Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov. and emendation of description.

A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458T and IAM 14160T shared 99% genetic relatedness, but were only 48-49% related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915T. The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915T and 32% with KMM 458T. From these results, it is concluded that strains KMM 458T and IAM 14160T comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T).

Characterization of microbial community structure in the surface sediment of osaka bay, Japan, by phospholipid Fatty Acid analysis.

Twenty-eight sediment samples collected from Osaka Bay, Japan, were analyzed for phospholipid ester-linked fatty acids (PLFA) to determine regional differences in microbial community structure of the bay. The abundance of three major groups of C(10) to C(19) PLFA (saturated, branched, and monounsaturated PLFA), which accounted for 84 to 97% of the total PLFA, indicated the predominance of prokaryotes in the sediment. The distribution of six clusters obtained by similarity analysis in the bay revealed a marked regional distribution in the PLFA profiles. Total PLFA concentrations (0.56 to 2.97 mug/g [dry weight] of the sediment) in sediments also showed marked variation among the stations, with higher concentrations of total PLFA in the central part of the bay. The biomass, calculated on the basis of total PLFA concentration, ranged from 0.25 x 10 to 1.35 x 10 cells per g (dry weight) of the sediment. The relative dominance of microbial groups in sediments was described by using the reported bacterial biomarker fatty acids. Very small amounts of the characteristic PLFA of microeukaryotes in sediments indicated the restricted distribution of microeukaryotes. By examining the distribution of clusters and groups of microorganisms in the bay, there were two characteristics of the distribution pattern: (i) the predominance of anaerobic bacteria and gram-positive prokaryotes, characterized by the high proportions of branched PLFA in the eastern and northeastern sides of the bay, where the reported concentrations of pollutants were also high, and (ii) the predominance of aerobic prokaryotes and eukaryotes, except for a few stations, in the western and southwestern sides of the bay, as evidenced by the large amounts of monounsaturated PLFA. Such significant regional differences in microbial community structure of the bay indicate shifts in microbial community structure.

Presence of tetrodotoxin and tetrodotoxin-producing bacteria in freshwater sediments.

The occurrence of tetrodotoxin (TTX) in sediment from Lake Suwa, Japan, was confirmed by a tissue culture assay, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Altogether, 17 TTX-producing bacteria belonging to five genera were isolated from Lake Suwa and Pond Inokasira. Our results indicate that TTX and TTX-producing bacteria occur even in freshwater environments.

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.

Identification of deep-sea-sediment bacteria which produce tetrodotoxin.

Forty-nine bacterial strains were isolated from deep-sea sediments. Among them, 22 strains were shown by the tissue culture assay method to produce sodium channel blockers. For some strains, high-performance liquid chromatography analysis confirmed that the blocker was tetrodotoxin. Tetrodotoxin-producing bacteria seem to be widespread in marine sediment.

A Photobacterium-like bacterium able to fix nitrogen.

A Photobacterium-like bacterium isolated from the roots of eelgrass (Zostera marina) was shown to fix nitrogen under anaerobic conditions. Nitrogen fixation by Photobacterium spp. has not been reported previous to this.

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed.

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10(2) to 4.6×10(2)/ml for seawater, 4.0×10(4) to 4.3×10(5)/g wet wt for eelgrass-bed sediment, and 2.1 × 10(5) to 1.2 × 10(7)/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.

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.

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.

Correlation of direct viable counts with heterotrophic activity for marine bacteria.

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.

Marine bacteria which produce tetrodotoxin.

A number of type strains of marine bacteria, including members of the family Vibrionaceae, were cultured and examined for tetrodotoxin productivity by high-performance liquid chromatography and gas chromatography-mass spectrometry. Most of the Vibrionaceae strains produced tetrodotoxin, anhydrotetrodotoxin, or both.

Habitat segregation and biochemical activities of marine members of the family vibrionaceae.

A comparative study of marine members of the family Vibrionaceae with the technique of numerical taxonomy revealed habitat segregation as well as a cosmopolitan nature of species distribution among the vibrios in different marine environments. The bacterial strains analyzed were isolated from seawater, sediments, phyto- and zooplankton, and fish in the Indian Ocean, the South and East China Sea, and West Pacific Ocean, and coastal areas of Japan. A total of 155 morphological, physiological, and biochemical tests were carried out for each of 405 strains examined. The results showed that most of the large taxonomical clusters which emerged from the computation corresponded to ecological groups which have particular niches. For instance, each group of seawater vibrios inhabited a particular water layer of limited depth range, in spite of the fact that strains of the group were isolated from sampling locations spread over a wide area from the Indian Ocean to Japanese coast. Various vibrio groups showed remarkable differences in their physiological and biochemical activities, and the activities of each group seemed to correspond with its ecological niche. The strains which inhabited surface-water layers grew fast and actively utilized many high-molecular-weight organic compounds and carbohydrates that are derived from fresh, easily degradable organic matter present in the surface waters, whereas the middle- and deep-water vibrios did not decompose most of the high-molecular-weight organic compounds except chitin but, rather, utilized some carbohydrates and organic acids which seemed to be derived from refractory particulate organic matter present in the deeper waters.

Purification and some properties of halophilic protease produced by a moderately halophilic marine Pseudomonas sp.

Halophilic protease in culture fluids of a moderately halophilic marine Pseudomonas sp. (A-14) was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 120 000. The optimum pH for activity was 8.0. The enzyme had maximal activity at 18% NaCl concentration. The enzyme was activated by Mg2+, Co2+, and Ca2+. Ca2+ increased the heat stability, and heavy metal ions such as Fe2+, Cu2+, and Hg2+ inactivated the enzyme. Thiol reagents and diisopropyl fluorophosphate did not affect the enzymatic activity of the protease. Metal-complexing reagents, such as ethylenediaminetetraacetic acid and o-phenanthroline, inhibited enzymatic activity, although citrate and oxalate did not affect it.

Distribution of viable marine bacteria in neritic seawater around Japan.

The direct viable court (DVC) method of determining the number of living bacteria in natural seawater was applied to samples collected from areas around Japan ranging from polluted to oligotrophic. In summer, the DVC comprised 1.5-39.8% (mean, 11.2%) of the total direct count (TDC) in Tokyo Bay, which was highly eutrophic. In offshore areas (Sagami Bay and the Kuroshio region), the percentages ranged from 0.7 to 7.9 (mean 2.8%). In winter, slightly higher percentages were observed in Sagami Bay. The majority of DVC cells were free-living. The distruction of bacteria correlated closely with particulate or dissolved organic matter.

A method of the selective isolation and enumeration of marine Vibrionaceae.

A noninhibitory medium and GasPack anaerobic culture system were employed for the selective enumeration and isolation of Vibrionaceae in seawater and marine sediments.Vibrio counts obtained by the new method for seawater and sediment samples were compared with vibrio numbers in the heterotrophic bacterial population appearing on a medium routinely employed in the laboratory for such counts. The ratio of the former to the latter counts ranged from 0.5 to 1.3, the average being 0.96. The seawater and sediment bacteria that grew and produced visible colonies on the medium under anaerobic conditions for 3 days at 20°C were almost exclusively vibrios.From the results reported here it is concluded that most of the vibrios present in seawater and sediment samples can be recovered by the new method developed in this study.

Distribution of aerobic bacteria which contain bacteriochlorophyll a.

Sixteen strains of aerobic bacteria which contain bacteriochlorophyll a were isolated from the samples collected in aerobic marine environments: thalli of Enteromorpha linza, Porphyra sp., Sargussum horneri; beach sand; and the surface seawater from Aburatsubo Inlet. When they occurred, their proportions among the aerobic heterotrophic populations ranged from 0.9 to 1.1% in the seaweed samples and from 1.2 to 6.3% in the beach sand samples and were 0.9% in the seawater sample. The results suggested that the aerobic photopigmented bacteria widely inhabit aerobic marine environments.

Comparative study of the aerobic, heterotrophic bacterial flora of Chesapeake Bay and Tokyo Bay.

A comparative study of the bacterial flora of the water of Chesapeake Bay and Tokyo Bay was undertaken to assess similarities and differences between the autochthonous flora of the two geographical sites and to test the hypothesis that, given similarities in environmental parameters, similar bacterial populations will be found, despite extreme geographic distance between locations. A total of 195 aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and Tokyo Bay water were examined for 115 biochemical, cultural, morphological, nutritional, and physiological characters. The data were analyzed by the methods of numerical taxonomy. From sorted similarity matrices, 77% of the isolates could be grouped into 30 phena and presumptively identified as Acinetobacter-Moraxella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio and Acinetobacter species were found to be common in the estuarine waters of Chesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predominated in Tokyo Bay waters, at the sites sampled in the study.