Taxonomy of the marine,luminous bacteria

Summary One hundred and seventy-three strains of marine, luminous bacteria isolated from sea water, surfaces and intestines of fish, as well as from the luminous organs of fish and squid were submitted to an extensive phenotypic characterization. A numerical analysis of the results grouped these strains into four clusters which were formed on the basis of overall phenotypic similarity. One cluster, which was given the designationBeneckea harveyi, consisted of strains which had a moles% GC content in their DNAs of 46.5±1.3 and a single, sheathed, polar flagellum when grown in liquid medium. Most of these strains had unsheathed, peritrichous flagella in addition to the sheathed, polar flagellum when grown on solid medium. The two phenotypically similar clusters which were assigned the species designationsPhotobacterium phosphoreum andP. mandapamensis consisted of strains which had 1–3 unsheathed, polar flagella and moles % GC contents in their DNAs of 41.5±0.7 and 42.9±0.5, respectively. The cluster designatedP. fischeri contained strains having 2–8 sheathed, polar flagella and a moles % GC content of 39.8±1.1. These four species could be further distinguished on the basis of a number of nutritional properties as well as other phenotypic traits. The assignment of the luminous, marine bacteria to four species was supported by differences in the properties of the luminous system as well as differences in the pattern of regulation of spartokinase activity which are discussed. The speciesB. harveyi was found to be phenotypically similar to a number of previously characterized, non-luminous strains ofBeneckea which should probably be assigned to this species.Non-Standard Abbreviations ASW artificial sea water - ATCC American Type Culture Collection - BM basal medium - BMA basal medium agar - GC guanine plus cytosine - LA luminous medium agar - LB luminous medium broth - MA Difco Marine Agar - NCMB National Collection of Marine Bacteria - PHB poly--hydroxybutyrate - S similarity coefficient - YEB yeast extract broth This paper is part of a dissertation submitted by the senior author to the Graduate Division of the University of Hawaii in partial fulfillment of the requirements for the Ph.D. Degree in Microbiology     

Phenotypic characterization ofPhotobacterium logei (sp. nov.), a species related toP. fischeri

Ten luminous marine bacteria having the diagnostic traits ofPhotobacterium fischeri but differing from this species in their ability to grow at 4°C have been subjected to a more extensive phenotypic characterization. The results indicated a high phenotypic similarity toP. fischeri; the major additional diagnostic trait was their inability to grow at 30°C. In these properties the isolates resembled a previously characterized nonluminous strain, ATCC 15382, which had a DNA homology of about 40% toP. fischeri. Immunological comparisons of the glutamine synthetases and Fe-superoxide dismutases of these strains as well as additional properties that are discussed indicated that the ten luminous isolates and strain ATCC 15382 constituted a new species, which we have designatedPhotobacterium logei sp. nov. (type strain 584, ATCC 29985).     

Chemical and Biological Properties of Lipopolysaccharides from Symbiotic Luminous Bacteria from Several Luminous Marine Animals

The chemical and biological properties of lipopolysaccharides (LPS) in five strains of symbiotic luminous bacteria isolated from four species of luminous marine fishes, Coelorhynchus kishinouyei (CK-1), Chlorophthalmus albatrossis (CA-1), Ventrifossa garmani (VG-1), and Acropoma japonicum (AJ-1b), as well as from a luminous squid, Doryteuthis kensaki (DK-1) were examined. The LPS isolated from these symbiotic luminous bacteria were characterized by the absence of 2-keto-3-deoxyoctonate, known to be a basic component of the usual gram-negative bacterial LPS. All LPS from these symbiotic luminous bacteria upon electrophoresis in sodium dodecylsulfate polyacrylamide gel exhibited one or two clear main bands with high mobility, and one or two obscure minor bands with low mobility when stained with periodate-Schiff reagent. LPS from CA-1 and VG-1 exhibited similar electrophoretic patterns, whereas the electrophoretic patterns of the LPS from CK-1, AJ-1b, and DK-1 were easily distinguishable from each other. All these LPS also had similarly potent and diverse biological activities in regard to their adjuvanticity, immunosuppression, polyclonal effect, B-cell mitogenicity, and activation of the phagocytic function of macrophages.     

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.     

Habitats and Distribution Patterns of Marine Luminous Bacteria in the Western Baltic Sea

Numbers of luminous bacteria were counted at three stations of the brackish water ecosystem of the western Baltic Sea from July 1985 to July 1986. Additional samples were taken during three cruises from stations at the North Atlantic Ocean, the Norwegian Sea and adjacent marine areas. — In Kiel Bight (western Baltic) values varied between 0 and 68,000 luminous cfu 1⁻¹. With exception of the coastal station a distinct seasonal distribution pattern was shown in a water depth of 20 m: high numbers found in summer were opposed to low numbers in winter, the peaks being rather high in comparison to those of other areas. Statistical analysis showed that the results of 20 m were significantly different from those of 0 and 10 m depth; however, there was no correlation with temperature and salinity. Taxonomic studies revealed that the population consisted primarily of the genus Photobacterium. — The optimum of salinity was not a brackish but a marine one and was about 30% for the majority of the strains tested. A smaller number of strains grew best at a salinity between 10 and 15%. Optima of temperature ranged from 15 to 20 °C for most of the test strains. — Taxonomic analysis was also performed with luminous strains from marine areas adjacent to the western Baltic Sea, Photobacterium being the dominant genus here, too. Luminous bacteria were also enriched from the external surface and the gut contents of whitings (Merlangius merlangus) and cods (Gadus morhua). A model is proposed which explains the distribution pattern found. According to this, the gut-dwelling luminous bacteria are transported by their hosts from the North Sea into the western Baltic Sea. Here they are released into the environment, thus inhabiting another niche.     

Epibiotic <Emphasis Type="Italic">Vibrio</Emphasis> Luminous Bacteria Isolated from Some Hydrozoa and Bryozoa Species

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA–DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.     

The absence of a correlation between plasmids and luminescence in marine luminous bacteria

Members of four species of marine luminous bacteria were examined for the presence of plasmids using gel electrophoresis of purified alkaline extracts. One to four plasmids, with molecular weights ranging from 5 to 120 megadaltons, were found to occur in 43% of the 58 bioluminescent strains examined. There was, thus, no correlation between the presence and absence of plasmids and luminescence, nor was there any single size of plasmid common to the different bacterial species. Spontaneous dark (dim) mutants were selected from five strains ofVibrio (Beneckea) harveyi; in no case was there any difference in the plasmid content between the bright parent strain and the dim isolate.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Cultivation and Characterisation of New Species of Apusomonads (the Sister Group to Opisthokonts), Including Close Relatives of Thecamonas (Chelonemonas n. gen.)

Apusomonads comprise an understudied and undersampled group of heterotrophic flagellates that is closely related to opisthokonts, the supergroup containing animals and fungi. We cultured representatives of a new clade of apusomonads, Chelonemonas n. gen., which is sister to marine forms of Thecamonas in SSU rRNA gene phylogenies. Scanning electron microscopy shows that members of Chelonemonas have a hexagonal patterning to their submembranous pellicle, which is not known to exist in other apusomonads. We propose that the subfamily Thecamonadinae refer to the marine Thecamonas/Chelonomonas clade. We also report two new strains of Multimonas, one of which is genetically divergent from previously described strains, and here described as a new species, Multimonas koreensis. Both strains of Multimonas have appendages on their dorsal surface that could be extrusomes, and a frilled appearance to the border of their pellicle. Explorations of taxon sampling in SSU rRNA gene phylogenies confirm the new strains' evolutionary affinities, but do not resolve relationships among the five main apusomonad clades. These phylogenies also separate the freshwater species “Thecamonas” oxoniensis from the marine members of the genus Thecamonas. The new strains described here may provide valuable genetic and morphological data for evaluating the relationships and evolution of apusomonads.     

Autoinduction of bacterial bioluminescence in a carbon limited chemostat

Several strains of four species of luminous marine bacteria were maintained in a chemostat at a constant dilution rate and a variety of steady state densities by carbon (glycerol) limitation in order to study the relationship between culture density and bioluminescence activity. In general, luminescence per cell was constant at high culture density, and decreased dramatically at low culture density. For Vibrio fischeri, luminescence decreased to nondectable levels when the culture was maintained at low density; such dark cells were stimulated to synthesize luciferase and became luminous within minutes when purified autoinducer was added to the chemostat. Two strains, Photobacterium phosphoreum NZ11D and Photobacterium leiognathi S1, did not show the decrease in light intensity at low culture density that was characteristic of all other strains tested; they appeared to be constitutive for bioluminescence.Abbreviations BCM basal salts glycerol medium - BM basal salts medium - BSA bovine serum albumin - D dilution rate - DTT dilitiothreitol - LU light unit=2×10¹⁰ quanta s^-1 - OD optical density - SWC sea water complete medium - specific growth rate     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium     

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates. Received: February 5, 1999 / Accepted: May 11, 1999     

Analysis of RNA Polymerase Beta Subunit (<Emphasis Type="Italic">rpoB</Emphasis>) Gene Sequences for the Discriminative Power of Marine <Emphasis Type="Italic">Vibrio</Emphasis> Species

In the present study, we sequenced the RNA polymerase beta subunit (rpoB) gene of marine Vibrio species and assessed its discriminative power in identifying vibrios. Both the rpoB and 16S rRNA sequences of 29 phenotypically different Vibrio strains isolated from coastal waters were determined. Molecular and phylogenetic comparisons of the sequences of these two genes classified the 29 strains into 11 different species. The resolution of the Vibrio spp. on the rpoB phylogenetic tree was approximately three times greater than that on the 16S rRNA phylogenetic tree. Moreover, by comparing the rpoB sequences of 98 marine γ-Proteobacteria, including 38 marine Vibrio species, Vibrio-specific primers were developed to amplify a 730-bp fragment of the rpoB gene. Using these primers, we successfully detected Vibrio signals in environmental samples and determined their relative abundances via comparisons with known standards. This rpoB-targeting polymerase chain reaction assay can be used efficiently to monitor relative Vibrio abundance in marine waters.     

Cloning, organization, and expression of the bioluminescence genes of Xenorhabdus luminescens.

The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli. The expression of these genes in E. coli was qualitatively similar to their expression in X. luminescens. The organization of the genes is similar to that found in the marine luminous bacteria. Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X. luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species. Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms.     

Cross-species induction of antibacterial activity produced by epibiotic bacteria isolated from Indian marine sponge Pseudoceratina purpurea

Four antibiotic producing bacteria were isolated from the surface of the marine sponge Pseudoceratina purpurea and exposed to living cells of two human pathogenic bacteria as well as some marine fouling bacteria to induce the production of antimicrobial activity. Experimental results showed that these four marine epibiotic bacteria enhanced their antibacterial production, when exposed to these test strains. The highest induction was exhibited by the sponge isolate PS79 against fouling bacterium FB-9 (from 3 mm to 7 mm inhibition zone). All the four strains were induced and showed increased activity specifically against the challenged pathogenic or fouling bacteria tested. Specific induction by these species suggests that the induction might be attributed to the response to the chemical signals received from potential challenger strains.     

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture‐dependent and ‐independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR‐43 exhibited cytotoxic activity against cancer cell lines. JBIR‐34 and ‐35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.     

Methylosulfonomonas methylovora gen. nov., sp. nov., and Marinosulfonomonas methylotropha gen. nov., sp. nov.: novel methylotrophs able to grow on methanesulfonic acid

Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the α-Proteobacteria. Methylosulfonomonas is a discrete lineage within the α-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the α-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera. Received: 19 July 1996 / Accepted: 7 October 1996     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

Luminous bacteria synthesize luciferase anaerobically

Four species of luminous bacteria, Photobacterium phosphoreum, P. leiognathi, P. fischeri and Beneckea harveyi (two strains of each), were shown to synthesize luciferase anaerobically. One of these, P. phosphoreum, produced as much luciferase anaerobically as it did aerobically, and all four species were found to grow almost equally rapidly under the two sets of conditions. Previous work with B. harveyi and P. fischeri had shown that aerobic luciferase synthesis can proceed only after an inhibitor in the complex medium has been removed and a species-specific autoinducer secreted. All strains tested also removed the inhibitor and secreted an autoinducer anaerobically. The small amount of luciferase produced anaerobically by some strains is thus apparently not due either to lack of removal of inhibitor or to insufficient production of autoinducer but may involve an oxygen-dependent control mechanism.Abbreviations LU light units - OD optical density