Phylogenetic resolution and habitat specificity of members of the Photobacterium phosphoreum species group

Substantial ambiguity exists regarding the phylogenetic status of facultatively psychrophilic luminous bacteria identified as Photobacterium phosphoreum, a species thought to be widely distributed in the world's oceans and believed to be the specific bioluminescent light-organ symbiont of several deep-sea fishes. Members of the P. phosphoreum species group include luminous and non-luminous strains identified phenotypically from a variety of different habitats as well as phylogenetically defined lineages that appear to be evolutionarily distinct. To resolve this ambiguity and to begin developing a meaningful knowledge of the geographic distributions, habitats and symbiotic relationships of bacteria in the P. phosphoreum species group, we carried out a multilocus, fine-scale phylogenetic analysis based on sequences of the 16S rRNA, gyrB and luxABFE genes of many newly isolated luminous strains from symbiotic and saprophytic habitats, together with previously isolated luminous and non-luminous strains identified as P. phosphoreum from these and other habitats. Parsimony analysis unambiguously resolved three evolutionarily distinct clades, phosphoreum, iliopiscarium and kishitanii. The tight phylogenetic clustering within these clades and the distinct separation between them indicates they are different species, P. phosphoreum, Photobacterium iliopiscarium and the newly recognized 'Photobacterium kishitanii'. Previously reported non-luminous strains, which had been identified phenotypically as P. phosphoreum, resolved unambiguously as P. iliopiscarium, and all examined deep-sea fishes (specimens of families Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae and Acropomatidae) were found to harbour 'P. kishitanii', not P. phosphoreum, in their light organs. This resolution revealed also that 'P. kishitanii' is cosmopolitan in its geographic distribution. Furthermore, the lack of phylogenetic variation within 'P. kishitanii' indicates that this facultatively symbiotic bacterium is not cospeciating with its phylogenetically divergent host fishes. The results of this fine-scale phylogenetic analysis support the emerging view that bacterial species names should designate singular historical entities, i.e. discrete lineages diagnosed by a significant divergence of shared derived nucleotide characters.     

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.     

Historical microbiology: revival and phylogenetic analysis of the luminous bacterial cultures of M. W. Beijerinck

Luminous bacteria isolated by Martinus W. Beijerinck were sealed in glass ampoules in 1924 and 1925 and stored under the names Photobacterium phosphoreum and 'Photobacterium splendidum'. To determine if the stored cultures were viable and to assess their evolutionary relationship with currently recognized bacteria, portions of the ampoule contents were inoculated into culture medium. Growth and luminescence were evident after 13 days of incubation, indicating the presence of viable cells after more than 80 years of storage. The Beijerinck strains are apparently the oldest bacterial cultures to be revived from storage. Multi-locus sequence analysis, based on the 16S rRNA, gapA, gyrB, pyrH, recA, luxA, and luxB genes, revealed that the Beijerinck strains are distant from the type strains of P. phosphoreum, ATCC 11040(T), and Vibrio splendidus, ATCC 33125(T), and instead form an evolutionarily distinct clade of Vibrio. Newly isolated strains from coastal seawater in Norway, France, Uruguay, Mexico, and Japan grouped with the Beijerinck strains, indicating a global distribution for this new clade, designated as the beijerinckii clade. Strains of the beijerinckii clade exhibited little sequence variation for the seven genes and approximately 6300 nucleotides examined despite the geographic distances and the more than 80 years separating their isolation. Gram-negative bacteria therefore can survive for many decades in liquid storage, and in nature, they do not necessarily diverge rapidly over time.     

The specificity of symbiosis: Pony fish and luminescent bacteria

Luminescent bacteria isolated from light organs of seven different species (3 genera) of fishes of the family Leiognathidae were subjected to taxonomic analysis. Of the 733 isolated all but seven were identified as Photobacterium leiognathi; the others are considered to be either chance contaminants of the sampling procedure or transients within the organ. In most fish, the luminous organ appeared to contain a single predominating strain of P. leiognathi with small numbers of one to three other strains of the same species, differing by only one or two characters.     

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.     

Isolation and identification of Photobacterium phosphoreum from an unexpected niche: migrating salmon

Six luminous bacteria were isolated from migrating salmon in the Yukon River, Alaska. All isolates were identified as Photobacterium phosphoreum. Previous studies suggest that P. phosphoreum is an exclusively marine bacterium, while our Alaskan isolates are from salmon which migrated up to 1,228 km from the marine environment.     

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).     

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.     

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.     

The bacterial flora of vacuum-packed cold-smoked salmon stored at 7 degrees C, identified by direct 16S rRNA gene analysis and pure culture technique

AIMS: The indigenous flora of freshly chilled cold-smoked salmon just after the vacuum packaging, and the spoilage flora after storage, in vacuum package at 7 degrees C for 19 days, were to be investigated with two different sampling strategies. METHODS AND RESULTS: Identification was performed using 16S rRNA sequencing of both isolated bacteria and bacterial DNA from tissue extract. The indigenous flora of fresh cold-smoked vacuum-packed salmon was dominated by, in order, Brochothrix thermosphacta, Yersinia ruckeri, Photobacterium and Carnobacterium, whereas the spoilage flora of the same product stored at 7 degrees C for 19 days was dominated by Lactobacillus and Photobacterium. The two sampling strategies showed similar results on the fish flora. Several new types of Photobacterium sequences, closely related to Photobacterium iliopiscarium and Photobacterium phosphoreum, were found from both the freshly processed and the stored salmon, indicating that smoked salmon harbours at least three different, as yet unknown, Photobacterium species. CONCLUSIONS: Ten per cent of the bacterial flora multiplying on chilled, vacuum-packed, cold-smoked salmon comprised unknown species. The two sampling strategies complement each other. SIGNIFICANCE AND IMPACT OF THE STUDY: As cold-smoked salmon is consumed without heat-treatment, the presence of undefined bacteria in high numbers should be considered in public health assessments.     

Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Phylogeny, genomics, and symbiosis of Photobacterium

Photobacterium comprises several species in Vibrionaceae, a large family of Gram-negative, facultatively aerobic, bacteria that commonly associate with marine animals. Members of the genus are widely distributed in the marine environment and occur in seawater, surfaces, and intestines of marine animals, marine sediments and saline lake water, and light organs of fish. Seven Photobacterium species are luminous via the activity of the lux genes, luxCDABEG. Much recent progress has been made on the phylogeny, genomics, and symbiosis of Photobacterium. Phylogenetic analysis demonstrates a robust separation between Photobacterium and its close relatives, Aliivibrio and Vibrio, and reveals the presence of two well-supported clades. Clade 1 contains luminous and symbiotic species and one species with no luminous members, and Clade 2 contains mostly nonluminous species. The genomes of Photobacterium are similar in size, structure, and organization to other members of Vibrionaceae, with two chromosomes of unequal size and multiple rrn operons. Many species of marine fish form bioluminescent symbioses with three Photobacterium species: Photobacterium kishitanii, Photobacterium leiognathi, and Photobacterium mandapamensis. These associations are highly, but not strictly species specific, and they do not exhibit symbiont-host codivergence. Environmental congruence instead of host selection might explain the patterns of symbiont-host affiliation observed from nature.     

Symbiotic association of Photobacterium fischeri with the marine luminous fish Monocentris japonica; a model of symbiosis based on bacterial studies.

Isolation of bacteria from the luminous organ of the fish Monocentris japonica has revealed that the organ contains a pure culture of luminous bacteria. For the four fish examined, all contained Photobacterium fischeri as their luminous bacterial symbiont. This is the first time that P. fischeri has been identified in a symbiotic association. A representative isolate (MJl) of the light organ population was selected for in vivo studies of its luminous system. Several physiological features suggest adaptation for symbiotic existence. First, MJl has been shown to produce and respond to an inducer of luciferase that could accumulate in the light organ. Secondly, the specific activity of light production was seen to be maximal under low, growth-limiting concentrations of oxygen. Thirdly, unlike another luminous species (Beneckea harveyi), synthesis of the light production system of these bacteria is not catabolite repressed by glucose--a possible source of nutrition in the light organ. Fourthly, when grown aerobically on glucose these bacteria excrete pyruvic acid into the medium. This production of pyruvate is a major process, accounting for 30-40% of the glucose utilized and may serve as a form of regulatory and nutritional communication with the host.     

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.     

Natural merodiploidy of the lux-rib operon of Photobacterium leiognathi from coastal waters of Honshu, Japan

Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis using lux genes, however, is based on the assumptions that the lux genes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains of Photobacterium leiognathi carry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes, luxCDABEG-ribEBHA. Merodiploid lux-rib strains of P. leiognathi were detected during sequence analysis of luxA. To define the gene content, organization, and sequence of each lux-rib operon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain, lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis of lnuch.13.1 defined two complete, physically separate, and apparently functional operons, designated lux-rib1 and lux-rib2. P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib1 and lux-rib2, whereas ATCC 25521T apparently carries only lux-rib1. In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521T, lux-rib1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib2 of lnuch.13.1, and the chromosomal location of lux-rib2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib1 and lux-rib2 are more closely related to each other than either one is to the lux and rib genes of other bacterial species, which rules out interspecies lateral gene transfer as the origin of lux-rib2 in P. leiognathi; lux-rib2 apparently arose within a previously unsampled or extinct P. leiognathi lineage. Analysis of 170 additional strains of P. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a single lux-rib operon and 68 that carry multiple lux-rib operons. Strains bearing a single lux-rib operon were obtained throughout the geographic sampling range, whereas lux-rib merodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy of lux or rib genes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.     

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     

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.     

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.     

Highly repetitive tRNA(Pro)-tRNA(His) gene cluster from Photobacterium phosphoreum.

A DNA fragment comprising the four tRNA gene sequences of the Escherichia coli argT locus hybridized with two Sau3A-generated DNA fragments from the vibrio Photobacterium phosphoreum (ATCC 11040). Detailed sequence analysis of the longer fragment shows the following gene organization: 5'-promoter-tRNA(Pro)-tRNAPro-tRNA(Pro)-tRNA(His)-tRNA(Pro)-tRNA(Pro)- tRNA(His)-tRNA(Pro)-five pseudogenes derived from the upstream tRNAPro interspersed by putative Rho-independent terminators. This sequence demonstrates the presence of highly repetitive, tandem tRNA genes in a bacterial genome. Furthermore, a stretch of 304 nucleotides from this cluster was found virtually unchanged in the other (shorter) fragment which was previously sequenced. The two clusters together contain eight tRNA(Pro) pseudogenes and eight fully intact tRNA(Pro) genes, an unusually high number for a single eubacterial isoacceptor tRNA. These results show that the organization of some tRNA operons is highly variable in eubacteria.