Four closely related species, Vibrio fischeri, Vibrio logei, Vibrio salmonicida and Vibrio wodanis, form a clade within the family Vibrionaceae; the taxonomic status and phylogenetic position of this clade have remained ambiguous for many years. To resolve this ambiguity, we tested these species against other species of the Vibrionaceae for phylogenetic and phenotypic differences. Sequence identities for the 16S rRNA gene were ≥97.4 % among members of the V. fischeri group, but were ≤95.5 % for members of this group in comparison with type species of other genera of the Vibrionaceae (i.e. Photobacterium and Vibrio, with which they overlap in G+C content, and Enterovibrio, Grimontia and Salinivibrio, with which they do not overlap in G+C content). Combined analysis of the recA, rpoA, pyrH, gyrB and 16S rRNA gene sequences revealed that the species of the V. fischeri group form a tightly clustered clade, distinct from these other genera. Furthermore, phenotypic traits differentiated the V. fischeri group from other genera of the Vibrionaceae, and a panel of 13 biochemical tests discriminated members of the V. fischeri group from type strains of Photobacterium and Vibrio. These results indicate that the four species of the V. fischeri group represent a lineage within the Vibrionaceae that is distinct from other genera. We therefore propose their reclassification in a new genus, Aliivibrio gen. nov. Aliivibrio is composed of four species: Aliivibrio fischeri comb. nov. (the type species) (type strain ATCC 7744T =CAIM 329T =CCUG 13450T =CIP 103206T =DSM 507T =LMG 4414T =NCIMB 1281T), Aliivibrio logei comb. nov. (type strain ATCC 29985T =CCUG 20283T =CIP 104991T =NCIMB 2252T), Aliivibrio salmonicida comb. nov. (type strain ATCC 43839T =CIP 103166T =LMG 14010T =NCIMB 2262T) and Aliivibrio wodanis comb. nov. (type strain ATCC BAA-104T =NCIMB 13582T =LMG 24053T).

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.

"Photobacterium mandapamensis" (proposed name) and Photobacterium leiognathi are closely related, phenotypically similar marine bacteria that form bioluminescent symbioses with marine animals. Despite their similarity, however, these bacteria can be distinguished phylogenetically by sequence divergence of their luminescence genes, luxCDAB(F)E, by the presence (P. mandapamensis) or the absence (P. leiognathi) of luxF and, as shown here, by the sequence divergence of genes involved in the synthesis of riboflavin, ribBHA. To gain insight into the possibility that P. mandapamensis and P. leiognathi are ecologically distinct, we used these phylogenetic criteria to determine the incidence of P. mandapamensis as a bioluminescent symbiont of marine animals. Five fish species, Acropoma japonicum (Perciformes, Acropomatidae), Photopectoralis panayensis and Photopectoralis bindus (Perciformes, Leiognathidae), Siphamia versicolor (Perciformes, Apogonidae), and Gadella jordani (Gadiformes, Moridae), were found to harbor P. mandapamensis in their light organs. Specimens of A. japonicus, P. panayensis, and P. bindus harbored P. mandapamensis and P. leiognathi together as cosymbionts of the same light organ. Regardless of cosymbiosis, P. mandapamensis was the predominant symbiont of A. japonicum, and it was the apparently exclusive symbiont of S. versicolor and G. jordani. In contrast, P. leiognathi was found to be the predominant symbiont of P. panayensis and P. bindus, and it appears to be the exclusive symbiont of other leiognathid fishes and a loliginid squid. A phylogenetic test for cospeciation revealed no evidence of codivergence between P. mandapamensis and its host fishes, indicating that coevolution apparently is not the basis for this bacterium's host preferences. These results, which are the first report of bacterial cosymbiosis in fish light organs and the first demonstration that P. leiognathi is not the exclusive light organ symbiont of leiognathid fishes, demonstrate that the host species ranges of P. mandapamensis and P. leiognathi are substantially distinct. The host range difference underscores possible differences in the environmental distributions and physiologies of these two bacterial species.

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.Horizontal gene transfer (HGT), the acquisition of genes by a member of one bacterial lineage from another lineage, is widely believed to play a major role in bacterial evolutionary divergence and speciation (29,39,40,46). As a possibly common process, HGT also is thought to result in reticulate evolution, precluding or at least seriously complicating reconstruction of bacterial phylogenies (6, 17). The actual frequency of HGT in nature and its influence on bacterial evolution, however, are controversial; many believe that HGT is uncommon, at best has only minor or infrequent effects on the process of bacterial speciation, and does not prevent reconstruction of ba...

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, luxCDABEGribEBHA. 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-rib 1 and lux-rib 2 . P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib 1 and lux-rib 2 , whereas ATCC 25521 T apparently carries only lux-rib 1 . In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521 T , lux-rib 1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib 2 of lnuch.13.1, and the chromosomal location of lux-rib 2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib 1 and lux-rib 2 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-rib 2 in P. leiognathi; lux-rib 2 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.Luminescence in Photobacterium leiognathi and other luminous bacteria is the product of bacterial luciferase, a mixedfunction oxidase that uses oxygen, reduced flavin mononucleotide, and a long-chain fatty aldehyde as substrates to produce blue-green luminescence. The genes for bacterial light production are present as an operon, luxCDABEG: luxA and luxB encode the ␣ and ␤ subunits of luciferase; luxC, luxD, and luxE specify the enzymatic components of a fatty acid reductase complex necessary for synthesis and recycling of the aldehyde substrate; and luxG encodes...

Six representatives of a luminous bacterium commonly found in association with deep, colddwelling marine fishes were isolated from the light organs and skin of different fish species. These bacteria were Gram-negative, catalase-positive, and weakly oxidase-positive or oxidase-negative. Morphologically, cells of these strains were coccoid or coccoid-rods, occurring singly or in pairs, and motile by means of polar flagellation. After growth on seawater-based agar medium at 22 6C for 18 h, colonies were small, round and white, with an intense cerulean blue luminescence. Analysis of 16S rRNA gene sequence similarity placed these bacteria in the genus Photobacterium. Phylogenetic analysis based on seven housekeeping gene sequences (16S rRNA gene, gapA, gyrB, pyrH, recA, rpoA and rpoD), seven gene sequences of the lux operon (luxC, luxD, luxA, luxB, luxF, luxE and luxG) and four gene sequences of the rib operon (ribE, ribB, ribH and ribA), resolved the six strains as members of the genus Photobacterium and as a clade distinct from other species of Photobacterium. These strains were most closely related to Photobacterium phosphoreum and Photobacterium iliopiscarium. DNA-DNA hybridization values between the designated type strain, Photobacterium kishitanii pjapo.1.1 T , and P. phosphoreum LMG 4233 T , P. iliopiscarium LMG 19543 T and Photobacterium indicum LMG 22857 T were 51, 43 and 19 %, respectively. In AFLP analysis, the six strains clustered together, forming a group distinct from other analysed species. The fatty acid C 17 : 0 cyclo was present in these bacteria, but not in P. phosphoreum, P. iliopiscarium or P. indicum. A combination of biochemical tests (arginine dihydrolase and lysine decarboxylase) differentiates these strains from P. phosphoreum and P. indicum. The DNA G+C content of P. kishitanii pjapo.1.1 T is 40.2 %, and the genome size is approximately 4.2 Mbp, in the form of two circular chromosomes. These strains represent a novel species, for which the name Photobacterium kishitanii sp. nov. is proposed. The type strain, pjapo

Use of inadequate methods for classification of bacteria in the so-called Harveyi clade (family Vibrionaceae, Gammaproteobacteria) has led to incorrect assignment of strains and proliferation of synonymous species. In order to resolve taxonomic ambiguities within the Harveyi clade and to test usefulness of whole genome sequence data for classification of Vibrionaceae, draft genome sequences of 12 strains were determined and analysed. The sequencing included type strains of seven species: Vibrio sagamiensis NBRC 104589 T , Vibrio azureus NBRC 104587 T , Vibrio harveyi NBRC 15634 T , Vibrio rotiferianus LMG 21460 T , Vibrio campbellii NBRC 15631 T , Vibrio jasicida LMG 25398 T , and Vibrio owensii LMG 25443 T . Draft genome sequences of strain LMG 25430, previously designated the type strain of [Vibrio communis], and two strains (MWB 21 and 090810c) from the 'beijerinckii' lineage were also determined. Whole genomes of two additional strains (ATCC 25919 and 200612B) that previously could not be assigned to any Harveyi clade species were also sequenced. Analysis of the genome sequence data revealed a clear case of synonymy between V. owensii and [V. communis], confirming an earlier proposal to synonymize both species. Both strains from the 'beijerinckii' lineage were classified as V. jasicida, while the strains ATCC 25919 and 200612B were classified as V. owensii and V. campbellii, respectively. We also found that two strains, AND4 and Ex25, are closely related to Harveyi clade bacteria, but could not be assigned to any species of the family Vibrionaceae. The use of whole genome sequence data for the taxonomic classification of the Harveyi clade bacteria and other members of the family Vibrionaceae is also discussed.

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