The classification of luminescent bacteria

Summary The luminescent bacteria are logically placed in two genera. The common coccoid and frequently non-motile species placed byBeije-rinck first in his genusPhotobacterium, 1889 under the namePhotobacterium phosphorescens syn.Bacterium phosphorescens Fischer, should be recognized as the type species ofPhotobacterium. Other characters indicate that this genus should be placed in the FamilyPseudomonadaceae Winslowet al.. and should include other straight, rod-shaped, luminescent, polar flagellate bacteria that ferment glucose without, however, necessarily producing gas (H₂ and CO₂) as does the type species. The species that have the form of vibrios should be accepted as members of the genusVibrio as suggested by several previous investigators. They have characters much like those ofVibrio comma, the type species of the genusVibrio.     

Systematic relationships within the Vibrionaceae (Bacteria: Gammaproteobacteria): steps toward a phylogenetic taxonomy

A phylogenetic hypothesis is presented for all 95 species of the family Vibrionaceae (Bacteria: Gammaproteobacteria) based on a combined analysis of eight molecular loci (16S rRNA, gyrB, recA, rpoA, gapA, mreB, topA, atpA) for up to 9337 nucleotide characters. Members of this taxon exhibit diverse life histories, including bioluminescence, pathogenicity to human and marine organisms, symbiosis, quorum sensing and extremophilic environment living, making a hypothesis of phylogenetic history important to studies addressing these traits from an evolutionary perspective. It is proposed that this phylogenetic set of relationships replaces previous phenetic hypotheses and be used to construct a phylogenetic taxonomy. Recent taxonomic proposals, including the validity of four, instead of one, families representing the 95 species and historical notions of genera within the group are compared with the presented phylogenetic hypothesis. Character support is traced through the tree and is used to address these taxonomic proposals. Photobacterium is not a monophyletic group as it is currently delimited. Aliivibrio is found within Photobacterium, suggesting a new definition for Photobacterium that includes all species of Aliivibrio. Enterovibrio, Salinivibrio and Grimontia, previously thought to be distinct from and basal to Photobacterium and Vibrio, are found nested deeply within a large Vibrio clade. © The Willi Hennig Society 2010.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

Organization of the lux genes of photobacterium phosphoreum

The lux genes from Photobacterium phosphoreum (NCMB844) have been cloned into Escherichia coli in a plasmid containing the T7-bacteriophage promoter. By specific expression in vivo under the T7 promoter, five structural genes (luxA-E) coding for the fatty acid reductase and luciferase polypeptides were identified as well as a new gene, designated as luxF, which codes for a 26kDa polypeptide. This new gene is located between luxB and luxE and thus disrupts the structural gene order of luxCDABE found in the Vibrio genus. The luxF gene and the protein it codes for have recently been identified in other Photobacterium species and so appears to be widely distributed within this genus. Nucleotide sequencing of the luxF gene has shown it to code for a protein homologous to the luciferase subunits, coded by the luxA and luxB genes. Although this gene is not necessary for light emission in all luminescent bacteria, it must play an essential role in the biochemistry, physiology, or ecology of the luminescent system in species of the Photobacterium genus.     

Phylogenetic identification of epibiotic bacteria possessing antimicrobial activities isolated from red algal species of Japan

We investigated the diversity of epibiotic bacteria possessing antimicrobial activity isolated from nine species of red algae, and identified their phylogenetic position. For the isolation of epibiotic bacteria, nine species of red algae, Pachymeniopsis lauceolata, Plocamium telfairiae, Gelidium amansii, Chondrus oncellatus, Grateloupia filicina, Ceramium kondoi, Lomentaria catenata, Schizymenia dubyi and Porphyra yezoensis, were collected from the intertidal zone of Awaji Island, Japan. In total 92 bacteria were collected from the above red algal species. Primary screening results using disc diffusion assay revealed that 33% of bacteria possess antibacterial activity. Ten bacteria that showed high antibacterial activity were further studied for their ability to inhibit a set of fouling bacteria, some luminescent Vibrio and Photobacterium species and a panel of pathogenic bacteria. In general, the inhibitory activities were high against fouling and luminescent bacteria, while low against various pathogenic bacteria tested. These results suggest that some epibiotic bacteria have adapted to defend their position in their surface environment through the production of antibacterial metabolites giving defense against a broad spectrum of bacterial competitors. The phylogenetic analysis using 16 S rRNA sequences identified 7 of the 10 strains as belonging to the genus Bacillus, and other strains each 1 belonging to genus Microbacterium, Psychrobacter, and Vibrio species.     

Composition en bases de l'ADN et position taxonomique de bactéries marines fermentant le glucose,du genre Vibrio et des genres voisins

Résumé L'analyse du GC% des souches marines appartenant au genre Vibrio confirme leur classement préalable, obtenu selon la méthode adansonnienne. La comparaison, tant par la valeur de leur GC% que par leur similitude phénotypique, des genres Photobacterium et Beneckea avec le genre Vibrio montre que la différenciation de ces trois genres n'est pas très nette.Le genre Vibrio se subdivise en quatre groupes de souches. Un premier groupe comprend les souches marines libres. Le second groupe rassemble les souches marines parasites des animaux poïkilothermes. Le troisième groupe est composé de souches, capables de parasiter les animaux poïkilothermes comme les animaux homéothermes. Enfin, le dernier groupe est constitué, contrairement aux trois premiers, par des souches ne nécessitant pas de NaCl pour croître.On constate que ces quatre groupes ont une localisation écologique différente.

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Deoxyribonucleic acid base composition and taxonomic relationships among marine usual characters and related bacteria

Summary DNA base composition of marine strains of genus Vibrio corroborate the results obtained by the adansonian method of classification.Comparisons of phenotypic and GC% values of genera Photobacterium and Beneckea with the genus Vibrio show that differentiation between these three genera is not clear.Genus Vibrio can be separated in four groups. The first contains free-living marine strains. The second group includes the marine strains pathogenic to poïkilothermic animals. The third group is composed of strains pathogenic to poïkilothermic and homeothermic animals. The last group is made up of strains which, contrary to the other groups, have shown no specific requirement of sodium for growth. We ascertain that each group has a typical ecologic localization.

     

Low oxygen is optimal for luciferase synthesis in some bacteria

The synthesis of the bioluminescent systems in many strains of two species of the genus Photobacterium which were isolated as symbionts is greater at low oxygen concentrations, where aerobic growth is blocked. In strains of two other species, one Photobacterium of symbiotic orgin, and one (genus Beneckea) whose luminous members are not known to be involved in symbiotic associations, a different response is observed. At low oxygen concentrations, where there is an inhibition of growth, there is also a similar decrease in the synthesis, of the luminescent system. These species-specific differences may indicate important ecological differences along with distinctive differences in the molecular control mechanisms involved in the synthesis of luciferase.     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

Reevaluation of the taxonomy ofVibrio,beneckea, andPhotobacterium: Abolition of the genusBeneckea

As a result of studies on the evolution of glutamine synthetase and superoxide dismutase, the genusBeneckea has been abolished and its constituent species, along withPhotobacterium fischeri andP. logei, assigned to the genusVibrio. The definitions ofVibrio andPhotobacterium have been modified accordingly.     

Characterization of the Bacterial Diversity in Indo-West Pacific Loliginid and Sepiolid Squid Light Organs

Loliginid and sepiolid squid light organs are known to host a variety of bacterial species from the family Vibrionaceae, yet little is known about the species diversity and characteristics among different host squids. Here we present a broad-ranging molecular and physiological analysis of the bacteria colonizing light organs in loliginid and sepiolid squids from various field locations of the Indo-West Pacific (Australia and Thailand). Our PCR-RFLP analysis, physiological characterization, carbon utilization profiling, and electron microscopy data indicate that loliginid squid in the Indo-West Pacific carry a consortium of bacterial species from the families Vibrionaceae and Photobacteriaceae. This research also confirms our previous report of the presence of Vibrio harveyi as a member of the bacterial population colonizing light organs in loliginid squid. pyrH sequence data were used to confirm isolate identity, and indicates that Vibrio and Photobacterium comprise most of the light organ colonizers of squids from Australia, confirming previous reports for Australian loliginid and sepiolid squids. In addition, combined phylogenetic analysis of PCR-RFLP and 16S rDNA data from Australian and Thai isolates associated both Photobacterium and Vibrio clades with both loliginid and sepiolid strains, providing support that geographical origin does not correlate with their relatedness. These results indicate that both loliginid and sepiolid squids demonstrate symbiont specificity (Vibrionaceae), but their distribution is more likely due to environmental factors that are present during the infection process. This study adds significantly to the growing evidence for complex and dynamic associations in nature and highlights the importance of exploring symbiotic relationships in which non-virulent strains of pathogenic Vibrio species could establish associations with marine invertebrates.     

中国北部湾光裸方格星虫可培养微生物的分离鉴定及其活性代谢物产生菌筛选

【目的】本研究从北部湾海域光裸方格星虫(Sipunculus nudus)肠道中分离鉴定可培养微生物,并对筛选菌株的代谢物活性进行研究,为后续开发和利用光裸方格星虫肠道微生物代谢产物提供理论支持。【方法】通过微生物培养、菌株分离纯化和16S rRNA基因序列分析,分析鉴定湛江、北海、防城港三地光裸方格星虫肠道可培养微生物;采用透明圈法、可见分光光度法、平板打孔法等对产胞外活性代谢物的菌株进行筛选和活性分析。【结果】中国北部湾不同海域光裸方格星虫肠道可培养微生物包括弧菌属(Vibrio)、希瓦氏菌属(Shewanella)、假交替单胞菌属(Pseudoalteromonas)、发光杆菌属(Photobacterium)和芽孢杆菌属(Bacillus)等12个细菌属。弧菌属(Vibrio)是3个地区样本共有的优势菌群。具有产胞外水解蛋白酶、壳聚糖酶、多糖以及抑菌活性等能力的菌株主要来自假交替单胞菌属(Pseudoalteromonas)、发光杆菌属(Photobacterium)和芽孢杆菌属(Bacillus)。【结论】中国北部湾不同海域光裸方格星虫肠道可培养微生物在属的种类上存在显著性差异,且光裸方格星虫肠道菌株具有产生多种胞外活性代谢物的能力,是一种良好的海洋活性代谢物来源。     

Electrophoretic mobilities of superoxide dismutases from species ofPhotobacterium,beneckea, vibrio,and selected terrestrial enterobacteria

Cell-free extracts of 93 strains from 23 species and biotypes ofPhotobacterium, Beneckea, andVibrio were electrophoresed on polyacrylamide gels and then stained for superoxide dismutase (SOD) activity. All strains exhibited a single activity band with the exception ofPhotobacterium leiognathi, which had one major and two minor bands. Strains representative of all three genera were found to have SOD activities that were sensitive to treatment with H₂O₂, suggesting that they were iron-containing enzymes. Examination of the effect of gel concentration on relative mobility (Rm) suggested that all the iron-containing SODs had very similar molecular weight. Most species could be distinguished on the basis of differences in the Rm values of their SODs.Vibrio was readily separable fromBeneckea andPhotobacterium. A limited electrophoretic analysis of the SODs fromAeromonas, Serratia, Proteus, Erwinia, and other species of terrestrial enterobacteria indicated groupings that were in agreement with previous studies.     

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes

Aims: To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation. Methods and Results: Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 × 10⁴ CFU ml^?1 (c. 200 CFU per PCR) to 2 × 10³ CFU ml^?1 (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species. Conclusions: The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus. Significance and Impact of the Study: This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.     

Characterization of nitrogen-fixing bacteria from a temperate saltmarsh lagoon,including isolates that produce ethane from acetylene

Nitrogen-fixing bacteria were isolated from sediments and water of a saltmarsh lagoon on the west coast of South Africa, and characterized according to factors that regulate nitrogen fixation in the marine environment. The majority of isolates were assigned to the Photobacterium or Vibrio genera on the basis of physiological and biochemical characteristics. One isolate was further assigned to the species Vibrio diazotrophicus. Carbohydrate utilization by each diazotrophic isolate was examined. Abilities of the isolates to utilize a range of mono-, di-, and polysaccharides largely reflected the predicted availability of organic carbon and energy in the lagoon, except that chitin was not utilized. Biochemical tests on the utilization of combined nitrogen showed that one isolate could utilize nitrate, and that this strain was susceptible to full repression of nitrogenase activity by 10mm nitrate. Urease activity was not detected in any of the isolates. In the absence of molybdenum two of the isolates, a Photobacterium spp. and V. diazotrophicus, reduced acetylene to ethylene and ethane, a property frequently associated with the activity of alternative nitrogenases. Addition of 25µM molybdenum inhibited ethane production by V. diazotrophicus, but stimulated ethylene and ethane production by the Photobacterium isolate. Addition of 28µM vanadium did not appear to regulate ethane production by either strain. Assays of nitrogenase activity in sediments from which some isolates were obtained indicated that molybdenum was not limiting nitrogenase activity at naturally-occurring concentrations. Southern hybridizations of the chromosomes of these strains with the anfH and vnfH genes of Azotobacter vinelandii and the nifH gene of Klebsiella pneumoniae indicated the presence of only one nitrogenase in these isolates.Correspondence to: B.J. Tibbles.     

Larval settlement of the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the surface of coralline algae

Bacterial biofilms are increasingly seen as important for the successful settlement of marine invertebrate larvae. Here we tested the effects of biofilms on settlement of the sea urchin Heliocidaris erythrogramma. Larvae settled on many surfaces including various algal species, rocks, sand and shells. Settlement was reduced by autoclaving rocks and algae, and by treatment of algae with antibiotics. These results, and molecular and culture-based analyses, suggested that the bacterial community on plants was important for settlement. To test this, approximately 250 strains of bacteria were isolated from coralline algae, and larvae were exposed to single-strain biofilms. Many induced rates of settlement comparable to coralline algae. The genus Pseudoalteromonas dominated these highly inductive strains, with representatives from Vibrio, Shewanella, Photobacterium and Pseudomonas also responsible for a high settlement response. The settlement response to different bacteria was species specific, as low inducers were also dominated by species in the genera Pseudoalteromonas and Vibrio. We also, for the first time, assessed settlement of larvae in response to characterised, monospecific biofilms in the field. Larvae metamorphosed in higher numbers on an inducing biofilm, Pseudoalteromonas luteoviolacea, than on either a low-inducing biofilm, Pseudoalteromonas rubra, or an unfilmed control. We conclude that the bacterial community on the surface of coralline algae is important as a settlement cue for H. erythrogramma larvae. This study is also an example of the emerging integration of molecular microbiology and more traditional marine eukaryote ecology.     

Characterization of marine luminous bacteria isolated off the Coast of China and description ofVibrio orientalis sp. nov.

Luminous strains of marine bacteria, isolated off the Coast of China, were subjected to a phenotypic characterization, which included a test of their ability to utilize 82 organic compounds as sole or principal sources of carbon and energy. A numerical analysis of the data revealed five clusters which were readily identified asPhotobacterium phosphoreum, P. leiognathi, Vibrio harveyi, andV. splendidus biotype I. The remaining cluster of luminous isolates was phenotypically distinct from all the previously described species ofVibrio andPhotobacterium and was given the species designation,Vibrio orientalis. This species differed from all the other luminous species ofVibrio by its ability to accumulate poly-β-hydroxybutyrate as an intracellular reserve product. Additional distinctive properties were the presence of an arginine dihydrolase system, growth at 4° but not 40°C, and the ability to utilize putrescine and spermine.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Rapid Phenotypic Characterization of <Emphasis Type="Italic">Vibrio</Emphasis> Isolates by Pyrolysis Metastable Atom Bombardment Mass Spectrometry

Pyrolysis mass spectrometry was investigated for rapid characterization of food-borne bacterial pathogens. Nine isolates of Vibrio parahaemolyticus and one isolate each of Vibrio fluvialis, Vibrio hollisae, and Vibrio vulnificus were analyzed. Pyrolysis mass spectra, generated via an alternative ionization method, metastable atom bombardment, were subject to principal component-discriminant analysis. The spectral patterns were used to distinguish Vibrio isolates differing in species, serotype and expression of the thermostable direct hemolysin gene. The patterns of similarity and dissimilarity amongst spectra in the Vibrio test set generally reflected those associated with species, serotype or hemolysin-producing genes, though the combined influence of these and other variables in the multi-dimensional data did not produce a simple clustering with respect to any one of these characteristics. These results suggested that with enough examples to model the most common combinations, the method should be able to characterize Vibrio isolates according to their phenotypic characteristics. Pyrolysis-mass spectrometry with metastable atom bombardment and pattern recognition appeared suitable for rapid infraspecific comparison of Vibrio isolates. This integrated analytical, pattern-recognition system should be examined further for potential utility in clinical and public health diagnostic contexts.     

Antibacterial activities of marine epibiotic bacteria isolated from brown algae of Japan

One hundred and sixteen epibiotic bacteria were isolated from the surface of nine species of brown algaeSargassum serratifolium, Sargassum fusiforme, Sargassum filicinum, Padina arborescens, Undaria pinnatifida, Petalonia fascia, Colpomenia sinuosa, Scytosiphon lomentaria andEcklonia cava which were collected at Awaji Island, Japan. Primary screening results using disc-diffusion assay revealed that, among the bacteria isolated 20% of epibiotic bacteria exhibited antibacterial activity. Among them, 10 bacteria which showed high antibacterial activity were further studied for their ability against (i) a set of fouling bacteria isolated from marine natural biofilm, (ii) some luminescentVibrio andPhotobacterium species and (iii) a panel of pathogenic bacteria. In general, inhibitory activities were high or moderate against fouling bacteria,Vibrio andPhotobacterium species, while they were found to be low against pathogenic bacteria tested. The phylogenetic analysis using 16S rRNA sequencing revealed that all of the bacteria with high antibacterial activity showed a close affiliation with genusBacillus. This result suggested that the genusBacillus is efficient producers of of antibacterial compounds and these epibiotic bacteria isolated are highly successful colonizers on macroalgal surfaces.     

Identification of environmental plasmid-bearing Vibrio species isolated from polluted and pristine marine reserves of Hong Kong, and resistance to antibiotics and mercury

Fifty environmental isolates of Vibrio species were isolated from water samples of Mai Po Nature Reserve and the Cape d’Aguilar Marine Reserve in Hong Kong and screened for the presence of plasmid. Mai Po is a wastewater-impacted area while the Cape d’Aguilar Marine Reserve is pristine natural marine water. Plasmid was found in Vibrio isolates from both sites at similar frequencies and each site showed distinctive plasmid profiles. These plasmid-bearing Vibrio isolates were identified as different species of the Vibrio genus by both biochemical test and subsequently full-length 16S rRNA sequences. Antibiotic resistance test showed that all these plasmid-bearing Vibrio isolates showed multiple resistance to 21 antibiotics tested. In addition, selective isolates also showed tolerance to 10 M Hg²⁺ in culture medium and they generally harbored large plasmid(s) (>‰30 kb). Our results show that the high frequency of plasmid in Vibrio species of both polluted and pristine environments may be ecologically important to the survival of these bacteria in the environment. The specific functioning of the cryptic plasmids remains the focus of current investigations.