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.     

Long-term preservation of active luminous bacteria by lyophilization

A simple method for long-term preservation of luminous bacteria is described. Cells of Vibrio fischeri, Photobacterium leiognathi and four strains of P. phosphoreum were suspended in a protective medium of low ionic strength (1% NaCI) supplemented with 15% lactose and 2% soluble starch, and lyophilized. The freeze-dried preparations were sealed under vacuum and stored at 4°C. Luminous bacteria were resuscitated affer six months by adding 2% NaCl up to the original volume. The rehydrated cells exhibited 16-28% of initial bioluminescence so that they could be used for a microbial test of toxicity (the Microtox test). This method is also useful for maintaining luminous bacteria in strain collections.     

Characterization ofAlteromonas hanedai (sp. nov.), a nonfermentative luminous species of marine origin

Eleven marine luminous isolates, which could not be identified with previously studied species of luminous marine bacteria, were subjected to an extensive characterization. The results indicated that these strains were phenotypically similar, had a G+C content in their DNA of 45 mol%, and differed from all previously characterized luminous species by their inability to ferment sugars. On the basis of these and other properties, the 11 luminous strains were assigned to the genusAlteromonas and given the species designationA hanedai. Strain 281 (ATCC 33224) has been designated as the type strain of this new species.     

Distribution and Identification of Luminous Bacteria from the Sargasso Sea

Vibrio fischeri and Lucibacterium harveyi constituted 75 of the 83 luminous bacteria isolated from Sargasso Sea surface waters. Photobacterium leiognathi and Photobacterium phosphoreum constituted the remainder of the isolates. Luminescent bacteria were recovered at concentrations of 1 to 63 cells per 100 ml from water samples collected at depths of 160 to 320 m. Two water samples collected at the thermocline yielded larger numbers of viable, aerobic heterotrophic and luminous bacteria. Luminescent bacteria were not recovered from surface microlayer samples. The species distribution of the luminous bacteria reflected previously recognized growth patterns; i.e., L. harveyi and V. fischeri were predominant in the upper, warm waters (only one isolate of P. phosphoreum was obtained from surface tropical waters).     

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     

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     

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     

Identification of Vibrio splendidus as a Member of the Planktonic Luminous Bacteria from the Persian Gulf and Kuwait Region with luxA Probes

Hybridization probes specific for the luxA genes of four groups of luminous bacteria were used to screen luminous isolates obtained from the Persian Gulf, near Al Khiran, Kuwait Nine of these isolates were identified as Vibrio harveyi, a commonly encountered planktonic isolate, while three others showed no hybridization to any of the four probes (V. harveyi, Vibrio fischeri, Photobacterium phosphoreum, or Photobacterium leiognathi) under high-stringency conditions. Polymerase chain reaction amplification was used to prepare a luxA probe against one of these isolates, K-1, and this probe was screened under high-stringency conditions against a collection of DNAs from luminous bacteria; it was found to hybridize specifically to the DNA of the species Vibrio splendidus. A probe prepared against the type strain of V. splendidus (ATCC 33369) was tested against the collection of luminous bacterial DNA preparations and against the Kuwait isolates and was found to hybridize only against the type strain and the three unidentified Kuwait isolates. Extensive taxonomic analysis by standard methods confirmed the identification of the 13 isolates.     

Genomic and phylogenetic characterization of luminous bacteria symbiotic with the deep-sea fish Chlorophthalmus albatrossis (Aulopiformes: Chlorophthalmidae)

Bacteria forming light-organ symbiosis with deep-sea chlorophthalmid fishes (Aulopiformes: Chlorophthalmidae) are considered to belong to the species Photobacterium phosphoreum. The identification of these bacteria as P. phosphoreum, however, was based exclusively on phenotypic traits, which may not discriminate between phenetically similar but evolutionarily distinct luminous bacteria. Therefore, to test the species identification of chlorophthalmid symbionts, we carried out a genomotypic (repetitive element palindromic PCR genomic profiling) and phylogenetic analysis on strains isolated from the perirectal light organ of Chlorophthalmus albatrossis. Sequence analysis of the 16S rRNA gene of 10 strains from 5 fish specimens placed these bacteria in a cluster related to but phylogenetically distinct from the type strain of P. phosphoreum, ATCC 11040(T), and the type strain of Photobacterium iliopiscarium, ATCC 51760(T). Analysis of gyrB resolved the C. albatrossis strains as a strongly supported clade distinct from P. phosphoreum and P. iliopiscarium. Genomic profiling of 109 strains from the 5 C. albatrossis specimens revealed a high level of similarity among strains but allowed identification of genomotypically different types from each fish. Representatives of each type were then analyzed phylogenetically, using sequence of the luxABFE genes. As with gyrB, analysis of luxABFE resolved the C. albatrossis strains as a robustly supported clade distinct from P. phosphoreum. Furthermore, other strains of luminous bacteria reported as P. phosphoreum, i.e., NCIMB 844, from the skin of Merluccius capensis (Merlucciidae), NZ-11D, from the light organ of Nezumia aequalis (Macrouridae), and pjapo.1.1, from the light organ of Physiculus japonicus (Moridae), grouped phylogenetically by gyrB and luxABFE with the C. albatrossis strains, not with ATCC 11040(T). These results demonstrate that luminous bacteria symbiotic with C. albatrossis, together with certain other strains of luminous bacteria, form a clade, designated the kishitanii clade, that is related to but evolutionarily distinct from P. phosphoreum. Members of the kishitanii clade may constitute the major or sole bioluminescent symbiont of several families of deep-sea luminous fishes.     

Growth and luminescence of luminous bacteria promoted by agents of microbial origin

The examination of four species of luminous bacteria Photobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri and Vibrio harveyi has enabled us to reveal some nutrient medium components effecting growth, luminescence intensity and luciferase synthesis. These agents are nucleic components (nucleotides, nucleosides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen-oxidizing, ironoxidizing and carboxydobacteria. The effect of promoting agents essentially alters the physiological state and ultrastructure of the cells of luminous bacteria and increases luciferase biosynthesis two- to three-fold compared to a control.     

Alicyclobacilli from an unexplored geothermal soil in Antarctica: Mount Rittmann

The isolation of three species from soil samples from an unexplored site in Antarctica, belonging to the Alicyclobacillus genus, is reported. The isolates, named MR1, MR2 and MR4, were from 30 samples of geothermal soil from Mount Rittmann. All the isolates had lipids based mainly on fatty acids possessing a terminal cyclohexane; hopanoid and quinones of MK-7 were also present. All isolates were characterized by phenotypic features and the isolate MR1 was subjected to genetic analyses. The isolates were thermoacidophilic, showing an optimum temperature of 63°C and an optimum pH of 3.5–4.0. They were able to grow in the temperature range 45–70°C. The percentage C+G of DNA of MR1 was 64.9% and showed a 16S rDNA similarity of 99.3% with Alicyclobacillus acidocaldarius ATCC 43034 and a DNA-DNA homology of 69.7% with A. acidocaldarius DSM 446. The strain MR1 is unable to produce amylolytic activity under different growth conditions. It is proposed to accommodate MR1 in the genus Alicyclobacillus as subspecies rittmannii of A. acidocaldarius. Received: 29 May 1997 / Accepted: 26 September 1997     

Analogs of the autoinducer of bioluminescence inVibrio fischer

The enzymes for luminescence inVibrio fischeri are induced only when a sufficient concentration of a metabolic product (autoinducer) specifically produced by this species accumulates. It has previously been shown that the autoinducer is 3-oxohexanoyl homoserine lactone and that it enters the cells by simple diffusion. To further study the mechanism of induction, we have synthesized several analogs of the autoinducer. The analogs were tested withV. fischeri for their inducing activity and for their ability to inhibit the action of the natural autoinducer. The compounds were found to display various combinations of inducing and inhibiting abilities. None of the compounds tested appeared to have any effect on cells ofV. harveyi strain MAV orPhotobacterium leiognathi strain 721, but several of the compounds decreased light output byP. phosphoreum strain 8265. These studies show that 1) the site of action of the autoinducer is not highly sterically constrained 2) the autoinducers of other species of luminous bacteria are likely to be quite different from that ofV. fischeri and 3) a simple mode in which one autoinducer molecule binds to a single receptor protein site and thus initiates luciferase synthesis is inadequate. The analogs should prove useful in the study of the binding site and mode of action of the autoinducer.Abbreviations SWC sea water complete     

Genomic and Phylogenetic Characterization of Luminous Bacteria Symbiotic with the Deep-Sea Fish Chlorophthalmus albatrossis (Aulopiformes: Chlorophthalmidae)

Bacteria forming light-organ symbiosis with deep-sea chlorophthalmid fishes (Aulopiformes: Chlorophthalmidae) are considered to belong to the species Photobacterium phosphoreum. The identification of these bacteria as P. phosphoreum, however, was based exclusively on phenotypic traits, which may not discriminate between phenetically similar but evolutionarily distinct luminous bacteria. Therefore, to test the species identification of chlorophthalmid symbionts, we carried out a genomotypic (repetitive element palindromic PCR genomic profiling) and phylogenetic analysis on strains isolated from the perirectal light organ of Chlorophthalmus albatrossis. Sequence analysis of the 16S rRNA gene of 10 strains from 5 fish specimens placed these bacteria in a cluster related to but phylogenetically distinct from the type strain of P. phosphoreum, ATCC 11040^T, and the type strain of Photobacterium iliopiscarium, ATCC 51760^T. Analysis of gyrB resolved the C. albatrossis strains as a strongly supported clade distinct from P. phosphoreum and P. iliopiscarium. Genomic profiling of 109 strains from the 5 C. albatrossis specimens revealed a high level of similarity among strains but allowed identification of genomotypically different types from each fish. Representatives of each type were then analyzed phylogenetically, using sequence of the luxABFE genes. As with gyrB, analysis of luxABFE resolved the C. albatrossis strains as a robustly supported clade distinct from P. phosphoreum. Furthermore, other strains of luminous bacteria reported as P. phosphoreum, i.e., NCIMB 844, from the skin of Merluccius capensis (Merlucciidae), NZ-11D, from the light organ of Nezumia aequalis (Macrouridae), and pjapo.1.1, from the light organ of Physiculus japonicus (Moridae), grouped phylogenetically by gyrB and luxABFE with the C. albatrossis strains, not with ATCC 11040^T. These results demonstrate that luminous bacteria symbiotic with C. albatrossis, together with certain other strains of luminous bacteria, form a clade, designated the kishitanii clade, that is related to but evolutionarily distinct from P. phosphoreum. Members of the kishitanii clade may constitute the major or sole bioluminescent symbiont of several families of deep-sea luminous fishes.     

Planktonic Luminous Bacteria of Vellar Estuary,South India

Distribution of planktonic luminous bacteria in relation to environmental parameters was investigated at two stations located in the Vellar Estuary. Luminous microflora showed a pronounced seasonal cycle with very low counts during monsoon months followed by an increase in post monsoon and peak counts during summer. The population density of these procaryotes was remarkably high ranging from 3.5 to 33.1 colony forming units per ml. They constituted 2.1 to 52.1 % of the total bacterial counts. Salinity appeared to govern the distribution of luminous procaryotes as their counts corresponded well with fluctuations in salinity. Taxonomic affiliation of the isolates revealed predominance of Vibrio harveyi. Vibrio fischeri and Photobacterium leiognathi exhibited sparse distribution.     

Photobacterium profundum sp. nov., a new, moderately barophilic bacterial species isolated from a deep-sea sediment

A novel, moderately barophilic bacterium was isolated from a sediment sample obtained from the Ryukyu Trench, at a depth of 5110 m. The isolate, designated strain DSJ4, is a Gram-negative rod capable of growth between 4°C and 18°C under atmospheric pressure, with optimum growth displayed at 10°C, and capable of growth at pressures between 0.1 MPa and 70 MPa at 10°C, with optimum growth displayed at 10 MPa. Strain DSJ4 is a moderately barophilic bacterium, and shows no significant change in growth at pressures up to 50 MPa. Phylogenetic analysis of the 16S rRNA sequence of strain DSJ4 places this strain within the Photobacterium subgroup of the family Vibrionaceae, closely related to the strain SS9 that was independently isolated from the Sulu Trough. The temperature and pressure ranges for growth, cellular fatty acid composition, and assorted physiological and biochemical characteristics indicate that these strains differ from other Photobacterium species. Furthermore, both SS9 and DSJ4 displayed a low level of DNA similarity to other Photobacterium type strains. Based on these differences, these strains are proposed to represent a new deep-sea-type species. The name Photobacterium profundum (JCM10084) is proposed. Received June 13, 1997 / Accepted: August 9, 1997     

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     

Reversible inhibition of bacterial bioluminescence by long-chain fatty acids

Long-chain unsaturated fatty acids, as well as certain saturated fatty acids such as lauric acid, are inhibitors of the in vivo luminescence of wild-type strains of four species of luminous bacteria (Beneckea harveyi, Photobacterium phosphoerum, P. fischeri, andP. leiognathi) as well as the myristic acid-stimulated luminescence in the aldehyde dim mutant M17 ofB. harveyi. Based on studies with the system in vivo, the principal site of action of all the fatty acids appears to be the reductase activity that converts myristic acid to myristyl aldehyde. This was confirmed by in vitro studies: Reductase activity in crude cell-free extracts is strongly inhibited by oleic acid.     

Genome sequence of Photobacterium mandapamensis strain svers.1.1, the bioluminescent symbiont of the cardinal fish Siphamia versicolor

Photobacterium mandapamensis is one of three luminous Photobacterium species able to form species-specific bioluminescent symbioses with marine fishes. Here, we present the draft genome sequence of P. mandapamensis strain svers.1.1, the bioluminescent symbiont of the cardinal fish Siphamia versicolor, the first genome of a symbiotic, luminous Photobacterium species to be sequenced. Analysis of the sequence provides insight into differences between P. mandapamensis and other luminous and symbiotic bacteria in genes involved in quorum-sensing regulation of light production and establishment of symbiosis.     

Thermal ecotypes of amphi-Atlantic algae. I. Algae of Arctic to cold-temperate distribution (Chaetomorpha melagonium,Devaleraea ramentacea andPhycodrys rubens)

Three species of Arctic to cold-temperate amphi-Atlantic algae, all occurring also in the North Pacific, were tested for growth and/or survival at temperatures of −20 to 30°C. When isolates from both western and eastern Atlantic shores were tested side-by-side, it was found that thermal ecotypes may occur in such Arctic algae.Chaetomorpha melagonium was the most eurythermal of the 3 species. Isolates of this alga were alike in temperature tolerance and growth rate but Icelandic plants were more sensitive to the lethal temperature of 25°C than were more southerly isolates from both east and west. With regard toDevaleraea ramentacea, one Canadian isolate grew extraordinarily well at −2 and 0°C, and all tolerated temperatures 2–3°C higher than the lethal limit (18–20°C) of isolates from Europe. ConcerningPhycodrys rubens, both eastern and western isolates died at 20°C but European plants tolerated the lethal high temperature longer, were more sensitive to freezing, and attained more rapid growth at optimal temperatures. The intertidal species,C. melagonium andD. ramentacea, both survived freezing at −5 and −20°C, at least for short time periods.C. melagonium was more susceptible thanD. ramentacea to desiccation. Patterns of thermal tolerance may provide insight into the evolutionary history of seaweed species.