrophic bacteria attached to POM changed not only from Pseudomonas-Alcaligenes group to Acinetobacter-Moraxella group, but also from the communities with high potential ability to decompose high molecular organic substrates to one with low ability, suggesting that there is a succession of bacterial communities involved in the decomposition of POM.

Scanning electron microscopy investigation of bacterial colonization of the marine copepod Acartia clausi

Association between copepods and bacteria was observed in many scanning electron micrographs. Particular sites on the copepods were selectively colonized by bacteria; the joints of segments and legs, swimming legs and depressed parts of the body surface were found to be densely covered with bacteria. In comparison, bacterial attachment to copepod skeletons in fecal pellets excreted by chaetognaths was not selective; bacteria were sparsely found all over the copepod. Between 9 to 30% of copepods in Tokyo Bay waters had attached bacteria in January and April 1983.