Tomoo Sawabe

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).

Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica

An aerobic, polarly flagellated marine bacterium that produces a prodigiosin-like pigment was isolated from the red-spotted culture beds of Laminaria japonica. Five isolates had unique bacteriolytic activity for both Gram-positive and -negative bacteria, which had never been observed among Alteromonas or related species. The isolates were identified as the causative agent of red spot disease of L. japonica seeds. The phenotypic features of the isolates were similar to these of Pseudoalteromonas rubra ATCC 29570T, but they could be differentiated using 10 traits (growth at 37 degrees C, requirement for organic growth factors, bacteriolytic activity, utilization of sucrose, N-acetylglucosamine, fumarate, succinate, D-galactose, L-proline and acetate). The G+C content of DNAs from the isolates was 44-46 mol%. The isolates constitute a new species, distinct from the other Alteromonas and Pseudoalteromonas species, as shown by DNA-DNA hybridization experiments and phylogenetic clustering of 16S rRNA gene sequences, for which the name Pseudoalteromonas bacteriolytica sp. nov. (type strain = IAM 14595T) is proposed. A set of phenotypic features which differentiate this new species from closely related Pseudoalteromonas and Alteromonas species is provided.

Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the “Porteresiae” clade.

Multilocus Sequence Analysis Reveals that Vibrio harveyi and V. campbellii Are Distinct Species

ABSTRACT Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.lncc.br/ ). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades.

Shewanella japonica sp. nov.

Two strains of agar-digesting bacteria, KMM 3299T and KMM 3300, respectively isolated from sea water and the mussel Protothaca jedoensis, have been characterized. Based on sequencing of the 16S rRNA gene, KMM 3299T showed the highest similarity (93-95%) to members of the genus Shewanella. The G+C contents of the DNAs of these strains were 43-44 mol%. The level of DNA homology between the two strains was conspecific (95%), indicating that they represent a distinct genospecies. These organisms were non-pigmented, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic, neutrophilic and able to degrade a wide range of high molecular mass polymers, including alginate, carrageenan, laminaran and agar. The novel organisms were susceptible to gentamycin, carbenicillin, lincomycin and oleandomycin. The predominant cellular fatty acids were i-15:0, 16:0, 16:1(n-7), 18:1(n-7). Eicosapentaenoic acid, 20:5(n-3), was detected in the two isolates at levels of 1-8%, depending on the temperature of cultivation. Phylogenetic evidence, together with phenotypic characteristics, showed that the two isolates studied constitute a novel species of the genus Shewanella. The name Shewanella japonica is proposed; the type strain is KMM 3299T(= LMG 19691T = CIP 106860T).

Bacillus horti sp. nov., a new Gram-negative alkaliphilic bacillus

Novel Gram-negative alkaliphilic strains were isolated from soil obtained from Atsuma, Hokkaido, Japan. The isolates were strictly aerobic rods that produced subterminally located ellipsoidal spores. Chemotaxonomic characteristics of the isolates included the presence of meso-diaminopimelic acid in the cell wall and a DNA G + C content of 40.2-40.9 mol%. The major isoprenoid quinone was menaquinone-7 and the cellular fatty acid profile consisted of a significant amount of 15-C branched-chain acids, iso-C15:0 and anteiso-C15:0. The growth rate was higher at pH 8-10 than at pH 7. Comparative sequence analysis of 16S rDNA of 14 alkaliphilic Bacillus strains indicates that the isolated strain has an equidistant relationship to three already defined rRNA groups of alkaliphilic Bacillus species. Based on the morphological and physiological characteristics, as well as phylogenetic position as determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these isolates should be designated as a new species, for which the name Bacillus horti is proposed. The type strain is K13T (= JCM 9943T).

Acetic acid production of Vibrio halioticoli from alginate: a possible role for establishment of abalone–V. halioticoli association

Acetic acid, which is converted from cellulose by means of the metabolism of their gut microbes, is an important oxidizable energy source and precursors of anabolism in ruminant animals and xylophagus insects. However, acetic acid production from algal polysaccharides by means of the metabolism of gut microbes of marine herbivorous invertebrates is not well studied. Abundance of Vibrio halioticoli, which is a dominant alginolytic gut microbe of abalone Haliotis discus hannai ,i n the gut of various marine herbivorous invertebrates and major fermentation products from alginate of these strains were investigated in this study. V. halioticoli strains were detected from the gut of three species of the Japanese abalone, Haliotis discus discus, Haliotis diversicolor aquatilis, and Haliotis diversicolor diversicolor, and a South African abalone, Haliotis midae, with a range of 40–65% amongst these microflora. The bacterium was also found in the gut of a turban shell, Turbo cornutus, of which occupation rate was 16%. Furthermore, acetic acid and formic acid were detected as major fermentation products of alginate in these V. halioticoli strains. It is suggested that the abundant populations of V. halioticoli in the gut of Haliotis abalone may have a great role for converting alginate to acetic acid. D 2003 Elsevier Science B.V. All rights reserved.

Novel alginate lyases from marine bacterium Alteromonas sp. strain H-4

A bacterium Alteromonas sp. strain H-4 isolated from Laminaria fronds produced extra- and intra-cellular alginate lyases and utilized alginate as its sole carbon source. An extracellular alginate lyase was purified from the culture supernatant of the strain and its substrate specificity was characterized. The estimated molecular mass of the enzyme was 32 kDa and the isoelectric point was 4.7. Both polyM and polyG block degrading activities were observed using the substrate-containing gel overlay technique after isoelectric focusing of the enzyme. By analyzing the reaction products from the polyM block, polyG block, MG random block and intact alginate, three major peaks containing unsaturated tri-uronide through octa-uronide were detected for each substrate. The results indicate that the enzyme of Alteromonas sp. H-4 can degrade both polyM and polyG blocks with a K(m) in mg/mL 20-times higher for the polyM block.

Metagenomic Analysis of Healthy and White Plague-Affected Mussismilia braziliensis Corals

Coral health is under threat throughout the world due to regional and global stressors. White plague disease (WP) is one of the most important threats affecting the major reef builder of the Abrolhos Bank in Brazil, the endemic coral Mussismilia braziliensis. We performed a metagenomic analysis of healthy and WP-affected M. braziliensis in order to determine the types of microbes associated with this coral species. We also optimized a protocol for DNA extraction from coral tissues. Our taxonomic analysis revealed Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Actinomycetes as the main groups in all healthy and WP-affected corals. Vibrionales, members of the Cytophaga–Flavobacterium–Bacteroides complex, Rickettsiales, and Neisseriales were more abundant in the WP-affected corals. Diseased corals also had more eukaryotic metagenomic sequences identified as Alveolata and Apicomplexa. Our results suggest that WP disease in M. braziliensis is caused by a polymicrobial consortium.

Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization

Aims:  To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae.