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1 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA
2 Laboratory of Microbiology and BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
3 Department of Genetics, Federal University of Rio de Janeiro, Ilha do Fundão, CEP 21944-970, Rio de Janeiro, Brazil
Correspondence
Jennifer C. Ast
jca{at}umich.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for gene sequences reported in this paper are EF415487–EF415631 and EF441349.
Supplementary material is available with the online version of this paper.
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Dunlap et al., 2007). Photobacterium phosphoreum (Beijerinck, 1889), the type species of the genus Photobacterium, was thought to be the light-organ symbiont of deep-water fishes (Hastings & Nealson, 1981), although the type strain of this species was isolated from the surface of a non-luminous fish (Ast & Dunlap, 2005). Recent analyses showed that P. phosphoreum was not recovered from light organ symbiosis; instead, the light-organ symbionts of deep-sea fishes formed a clade distinct from P. phosphoreum, designated previously as Photobacterium kishitanii (Dunlap & Ast, 2005; Dunlap et al., 2007). P. kishitanii is closely related to P. phosphoreum and Photobacterium iliopiscarium, although based on phylogenetic analysis, the latter two species are more closely related to each other than either is to P. kishitanii (Ast & Dunlap, 2005). Here, we present comprehensive phylogenetic, genomic and phenotypic evidence that strains of P. kishitanii represent a novel species, within the genus Photobacterium, for which the name Photobacterium kishitanii sp. nov. is proposed. The type strain is pjapo.1.1T (=ATCC BAA-1194T=LMG 23890T).
Six strains representing P. kishitanii sp. nov. were examined here (Table 1), three isolated from fish light-organs (Ast & Dunlap, 2005; Dunlap & Ast, 2005; Haygood et al., 1992) and three from enrichments of fish skin (Ast & Dunlap, 2005; Hendrie et al., 1970; Georgala, 1958; Reichelt & Baumann, 1973). To obtain DNA sequences for phylogenetic analysis, genomic DNA was purified using a DNA extraction kit (Qiagen) following the manufacturer's protocol for Gram-negative bacteria. Seven housekeeping genes (16S rRNA, gapA, gyrB, pyrH, recA, rpoA and rpoD) were amplified from the six strains and from eleven representative strains of the genus Photobacterium. In addition, the genes of two contiguous operons, the lux operon (luxC, luxD, luxA, luxB, luxF, luxE and luxG, the products of which are responsible for the luminescent phenotype) and the rib operon (ribE, ribB, ribH and ribA, the products of which are involved in riboflavin synthesis) were amplified from luminous strains, for a total of more than 17 kbp of sequence. See Supplementary Tables S1–S4 for PCR amplification profiles and primer sequences (available in IJSEM Online). Amplified products were purified by using a PCR clean-up kit (Millipore) and directly sequenced by using the University of Michigan Sequencing Core. Sequences for housekeeping, lux and rib genes were obtained for two strains of Vibrio fischeri to serve as outgroups. GenBank accession numbers for all DNA sequences, including those obtained previously, are listed in Supplementary Table S5.
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) using the 18 genes listed above (see Supplementary Material for details of phylogenetic analysis). Support values were calculated using 5000 jackknife resampling replicates. The resulting most parsimonious phylogenetic hypothesis clearly demonstrates that the novel strains identified as P. kishitanii represent a lineage separate from other species of Photobacterium (Fig. 1), with robust resampling support. Within the genus Photobacterium, the representatives of P. kishitanii form a clade with the species P. phosphoreum and P. iliopiscarium.
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; Wheeler et al., 2006), as implemented by the program POY (Wheeler et al., 2003). Direct optimization iteratively evaluates alignment in the context of phylogeny; it therefore produces a rigorously tested alignment, unlike single-pass multiple sequence alignment algorithms used by other alignment programs. Details of the POY analysis can be found in Supplementary Material. Identities of P. kishitanii pjapo.1.1T to other species were 97.7 % to P. indicum LMG 22857T, 99.6 % to P. iliopiscarium LMG 19543T and 99.7 % to P. phosphoreum LMG 4233T. These values are within the range of per cent identities between other recognized species of Photobacterium; for example, the identity between P. indicum LMG 22857T and P. phosphoreum LMG 4233T is 97.9 %.
To characterize genomic features that distinguish the strains of P. kishitanii from other species of Photobacterium, we performed two tests of genomic similarity, DNA–DNA hybridization and amplified length fragment polymorphism (AFLP) analysis. For DNA–DNA hybridization, high molecular mass DNA was prepared by the method of Wilson (1987) with minor modifications (Cleenwerck et al., 2002). DNA quality and quantity were determined by measuring absorptions at 260, 280 and 234 nm. Only high quality DNA with A260/A280 and A234/A260 ratios of 1.8–2.0 and 0.40–0.60 was used. Hybridizations were performed using a modification (Goris et al., 1998; Cleenwerck et al., 2002) of the microplate method described by Ezaki et al. (1989) with a hybridization temperature of 37 °C. Reported values are the mean of a minimum of four hybridizations. The values for hybridization between strain P. kishitanii pjapo.1.1T and other species of Photobacterium were 51 % to P. phosphoreum LMG 4233T, 43 % to P. iliopiscarium LMG 19543T and 19 % to P. indicum LMG 22857T. These values, which are below the current level that delimits separate species, demonstrates that strains of P. kishitanii are distinct from other species of Photobacterium.
For AFLP analysis, DNA was prepared as above, and template preparation, PCR reactions and PAGE were performed as described by Thompson et al. (2001). Electrophoretic patterns were tracked and normalized using the GENESCAN 3.1 software (Applera). Normalized tables of peaks, containing fragments of 50–539 bp were transferred to the BioNumerics 4.5 software (Applied Maths) for numerical analysis. Patterns were clustered using the Dice coefficient and the UPGMA algorithm. A band position tolerance value of 0.3 % was allowed to compensate for misalignment of similarly sized bands due to technical imperfections. The profiles were compared with the profiles of other species of Vibrionaceae using the database at the BCCM/LMG Bacteria Collection. The dendrogram of the AFLP profiles demonstrates that the strains of P. kishitanii cluster together and are clearly distinct from other analysed species of Photobacterium and Vibrio (Fig. 2).
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Phenotypic characterizations, e.g. cell morphology, response to Gram staining, motility, oxidase and catalase tests were performed using standard methods. Additional biochemical tests were performed using API 20E and API 20NE tests (bioMérieux). Cells for inoculation of the strips were grown for 24 h at 20 °C on M12 medium and results were visually interpreted according to the manufacturer's instructions. On the basis of the arginine dihydrolase test, novel strains of P. kishitanii can be differentiated from strains of P. phosphoreum, and the lysine decarboxylase test differentiates novel strains of P. kishitanii from strains of P. phosphoreum and P. indicum. However, no single biochemical trait or combination of traits distinguishes strains of P. kishitanii from strains of P. iliopiscarium. Complete phenotype data may be found in Supplementary Table S7.
To characterize further P. kishitanii pjapo.1.1T, DNA base composition was determined by HPLC according to the method of Mesbah et al. (1989). Non-methylated phage 
was used as a reference. The DNA G+C content of P. kishitanii pjapo.1.1T is 40.2 mol%, which is consistent with other species of Photobacterium (Baumann & Baumann, 1984).
To estimate genome size and chromosome composition, genomic DNA inserts were prepared according to Lucangeli et al. (2000) with modifications. DNA fragments of undigested inserts and inserts digested with NotI or I-CeuI enzyme (New England Biolabs) were separated by PFGE using standard conditions (see Supplementary Material). Based on the electrophoretic banding patterns, the P. kishitanii pjapo.1.1T genome is approximately 4.2 Mbp, configured in two circular chromosomes of sizes about 2.8 and 1.4 Mbp (Fig. 3). Strains B-421, ckamo.1.1, FS-8.1 and NCIMB 844 have genome sizes ranging from 4.0 to 4.7 Mbp (data not shown). Similar analyses of the genomes of 27 additional strains of P. kishitanii (data not shown) revealed that genomes can range in size from 3.9 to 4.9 Mbp with an average of 4.2 Mbp; all analysed strains were found to have two circular chromosomes, as in other species of Vibrionaceae (Okada et al., 2005). The I-CeuI enzyme digestion resulted in eight fragments, suggesting that P. kishitanii pjapo.1.1T has at least eight rrn loci (I-CeuI cuts most bacterial genomes only within rrn loci), a result consistent with the high rrn copy number in other species of Vibrionaceae (Klappenbach et al., 2001).
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; analysis details can be found in Supplementary Material). These results indicate that the following strains should be reclassified as P. kishitanii sp. nov.: ATCC 35080, ATCC 35081, ATCC 35082, NCIMB 61, NCIMB 62, NCIMB 64, NCIMB 65, NCIMB 66, NCIMB 68, NCIMB 69, NCIMB 70, NCIMB 71, NCIMB 844, NCIMB 1276, NCIMB 1277, NCIMB 12838 and NCIMB 12839. The following strains were confirmed as belonging to P. phosphoreum: NCIMB 7, NCIMB 188, NCIMB 193, NCIMB 395, NCIMB 1275 and NCIMB 1279. In previous work (Ast & Dunlap, 2005), three other strains from NCIMB identified as belonging to the species P. phosphoreum were resolved as belonging to the species P. iliopiscarium (NCIMB 13476, NCIMB 14378 and NCIMB 13481). A phylogenetic tree showing the results from the analysis including all NCIMB and ATCC strains mentioned above is available as Supplementary Fig. S1. All of these strains that originated from fish light-organs are P. kishitanii sp. nov., and to date, no strain identified by these criteria as P. phosphoreum has been isolated from the light organ of a fish or a squid (Ast & Dunlap, 2005; Dunlap & Ast, 2005; Dunlap et al., 2007; this study). Therefore, in contrast to P. kishitanii sp. nov., which can be isolated from light organs of several deep, cold-dwelling fishes, strains of P. phosphoreum apparently do not occur as a bioluminescent symbiont of marine animals. On the basis of these phylogenetic, genomic and taxonomic analyses, strains identified as P. kishitanii clearly represent a separate species of Photobacterium, for which the name Photobacterium kishitanii sp. nov. is proposed.
Description of Photobacterium kishitanii sp. nov.
Photobacterium kishitanii (ki.shi.tan'i.i. N.L. gen. n. kishitanii of Kishitani, to honour the deceased Japanese scientist Teijiro Kishitani, who first isolated luminous bacteria from the light organ of Physiculus japonicus). The following description is based on analyses of six strains (Table 1
).
Cells are Gram-negative, coccoid or coccoid-rods, motile, occurring singly or in pairs, 0.9 µm in width by 1.2–3.0 µm in length. After 18 h, colonies grown on LSW-70 at 22 °C are small, round, white and strongly luminous. Catalase-positive. Oxidase-negative or weakly positive. Genome size of the type strain is approximately 4.2 Mbp (ranging within the six strains from 4.0 to 4.7 Mbp), consisting of two circular chromosomes. Cells produce the fatty acid C17 : 0 cyclo. Light-organ symbiont of many fishes, may also be found on the surfaces of fishes and in seawater. The DNA G+C content of the type strain is 40.2 mol%.
The type strain, pjapo.1.1T (=ATCC BAA-1194T=LMG 23890T), was isolated in 1982 from the light organ of the deep-sea fish Physiculus japonicus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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