Kumiko Kita-Tsukamoto

Inferring the Evolutionary History of Vibrios by Means of Multilocus Sequence Analysis

We performed the first broad study aiming at the reconstruction of the evolutionary history of vibrios by means of multilocus sequence analysis of nine genes. Overall, 14 distinct clades were recognized using the SplitsTree decomposition method. Some of these clades may correspond to families, e.g., the clades Salinivibrio and Photobacteria, while other clades, e.g., Splendidus and Harveyi, correspond to genera. The common ancestor of all vibrios was estimated to have been present 600 million years ago. We can define species of vibrios as groups of strains that share >95% gene sequence similarity and >99.4% amino acid identity based on the eight protein-coding housekeeping genes. The gene sequence data were used to refine the standard online electronic taxonomic scheme for vibrios (http://www.taxvibrio.lncc.br).

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.

Microbial diversity in marine sediments from Sagami Bay and Tokyo Bay, Japan, as determined by 16S rRNA gene analysis

16S rDNA clone libraries were analysed to investigate the microbial diversity in marine sediments from Sagami Bay (stations SA, water depth of 1159 m, and SB, 1516 m) and Tokyo Bay (station TK, 43 m). A total of 197 clones was examined by amplified rDNA restriction analysis (ARDRA) using three four-base-specific restriction enzymes (Hhal, Rsal and Haelll). In SA, 57 RFLP types were detected from 77 clones. In SB, 17 RFLP types were detected from 62 clones. In TK, 21 RFLP types were detected from 58 clones. The genotypic diversity among the three sampling sites was 0.958, 0.636 and 0.821, respectively, indicating that the microbial diversity of SA was higher than at the other two stations. At SA, the most abundant RFLP type constituted 10% of all clones. The samples from SB and TK had dominant RFLP types which constituted 60% and 38% of the total clone libraries, respectively. The community structure of SA included many single-type clones, which were found only once in the clone libraries. This structure contrasted with that of the other two stations. Thirty-seven clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the microbial diversity in the clone libraries. No clones were identical to any of the known 165 rRNA sequences or to each other. All sequences had >84.8% similarity to rDNA sequences retrieved from the DNA databases. Sequenced clones fell into five major lineages of the domain Bacteria: the gamma, delta and epsilon Proteobacteria, Gram-positive bacteria and the division Verrucomicrobia. At SA, the Verrucomicrobia and the three subclasses of the Proteobacteria were found. Most clone sequences belonged to the gamma Proteobacteria. The high-GC Gram-positive bacteria and the gamma subclass of the Proteobacteria were common at both SB and TK. Although the depths of SB and TK were very different, the community diversity inferred from ARDRA and the taxonomic position of the dominant clones were similar. All clones belonging to the highGC Gram-positive bacteria collected from both SB and TK fell into the same cluster and are regarded as members of an unknown actinomycete group. The clone compositions were different at each sampling site, and clones of the gamma Proteobacteria and high-GC Gram-positive bacteria were dominant.

Vibrio azureus sp. nov., a luminous marine bacterium isolated from seawater

Two luminous marine bacterial strains, LC2-005(T) and LC2-102, were isolated from seawater at Kuroshio Region and Sagami Bay in Japan, respectively. These bacteria were Gram-negative, oxidase-positive, catalase-positive, motile and rod-shaped. On the basis of 16S rRNA gene sequence analysis, strains LC2-005(T) and LC2-102 formed a cluster within the Vibrio harveyi species group. However, multilocus sequence analysis using five loci (pyrH, ftsZ, mreB, gyrB and gapA) and DNA-DNA hybridization experiments indicated that these strains were distinct from the currently known Vibrio species. Additionally, these strains differ from related Vibrio species in utilization of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose and arabinose, production of lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, esterase (C4), lipase (C4), chymotrypsin, acid phosphatase, alpha-glucosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase and the ability to reduce nitrate to nitrite. The major fatty acids were C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c, C(16 : 0), C(18 : 1)omega7c and C(14 : 0). The DNA G+C contents of strains LC2-005(T) and LC2-102 were 45.2 and 45.5 mol%, respectively. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains LC2-005(T) and LC2-102 belong to the same genospecies and represent a novel species of the genus Vibrio, for which the name Vibrio azureus sp. nov. is proposed. The type strain is LC2-005(T) (=NBRC 104587(T) =KCTC 22352(T)).

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.

Photobacterium aquimaris sp. nov., a luminous marine bacterium isolated from seawater.

Two luminous marine bacteria, strains LC2-065(T) and LC2-102, were isolated from seawater at Sagami Bay in Japan. These bacteria were Gram-negative, oxidase-negative, catalase-positive, motile and coccoid-rods. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA) using six loci (ftsZ, gapA, gyrB, mreB, pyrH and topA) and sequence analysis of the alpha subunit of luciferase (luxA) gene revealed that these bacteria were distinct from other species of the genus Photobacterium. These novel strains were most closely related to Photobacterium kishitanii. The DNA-DNA hybridization value between strain LC2-065(T) and Photobacterium kishitanii ATCC BAA-1194(T) was 42.1 %. The major fatty acids were C(12 : 0,) C(14 : 0), C(16 : 0), C(18 : 0) and C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c (summed feature 3). The DNA G+C contents of strains LC2-065(T) and LC2-086 were 42.2 and 42.9 mol%, respectively. The phenotypic features of the novel strains were similar to those of P. kishitanii and P. phosphoreum, but there were sufficient physiological differences for the novel strains to be easily differentiated. On the basis of these results, these new strains represent a novel species, for which the name Photobacterium aquimaris sp. nov. is proposed. The type strain is LC2-065(T) (=NBRC 104633(T)=KCTC 22356(T)).

Characterization of Psychrotrophic Bacteria in the Surface and Deep-Sea Waters from the Northwestern Pacific Ocean Based on 16S Ribosomal DNA Analysis

Abstract: Seventy-eight 4°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°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.

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).

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).