Flagellar motors of marine bacteria Halomonas are driven by both protons and sodium ions.

Bacterial cells in aquatic environments are able to reach or stay near nutrient patches by using motility. Motility is usually attained by rotating flagellar motors that are energized by electrochemical potential of H+ or Na+. In this paper, the ion specificity for flagellar rotation of two marine isolates Halomonas spp. strains US172 and US201 was investigated. Both isolates require sodium for growth and possess a respiratory-driven primary sodium pump. They are motile because of lateral flagella regardless of the presence of sodium ions. Their swimming speed under various concentrations of sodium ions with and without carbonylcyanide m-chlorophenylhydrazone, a proton conductor, and with and without phenamil, a specific inhibitor for the sodium-driven flagellar motors, was examined. The effect of carbonylcyanide m-chlorophenylhydrazone on the transmembrane proton gradient was also determined. Our results showed that the flagellar motors of the Halomonas strains were energized by both H+ and Na+ in one cell. The bimodal nature of Halomonas spp. motility with respect to the driving energy source may reflect ecophysiological versatility to adapt to a wide range of salt conditions of the marine environment.

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 psychrotrophic bacteria in the surface and deep-sea waters from the northwestern Pacific Ocean based on 16S ribosomal DNA analysis.

Seventy-eight 4 degrees C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0-200 m) and deep-sea (1000-9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4 degrees C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats.

Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis.

A new Pseudomonas species, for which the name Pseudomonas plecoglossicida is proposed, was isolated from cultured ayu (Plecoglossus altivelis) with bacterial haemorrhagic ascites. The causative agent was similar to Pseudomonas putida biovar A in its phenotypic characteristics and on the basis of 16S rRNA gene sequence analysis, but it reduced nitrate to nitrite. Furthermore, it was distinguished phenotypically from Pseudomonas putida biovar A by utilization of D-malate, L-(+)-tartrate, m-tartrate and nicotinate. The levels of DNA-DNA hybridization between the isolate strain FPC 951T and other reference strains of Pseudomonas species, including Pseudomonas putida, were less than 50%. The G+C content of the DNA of FPC 951T was 62.8 mol%. Strain FPC 951T (= ATCC 700383T) is designated the type strain of the new species.

Reassessment of the taxonomic position of Vibrio iliopiscarius (Onarheim et al. 1994) and proposal for Photobacterium iliopiscarium comb. nov.

The phylogenetic position of Vibrio iliopiscarius was inferred by the maximum-likelihood, maximum-parsimony and neighbour-joining methods on the basis of almost complete 16S rRNA gene sequences. The results showed that this species falls into the same cluster as Photobacterium species and is clearly distinct from other Vibrio species. Its nearest phylogenetic neighbour is Photobacterium phosphoreum. From these results, it is concluded that V. iliopiscarius should be reclassified as Photobacterium iliopiscarium comb. nov., the type strain of which is PS1T (= ATCC 51760T).

16S rDNA restriction fragment length polymorphism analysis of psychrotrophic vibrios from Japanese coastal water.

Restriction fragment length polymorphism (RFLP) analysis was carried out for 136 natural isolates belonging to the family Vibrionaceae. These were collected from inshore areas of Japan, mainly in winter. Twenty-eight 16S rDNA genotypes were obtained by digestion with four restriction endonucleases (HhaI, DdeI, RsaI, and Sau3AI). To estimate the genetic relationships, 53 informative fragments were scored by their presence or absence. A dendrogram was constructed using the unweighted pair group method with the arithmetic averages algorithm. Five RFLP groups (groups I to V) were obtained. Group I corresponded to Vibrio splendidus-like strains. It was confirmed that this group was not only found in Otsuchi Bay, but also in broad coastal areas of Japan. Group II strains were not identified as previously known Vibrio species. Group III strains were regarded as members of the Vibrio main group, which is a major phylogenetic group deduced from 16S rRNA gene analysis in the family Vibrionaceae. The RFLP profile indicated that Group IV strains were closely related to V. hollisae. Group V strains showed RFLP patterns which have not been observed previously. From the clustering analysis, it was concluded that group V strains were not Vibrio species. Most of the isolates studied were not identified as previously described species. It suggests that many psychrotrophic vibrios in cold marine environments remain as unknown species.

A proposal to transfer Vibrio marinus (Russell 1891) to a new genus Moritella gen. nov. as Moritella marina comb. nov.

The taxonomic positions of Vibrio marinus and 11 related natural isolates were examined. Their phylogenetic positions on the basis of almost complete 16S rRNA sequences were determined by maximum likelihood, maximum parsimony and neighbor-joining analyses. V. marinus and the 11 isolates fell into a single cluster that was clearly distinct from other genera. There is now strong evidence that V. marinus should be reclassified as Moritella marina gen. nov., comb. nov.

A new approach to separate the genus Photobacterium from Vibrio with RFLP patterns by HhaI digestion of PCR-amplified 16S rDNA.

A new approach to separate members of the genus Photobacterium from the genus Vibrio with RFLP (Restriction Fragment Length Polymorphism) patterns by HhaI digestion of PCR-amplified 16S rDNA was developed in the present study. It was clearly shown that these patterns of the genus Photobacterium were unique and distinguishable from Vibrio species. This method is very simple and does not need other supporting procedures, such as Southern transfer and probe hybridization. It can be applied not only to luminous species, but also to non-luminous Photobacterium spp. This result promises a rapid tool to distinguish the genus Photobacterium from Vibrio and should be useful in routine identification system.

16S rDNA genotyping using PCR/RFLP (restriction fragment length polymorphism) analysis among the family Vibrionaceae.

The 16S rDNA genotypes among the family Vibrionaceae were determined using PCR/RFLP analysis. Five tetrameric restriction enzymes (HhaI, DdeI, RsaI, Sau3AI and MspI) were used for RFLP analysis and adequate numbers of informative bands were obtained from each enzyme. Twenty-seven genotypes were obtained from 49 type and reference strains including 35 species. Nineteen species could be assigned to specific 16S rDNA genotypes, supporting the application of this analysis for identification. Trees constructed using five endonucleases resolved groups almost identical to those inferred from 16S rRNA gene sequencing. However, the branch lengths and detailed relationships among strains within a group differed from those inferred from sequence comparisons. The results of this study should be useful for genotyping, identification and approximate classification of natural isolates belonging to the family Vibrionaceae.

The 16S rRNA sequence and genome sizing of tributyltin resistant marine bacterium, strain M-1.

The 16S rRNA of the tributyltin resistant marine bacterium, strain M-1, was partly sequenced to confirm the taxonomic status. The results indicated that this bacterium should be classified under the genus Alteromonas, instead of a previous report in which this strain was identified as a Vibrio. The genome size of this strain was also measured by pulsed field gel electrophoresis (PFGE) using a contoureclamped homogeneous electric field. The strain was found to contain a genome size of 2,240 kilo base pairs, whereas Alteromonas nigrifaciens and Shewanella putrefaciens had 2,040 and 2,383 kilo base pairs, respectively. This is the first report of the genome sizing of the genus Alteromonas by PFGE.

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.