INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1991, p. 529-534 Vol. 41, No. 4 0020-7713/91/040529-06$02.OO/O Copyright 0 1991, International Union of Microbiological Societies

Evaluation of the Genus and Reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the Genus Photobacterium as Photobacterium damsela comb. nov. with an Emended Description S. K. SMITH,l* D. C. SUTTON,l J. A. FUERST,' AND J. L. REICHELT' Sir George Fisher Centre for Tropical Marine Studies, James Cook University of North Queensland, Townsville, Queensland 481 I ,' and Department of Microbiology, University of Queetzsland, St. Lucia, Queensland 4067,2 Australia

The genus Listonella, which was recently described on the basis of 5s rRNA sequence data, was found to be of dubious value on the basis of the results of a comparison of a number of taxonomic studies involving members of the . The available data suggest that 5s rRNA sequences may be of limited taxonomic use at the intra- and intergeneric levels, at least for apparently recently evolved groups, such as the Vibrionaceae. In this light, we assessed the generic assignment of the species Listonella damsela. Phenotypic characterization of 12 strains of assigned to L. damsela, including type strain ATCC 33539, revealed a strong resemblance to members of the genus Photobacterium. All of the strains conformed to major characteristics common to all known Photobacterium species. The characteristics of these organisms included the absence of a flagellar sheath and accumulation of poly-P-hydroxybutyrateduring growth on glucose coupled with the inability to utilize DL-P-hydroxybutyrate as a sole carbon source. On the basis of the phenotypic data, we propose that L. damsela should be reassigned to the genus Photobacterium as Photobacterium damsela comb. nov.

~- ._ Listonella damsela, which was initially described as present an extended description of L. damsela, which in- damsela, is recognized as an opportunistic pathogen cludes evidence suggesting that the species should be reas- that is capable of causing disease in a variety of hosts, signed to the genus Photobacterium. This description was including damselfish (22), sharks (18), dolphins (17), and based on phenotypic data, which were analyzed in light of all humans (26). The soft tissue infection which most often currently available information (phenetic and genetic) on the occurs may be due to the production of a cytolysin (20). positions of the genera of the family Vibrionaceae. Despite recognition of the potential pathogenicity of this species, previously published taxonomic data on L. damselu are scattered and incomplete. Aside from the initial pheno- MATERIALS AND METHODS typic description (22), information useful in characterization Bacterial strains and bacteriological media. has generally been presented only in conjunction with re- The bacterial ports of disease incidence (14, 18). As a result, the generic strains used in this study and their sources are listed in Table position of L. damsela has not been adequately assessed. 1. The bacterial strains were maintained on luminous me- The current validly published generic designation of this dium (LM) at 25°C. species is the new genus Listonella (23). Establishment of The artificial seawater which we used was a modification this genus was one of a number of taxonomic changes of that described by MacLeod (25) and contained per liter of distilled water 17.55 g of NaC1, 0.75 g of KC1, 0.285 g of proposed for the family Vibrionaceae that were based on a . review of phylogenetic relationships within the family in Na,SO,, 5.10 g of MgCI, 6H,O, and 0.145 g of CaCI,. which 5s rRNA sequence data were used (23). The species The basal medium which we used was the basal medium (8), and Listonella pelagia were also described by Baumann et al. except that it was modified described as members of this new genus, and subsequently with a lower concentration of Tris HCI (20 mM) and con- tained ferric ammonium citrate (0.025 g/liter) in place of workers have suggested that the genus Listonella may be extended to include the species currently designated Vibrio FeSO, - 7H,O. Yeast extract broth and LM were prepared tubiashii, Vibrio ordalii, Vibrio aestuarianus as described by Baumann et al. (8). and (30, 31). Phenotypic characterization. Our consideration of the generic position of L. damsela led The majority of the methods us to examine the new genus Listonella and also the appro- used in this study were methods described by Stanier et al. (8). priateness of 5s rRNA data for establishing phylogenetic (36), modified for heterotrophic marine bacteria Only significant modifications of these methods and methods from relationships within and between genera of the Vibrion- aceae. other sources are described below. Strains were incubated at In this study we took a polyphasic approach and compared 25°C for all of the tests unless otherwise stated. the relationships within the Vibrionaceae derived from 5s Cell shape, motility, and Gram stain reaction were deter- mined by using 24-h yeast extract broth cultures as previ- rRNA sequence data (with particular reference to the genus ously described Listonella) with the relationships determined by other meth- (8). ods in previous taxonomic studies. In this paper we also Flagellum characteristics were determined by using cells grown on LM for 24 h. The cells were negatively stained with membrane-filtered 1% uranyl acetate containing 0.4% sucrose, in a manner similar to that used by Allen and * Corresponding author. Baumann (l),and were examined with a Hitachi model

5 29 530 SMITH ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Strains of L. darnsela used in this study described by Reichelt and Baumann (32) and was assessed by examining cells for PHB by phase-contrast microscopy. Strain Source" Isolated from: Fermentation of glucose and production of acid and/or gas 2588-80T (= ATCC 33539T) ATCC Fish ulcer were determined by using the F1 medium of Baumann et al. 0183-79 (= ACMM 625) SPHTM Human puncture wound (S), modified with a lower concentration of Tris HCI (20 mM) PD9 (= ACMM 624) SPHTM Diseased shark and the addition of bromcresol purple (0.001%). The growth PDlO (= ACMM 623) SPHTM Diseased shark 86.665H (= ACMM 620) DOAT Diseased turtle of strains in the absence of sodium ions was tested in the 86.665NH (= ACMM 621) DOAT Diseased turtle media described by Baumann and Baumann (5). Oxidase BKG/B (= ACMM 630) TVS Diseased fish reactions were tested with tetramethyl-p-phenylenediamine YC3 (= ACMM 627) ACMM Diseased fish dihydrochloride by using the technique of Stanier et al. (36). 14 (= ACMM 626) ACMM Aquarium seawater The Voges-Proskauer test was performed as described by 17 (= ACMM 632) ACMM Aquarium seawater Lee et al. (21). The ability of strains to convert arginine to PT3 (= ACMM 628) ACMM Aquarium seawater ornithine anaerobically was tested in modified Thornley FLAd (= ACMM 622) ACMM Fish surface arginine dihydrolase medium as described by Baumann and a ATCC, American Type Culture Collection, Rockville, Md.; SPHTM, P. Baumann (5). Production of the extracellular enzymes gelat- Desmarchelier, School of Public Health and Tropical Medicine, Sydney, New inase, amylase, lipase, and alginase was determined by using South Wales, Australia; DOAT, J. Carson, Department of Agriculture, Tasmania, Australia; TVS, J. Glazebrook, School of Tropical Veterinarian the methods of Baumann et al. (8). Urease activity was Science, James Cook University, Townsville, Queensland, Australia; tested in the medium of Christensen (13). Utilization of 51 ACMM, Australian Collection of Marine Microorganisms, James Cook Uni- organic carbon compounds (Table 2) as sole sources of versity, Townsville, Queensland, Australia. carbon and energy was tested as described by Baumann et al. (8). The minimal medium base used was prepared both with and without 0.05% yeast extract as a source of growth H-800 transmission electron microscope operated at an factors. The compounds tested were selected from the 150 accelerating voltage of 100 kV. compounds used by Baumann et al. (S), which were utilized The ability to accumulate poly-f3-hydroxybutyrate (PHB) by 1 to 90% of the strains examined in that study. Com- as an intracellular reserve product was tested in the media pounds that were utilized by all or no strains were not

TABLE 2. Numbers of L. darnsela strains that utilize various organic compounds as sole or principal sources of carbon and energy in the presence and in the absence of 0.05% yeast extract and numbers of Photobacterium strains that utilize the compounds in the presence of 0.05% yeast extractu

L. darnsela (n = 12)6 No. of strains positive Presence of Compound 0.05% yeast NO. of P. angusturn P. phosphorertm P. leiogrzuthi extract strains Atypical strain(s)' (n = 51d (n = 74)' (n = 28)' positive Cellobiose 12 0 0 0 1 ACMM 623 D-Galactose 12 5 73 28 8 ACMM 620, ACMM 621, ACMM 625, ACMM 632 D-Gluconate 0 5 63 28 0 D-Mannose 12 5 74 28 9 ACMM 620, ACMM 623, ACMM 632 Xylose 0 4 0 0 0 DL-Glycerate 12 0 68 21 0 m-Lactate 12 5 13 28 2 ATCC 33539T, ACMM 622 a-Ke tog lutarate 1 ATCC 33539T 0 0 1 0 Pyruvate 12 5 0 27 2 ATCC 33539=. ACMM 630 L-Glutamate 12 0 28 19 2 ATCC 33539=, ACMM 627 L-Serine 9 ACMM 620, ACMM 622, ACMM 625 5 26 21 0 Caprate 0 0 0 12 0 The following compounds were not utilized by any of the L. danlsela strains examined or by Photobacterium species: L-arabinose, L-rhamnose, salicin, caproate, butyrate, caprylate, heptanoate, isobutyrate, isovalerate, pelargonate, propionate, valerate, DL-P-hydroxybutyrate, aconitate, citrate, betaine, hippurate, sarcosine, glutarate, malonate, mannitol, sorbitol, ethanol, n-propanol, benzoate, p-hydroxybenzoate, quinate, tyrosine, D-a-alanine, p-alanine, y-aminobutyrate, 6-aminovalerate, arginine, citrulline, glycine, L-leucine, L-ornithine, putrescine, and spermine. n is the number of strains tested. ' Atypical strains are those isolates examined in this study whose reaction (positive or negative) was the opposite of the reaction of the majority of the strains. Data from reference 8. Data from reference 32. VOL.41. 1991 REASSIGNMENT OF LISTONELLA DAMSELA 531

FIG. 1. (a) Negatively stained strain ATCC 33539', showing one unsheathed polar flagellum. Bar = 1.0 pm. (b) Flagella of strain ACMM 621, showing the regular substructure that is typical of the unsheathed state. Bar = 0.1 pm. (c) Negatively stained strain ACMM 621, showing multitrichous unsheathed polar flagella. Bar = 0.5 Fm. (d) Phase-contrast micrograph showing bright refractile intracellular PHB granules present in cells of strain ATCC 33539' following growth on glucose. Bar = 2.0 pm.

included. The ability of strains to bioluminesce was exam- there have been more than 18 major numerical taxonomic ined visually by using 24- and 48-h cultures grown on LM studies (24) and a substantial number of genetic studies of (32). The possibility of low-level bioluminescence was tested this taxon (2-4, 12, 23, 33). As a result, the family has by examining LM broth cultures through one entire growth undergone a number of taxonomic changes as new tech- cycle, using a photometer. Growth at 4 and 35°C was niques and, therefore, new data have become available (7). determined as described by Baumann et al. (8). Remarkably, the variety of approaches which have been used have produced results that generally have demon- RESULTS strated good agreement (9). An exception to this is the most recent phylogenetic examination of the family in which 5s All of the isolates which we examined were facultatively rRNA sequence data were used (23). There are incongruities anaerobic, gram-negative, straight rods. Transmission elec- between the results of this study and those of other taxo- tron microscopy revealed the presence of one or more nomic studies in which different approaches were used at unsheathed polar flagella that were 12 to 17 nm wide. both the intrageneric and intergeneric levels. These incon- Photomicrographs of representative isolates, including strain gruities provide several good reasons for concern about the ATCC 33539T (T = type strain), are shown in Fig. la through changes in generic designations within the Vibrionaceae c. All of the isolates accumulated PHB granules (Fig. Id) proposed on the basis of the 5s rRNA data, particularly the when they were grown on medium containing glucose but establishment of the genus Listonellu. did not utilize the exogenous monomer DL-P-hydroxybu- At the intrageneric level there are a number of discrepan- tyrate. We observed strong positive biochemical reactions cies between the relationships determined on the basis of 5s for arginine dihydrolase and urease production. Weak posi- rRNA sequence data and the relationships determined in tive results were detected in the Voges-Proskauer test, in the other taxonomic studies. First, the results of recent geno- oxidase reaction test, and in the test for production of typic and phenotypic research have indicated that there is extracellular lipase. Gas production in fermentation medium significant variability within and between the species cur- was also weak and could not be detected in some strains rently assigned to the genus Listonella. Wiik and Egidius after several subcultures. None of the strains was biolumi- (38) demonstrated that there is host-dependent strain heter- nescent, was able to grow in the absence of sodium ions, or ogeneity in L. anguillarum (), and Urdaci produced the extracellular enzymes amylase, alginase, and et al. (37) showed that the fatty acids profiles of the three gelatinase. The nutritional versatility of the strains is shown Listonella species (V. damsela, Vibrio pelagius, and V. in Table 2, which includes data for the Photobacterium anguillarum) are distinct and very different in a principal- species for comparison. Table 2 also shows that for all of the components analysis (37). These results suggest that these strains, addition of 0.05% yeast extract to the minimal three species represent a heterogeneous group. medium base as a source of growth factors was stimulatory Second, intrageneric discrepancies are evident between and increased the range of carbon compounds utilized. By relationships determined from 5s rRNA sequence data and using control media consisting of the minimal medium base relationships determined from DNA-DNA hybridization plus 0.05% yeast extract without an added carbon substrate, data. Vibrio harveyi was closely related to Vibrio alginolyti- we found that this supplement by itself was unable to cus and Vibrio parahuemolyticus as measured by levels of support growth. DNA homology (50 to 58% at 63°C [33] and 52 to 55% at 60°C [12]). but V. hurveyi did not cluster with either of these DISCUSSION species in the 5s rRNA analysis (23). Similarly, Vibrio cholerae and Vibrio mimicus, which are also closely related Evaluation of the genus ListoneZZa. The Vibrionaceae is one as determined by DNA homology levels (40 to 67% at 63°C of the most extensively characterized prokaryotic families; [15] and 55 to 67% at 60°C [12]), also failed to cluster in the 332 SMITH ET AL. INT. J. SYST.BACTERIOL.

5s rRNA analysis (23). In contrast, Photobacterium leiog- exhibit limited genetic variability because of their more nathi and Photobacterium angustum, which exhibited levels recent evolution (35). of DNA homology similar to those described above (53 to Therefore, given that there is some uncertainity about the 61% at 63°C [33]), were found to have identical 5s rRNA genus Listonella and recognizing the need for distinctive sequences (23). phenotypic properties at the generic level, we suggest that Third, there are discrepancies concerning the assignment the characteristics of the genera Vibrio and Photobacterium of Photobacterium Jischeri and Photobacterium logei to the as outlined in Bergey ’s Manual of Systematic Bacteriology genus Photobacterium on the basis of 5s rRNA sequence (6, lo), which have demonstrated good agreement with the data, which indicated an apparently close relationship (ca. results of previous molecular studies (9), should remain as !J6% homology as determined by average linkage analysis the framework for establishing generic assignments for mem- 1231). Although the DNA-rRNA hybridization data of Bau- bers of the Vibrionaceae until enough additional substantial inann and Baumann (3) initially supported this assignment, phylogenetic evidence and complementary phenotypic char- these authors subsequently placed P. jscheri and P. logei acteristics are available to warrant significant changes to the iin the genus Vibrio on the basis of 16s rRNA data (provided taxonomy of this family. This would most likely require a by C. R. Woese) (for a discussion, see reference 5); this major study of 16s rRNA sequence data, which contain placement was supported by the result of immunological substantially more phylogenetic information (29) and are studies of certain enzymes (2, 4). In addition, Reichelt et al. significantly more robust with respect to nonrandom changes ((33)obtained DNA homology values of only 0 to 11%(under (39). (optimalconditions) between either P.Jischeri or P. logei and Reassignment of Listonella damsela. A comparison of the other Photobacterium and Vihrio species. Such levels pro- phenotypic characteristics of L. damsela, as determined in vide no indication of a close relationship, and it has been this study, with those of the genera Vibrio and Photobacte- suggested that these two species may deserve a separate rium suggests that L. damsela should be reassigned to the generic designation (5, 32, 33). genus Photobacterium. The principal characteristics that At the intergeneric level the analyses of Baumann and distinguish the genus Photobacterium from the genus Vibrio co-workers involving DNA-rRNA hybridization (3) and im- are the absence of a flagellar sheath in the genus Photobac- munological studies of certain enzymes (2, 4) provide little terium and the accumulation of the storage product PHB support for the separation of two members of the genus during growth of Photobacterium spp. on glucose coupled Listonella, L. anguillarum and L. pelagia, from members of with the inability to utilize the exogenous monomer of PHB the genus Vibrio sensu strict0 (as defined by MacDonell and (m-p-hydroxybutyrate) for growth (6). All of the strains Colwell [23]). The results reported for these species were tested in this study possessed these characteristics, as other well within the range demonstrated for other Vibrin species members of the genus Photobacterium do. The reassignment (2, 3). Furthermore, L. anguillarum and L. pelagia have proposed here is in accordance with the description of the exhibited at least 20 to 30% DNA homology (under optimal genus Photobacterium in Bergey ’s Manual (6). Molecular conditions) with most other Vibrio species tested in previous sequence data are incomplete and have not yet been incor- studies (12, 33). This level is considered by some authors to porated into the generic definition, and so the genera still be appropriate for members of the one genus (34). need to be characterized by using phenotypic traits. This We must conclude that the polyphasic approach reveals view is consistent with the view expressed by the Ad Hoc significant discrepancies between the relationships derived Committee on Approaches to Taxonomy within the Proteo- from 5s rRNA sequence data and the relationships derived bacteria on the need for phenotypic data to be included in from other taxonomic studies of Vibrio and Photobncterium making generic taxonomic decisions (27). species. Although rRNAs are probably the most suitable The following other traits possessed by all of the strains macromolecules for measuring relatedness between taxo- used in this study are typical of the genus Photobacterium nomically distant organisms (39) and provide extensive (6) but atypical of the genus Vibrio (10): weak oxidase evolutionary insights (16), 5s rRNA has limitations because reaction, thought to be due to low levels of cytochrome c; of the small size of the molecule (ca. 120 nucleotides for 5s limited nutritional versatility, with only seven to nine of the rRNA, compared with ca. 1,600 nucleotides for 16s rRNA tested compounds utilized (Table 2); inability to utilize and ca. 3,300 nucleotides for 23s rRNA [28]). This limits the mannitol as a sole source of carbon and energy; and the information content (28) and therefore limits the accuracy of absence of extracellular amylase or alginase. phylogenetic inferences when there is variance at too few To our knowledge, few molecular genetic studies of L. positions (29). 5s rRNA is also susceptible to nonrandom damsela with other Photobacterium species have been un- (selected) sequence changes which add to random changes dertaken. The previously published G + C contents of L. and so artifically increase phylogenetic distances (39). In the damsela (43.6 mol% [22] and 42 mol% [18]) fall within the larger rRNAs these changes are obscured by the presence of range reported for the genus Photobacterium (40 to 44 long stretches of conserved sequences (39). mol%) (6). This is also consistent with the range for the Given these features of 5s rRNA molecules, the observed genus Vibrio; however, most Vibrio species have G + C discrepancies can be understood in terms of insufficient contents of 44 mol% or more (10). One DNA-DNA hybrid- discrimination for close relationships in recently evolved ization study (12) which involved L. damsela and Photobac- groups and the potentially disruptive effects of nonrandom terium species has been described; however, only very low changes in such small molecules. Yet 5s rRNA sequence levels of DNA homology were found between L. damsela data have resolved successfully more distant phylogenetic and both Vibrio and Photobacterium species (5% or less at relationships (i.e., the families Vibrionaceae, Aeromona- 75°C [12]). Such levels are considered to be insufficiently daceae, and Enterobacteriaceae [23]) in agreement with the accurate to be conclusive (19). results of previous molecular studies (11). It appears that 5s In conclusion, given that there is some uncertainity about rRNA data may be of limited taxonomic use at the generic the genus Listoneflu, the phenotypic data presented here level, at least for what is believed to be a recently evolved provide evidence for reassignment of L. damsela to the group such as the Vibrionaceae (34), whose members tend to genus Photobacterium. Therefore, we propose that L. dam- VOL. 41, 1991 REASSIGNMENT OF LISTONELLA DAMSELA 533

TABLE 3. Characteristics useful for differentiating Photobacterium species

Utilization (in the presence of 0.05% yeast extract) of Growth at: Species Bioluminescence Lipase Gelatinase Cellobiose Gluconate Xylose Glycerate Glutamate 4°C 35°C P. damsela - U + - + - - + + - + P. angustumb - V + - + + - - + + P. phosphoreum‘ + - - - + - + V + - P. leiognathi‘ + + - - + - V V - +

a +, 80% or more of the strains are positive: -. 20% or more of the strains are negative; V, 21 to 79% of the strains are positive. Data from reference 8. Data from reference 32. sela should be reassigned to the genus Photobacterium as DNAhibosomal RNA hybridization. Microbios Lett. 3:ll-20. Photobacterium damsela. 4. Baumann, L., and P. Baumann. 1980. Immunological relation- Table 3 shows the characteristics that are useful for ships of glutamine synthetases from marine and terrestrial differentiating Photobacterium species, including P. dam- enterobacteria. Curr. Microbiol. 3:191-196. sela. 5. Baumann, P., and L. Baumann. 1981. The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Emended description of Photobacterium damsela (Love et al. Pseudomonas, and Alcaligenes, p. 1302-1331. In M. P. Starr, 1981) Smith, Sutton, Fuerst, and Reichelt comb. nov. Photo- H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), bacterium damsela (dam.se’la. M.L. n. damsela, pertaining The prokaryotes. A handbook on habitats, isolation, and iden- to damselfish). Gram-negative, straight rods that are motile tification of bacteria. Springer-Verlag, New York. by means of one or more unsheathed polar flagella. Facul- 6. Baumann, P., and L. Baumann. 1984. Genus 11. Photobacterium tatively anaerobic, often with weak gas production. Able to Beijerinck 1889, p. 539-545. In N. R. Kreig and J. G. Holt (ed.), accumulate the storage product PHB when grown on me- Bergey’s manual of systematic bacteriology, vol. 1. The dium containing glucose, but unable to utilize the exogenous Williams & Wilkins Co., Baltimore. monomer DL-P-hydroxybutyrate. Arginine dihydrolase and 7. Baumann, P., L. Baumann, S. S. Bang, and M. J. Woolkalis. 1981. Reevaluation of the taxonomy of Vibrio, Beneckea, and urease tests are strongly positive. Oxidase, Voges-Proskauer, Photobacterium: abolition of the genus Beneckea. Curr. Micro- and extracellular lipase tests are weakly positive. No growth biol. 4:127-132. occurs in the absence of sodium ions. Not bioluminescent. 8. Baumann, P., L. Baumann, and M. Mandel. 1971. Taxonomy of Negative for the production of the extracellular enzymes marine bacteria: the genus Beneckea. J. Bacteriol. 107:268-294. amylase, alginase, and gelatinase. The following carbon 9. Baumann, P., L. Baumann, M. J. Woolkalis, and S. S. Bang. compounds are utilized as sole carbon and energy sources 1983. Evolutionary relationships in Vibrio and Photobacterium: in the presence of 0.05% yeast extract; pyruvate, lactate, a basis for a natural classification. Annu. Rev. Microbiol. glutamate, mannose, galactose, cellobiose, and glycerate. 37:369-398. Utilization of serine and utilization of a-ketoglutarate in the 10. Baumann, P., A. L. Furniss, and J. V. Lee. 1984. Genus I. Vibrio Pacini 1854, p. 518-538. In N. R. Kreig and J. G. Holt (ed.), presence of 0.05% yeast extract are variable. 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