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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1993, p. 8-19 Vol. 43, No. 1 0020-7713/93/010008-12$02.00/0 Copyright 0 1993, International Union of Microbiological Societies

Phylogenetic Relationships of Marine , Mainly Members of the Family Vibrionaceae, Determined on the Basis of 16s rRNA Sequences

KUMIKO KITA-TSUKAMOTO,l* HIROSHI OYAIZU,2 KENJI NANBA,l AND US10 SIMIDU’ Ocean Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164, and Faculty of Agn’culture, University of Tokyo, Hongo, Bunkyou, Tokyo 113, Japan

The phylogenetic relationships of 50 reference strains, mostly marine bacteria which require Na+ for growth, were determined on the basis of 600 16s rRNA nucleotides by using reverse transcriptase sequencing. Strains belonging to 10 genera were included (four genera of the family Vibrionaceae, the genus of the family Aeromonaduceae, and the genera Alteromonas, Marinomonas, Shewanella, Pseudomonas, and Deleya). The sequences were aligned, the similarity values and evolutionary distance values were determined, and a phylogenetic tree was constructed by using the neighbor-joining method. On the basis of our results, the family Vibrionaceae was separated into at least seven groups (genera and families). Kbrio murinus clearly was on a line of descent that was remote from other . As determined by the similarity and evolutionary distance values, V. marinus is more distantly related to the family Vibrionaceae than the members of the Aeromona- daceae are. Also, cholerae strains formed a separate group with at the genus level. Of 30 species of the Vibrionaceae, 17 formed a large phylogenetic cluster. The genus was found to be a heterogeneous group, and the species were distributed in various subgroups of the Vibrionaceae. The separation of the family Aeromonadaceae from the family Vibrionaceae and the separation of the genera Marinomonas and Shewanella from the genus Alteromonas were confirmed in this phylogenetic study. However, a marine Pseudomonas species, Pseudomonas rtuutica, was clearly separated from two terrestrial Pseudomonas species. Each group that was separated by the phylogenetic analysis had characteristic 16s rRNA sequence patterns that were common only to species in that group. Therefore, the characteristic sequences described in this paper may be useful for identification purposes.

Heterotrophic bacteria which are gram negative and mo- the expansion of the family Vibrionaceae, it is important to tile by means of flagella are commonly isolated from marine establish the phylogenetic relationships among its members. environments and apparently are a major component of the Although methods based on molecular biology, including bacterial flora of the sea. On the basis of their ability to DNA-DNA hybridization and DNA-rRNA hybridization, ferment carbohydrates, these organisms can be divided into have been used for bacterial for a long time, the two groups. The fermentative strains have been assigned to recent development of techniques for sequencing DNA and the genera Vibrio, Listonella, , Colwellia RNA is expected to lead to a fundamental revision of (lo), and Aeromonas, and the nonfermentative strains have microbial systematics. MacDonell and Colwell have applied been included in the genera Alteromonas, Pseudomonas, the results of a sequence analysis of 5s rRNAs to the Alcaligenes, Deleya, Marinomonas, Shewanella, and Fla- taxonomy of marine bacteria, mainly members of the genus vobacterium (1). Kbrio (22). The family Hbrionaceae is one of the most important Recently, many reports have been published on the 16s bacterial groups in marine environments. Members of this rRNA sequences of bacteria and the phylogenetic relation- family often predominate in the bacterial flora of seawater, ships deduced from analyses of these sequences (6,7,11,19, plankton, and . In a survey carried out in the West Pacific 25). Most of the results indicate that phylogenetic relation- Ocean, vibrios accounted for nearly 80% of the bacterial ships based on 16s rRNA sequences support the distinction population in surface seawater (31). Members of the Vib- among eubacteria, archaeobacteria, and eucaryota and are rionaceae are also the main organisms present in the intes- generally in accord with the present status of bacterial tinal flora of marine fish (30). In addition, some members of classification. Also, the phylogenetic significance of bacte- the Vibrionaceae are important pathogens for humans and rial classification based on 16s rRNA sequences has been animals. recognized by many workers, and an ad hoc committee has The marine members of the Kbrionaceae have been the recently recommended changes in classification (36). subject of many taxonomic studies, and this group is now the In this paper we describe the phylogenetic relationships best-established marine bacterial taxon. The number of among 50 reference strains that are representative of marine described species in the family Vibrionaceae, particularly bacteria, including 34 strains of the family Vibrionaceae. the genus Vibrio, has been expanding rapidly. The number of Most of the marine bacterial strains which we studied Kbrio species increased from 5 in Bergey’s Manual of require Na+ for growth. Determinative Bacteriology in 1974 (29) to 20 in Bergey’s Manual of Systematic Bacteriology in 1984 (2) and now exceeds 34 (3,4,9,12-16,20,26,28,33,34,37). Because of MATERIALS AND METHODS Strains and cultivation. The 50 reference strains used in this study are listed in Table 1. These organisms were * Corresponding author. obtained from several culture collections, including the

8 VOL. 43, 1993 PHYLOGENETIC RELATIONSHIPS OF MARINE BACTERIA 9

TABLE 1. Strains used in this study imately early stationary phase by centrifugation, washed with sterile TMK buffer (10 mM magnesium acetate, 25 mM Taxon Strain Abbreviation" KCl, 50 mM Tris-HC1 [pH 7.6]), and stored at -25°C. Aeromonas caviae JCM 1060+ ACAV Extraction and determination of 16s rRNA sequences. Cells subsp. IF0 13282T AHAN were suspended in TMK buffer, and Macoloid solution (23) anaerogenes Aeromonas media JCM 2385T WED Aeromonas salmonicida subsp. IF0 13784 ASMA masoucida TABLE 2. Accession numbers for partial 16s rRNA sequences ATCC 35624T AVER of strains included in this study Alteromonas espejiana IAM 12640 ALES Alteromonas haloplanktis ATCC 14393T ALHA Accession no. Alteromonas nigrifaciens IAM 13010T ALNI Taxon Deleya marina IAM 12928 DMAR Primer Primer Primer R520 R920 R1400 ECOL Listonella anguillara IF0 13266T LANG Aeromonas caviae JCM 1060T D11169 D11218 D11267 Listonella damsela ATCC 33539T LDAM Aeromonas hydrophila subsp. anaero- D11170 D11219 D11268 Listonella pelagia ATCC 25916T LPEL genes IF0 13282T Marinomonas communis IAM 12914T MCOM Aeromonas media JCM 238ST D11174 D11223 D11272 Marinomonas vaga IAM 12923T MVAG Aeromonas salmonicida subsp. ma- D11175 D11124 D11273 Photobacterium angustum PANG soucida IF0 13784 Photobacterium leiognathi ATCC 25521T PLEI Aeromonas veronii ATCC 35624T D11176 D11225 D11274 Photobacterium phosphoreum ATCC 11040T PPHO Alteromonas espejiana IAM 12640 D11171 D11220 D11269 ATCC 14029T PLSH Alteromonas haloplanktis ATCC D11172 D11221 D11270 IAM 1514T PSAE 14393T Pseudomonas fluorescens IAM 12022T PSFL Alteromonas nignyaciens IAM 13O1OT D11173 D11222 D11271 Pseudomonas nautica IAM 12929= PSNA Deleya marina IAM 12928 D11177 D11226 D11275 Shewanella putrefaciens IAM 12079 SPUT Listonella anguillara IF0 13266T D11178 D11227 D11276 Vibrio aestuarianus ATCC 35048T VAES Listonella damsela ATCC 33539T D11179 D11228 D11277 NCMB 1903T VALG Listonella pelagia ATCC 259MT D11180 D11229 D11278 ATCC 25920T VCAM Marinomonas communis IAM 12914T D11181 D11230 D11279 Mbrio carchariae ATCC 35084T VCAR Marinomonas vaga IAM 12923T D11182 D11231 D11280 Mbrio cholerae Eltor Ogawa IID 936 VCHOl Photobacterium angustum D11183 D11232 D11281 Classical Inaba IID 467 VCHO2 Photobacterium leiognathi ATCC D11184 D11233 D11282 Vibrio cholerae Eltor Inaba IID 1655 VCHO3 25521T Vibrio cholerae Classical lnaba IID 935 VCHO4 Photobacteriumphosphoreum ATCC D11186 D11235 D11284 Vibrio cholerae Classical Ogawa IID 934 VCHO5 11040T Vibrio costicola NCMB 701T vcos Plesiomonas shigelloides ATCC D11185 D11234 D11283 Vibrio diarotrophicus ATCC 33466T VDIA 14029T Vibrio ficheri ATCC 7744T VFIS Pseudomonas aeruginosa IAM 5 14T D11187 D11236 D11285 NCTC 11328 VFLU Pseudomonas fluorescens IAM 12022T D11188 D11237 D11286 Vibriogazogenes ATCC 29988T VGAZ Pseudomonas nautica IAM 12929T D11189 D11238 D11287 ATCC 14126T VHAR Shewanella putrifaciens IAM 12079 D11190 D11239 D11288 Vibrio hollisae JCM 1283T VHOL Vibrio aestuarianus ATCC 35048T D11191 D11240 D11289 Vibrio logei ATCC 15382 VLOG Vibrio alginolyticus NCMB 1903T D11192 D11241 D11290 Vibrio marinus ATCC 15381T VMAR Vibrio campbellii ATCC 25920T D11193 D11242 D11291 Vibrio metschnikovii NCTC 8443T VMET Vibrio carchariae ATCC 35084T D11194 D11243 D11292 Hbrio mimicus ATCC 33655 VMIM Vibrio cholerae Eltor Ogawa IID 936 D11195 D11244 D11293 Mbrio natriegens CCM 2575T VNAT Virio cholerae Classical Inaba IID 467 D11196 D11245 D11294 Vibrio nereis ATCC 25917T VNER Vibrio cholerae Eltor Inaba IID 1655 D11197 D11246 D11295 Vibrio nignpulchritudo ATCC 27043T VNIG Vibrio cholerae Classical Inaba IID D11198 D11247 D11296 ATCC 33509T VORD 935 Vibrio orientalis ATCC 33934T VORI Mbrio cholerae Classical Ogawa IID D11199 D11248 D11297 Wbrioparahaemolyticus ATCC 17802T VPAR 934 Vibrio proteolyticus NCMB 1326T VPRO Vibrio costicola NCMB 701T D11200 D11249 D11298 ~ ____~ Hbrio diazotrophicus ATCC 33466T D11201 D11250 D11299 a Abbreviations are used Fig. 1 through 3. Viriojbcheri ATCC 7744T D11202 D11251 D11300 T = type strain. Vibriofluvialis NCTC 11328 D11203 D11252 D11301 Vibriogazogenes ATCC 29988T D11204 D11253 D11302 Vibrio harveyi ATCC 14126T D11205 D11254 D11303 Vibrio hollisae JCM 1283T D11206 D11255 D11304 American Type Culture Collection (ATCC), Rockville, Md.; Wbrio logei ATCC 15382 D11207 D11256 D11305 the Czechoslovak Collection of Microorganisms (CCM); the Vibrio marinus ATCC 15381T D11208 D11257 D 11306 Institute of Applied Microbiology (IAM), Tokyo, Japan; the Vibrio metschnikovii NCTC 8443T D11209 D11258 D11307 Institute for Fermentation, Osaka (IFO), Osaka, Japan; The Vibrio mimicus ATCC 33655 D11210 D11259 D11308 Institute of Medical Science (IID), Tokyo, Japan; the Japan CCM 2575T D11211 D11260 D11309 Collection of Microorganisms (JCM), Tokyo, Japan; the Mbrio nereis ATCC 25917T D11212 D11261 D11310 National Collection of Marine Bacteria (NCMB), Alberdeen, Vibrio nignpulchritudo ATCC 27043T D11213 D11262 D11311 Vibrio ordalii ATCC 33509T D11214 D11263 D11312 Scotland; and the National Collection of Type Cultures Vibrio orientalis ATCC 33934T D11215 D11264 D11313 (NCTC), London, England. Virioparahaemotyticus ATCC 17802T D11216 D11265 D11314 Strains were grown with shaking in half-strength %Bell Hbrio proteoEyticus NCMB 1326T D11217 D11266 D11315 2216E medium (38) at 20°C. Cells were harvested at approx- 10 KITA-TSUKAMOTO ET AL. Im. J. SYST.BACTERIOL.

VAL0 VCAM VCAR MIA VFLU MLAR VNAT VNER VNI G WAR VPRO

AHAN ACAV AMED ASMA AVER

ALES ALHA ALNI

FIG. 1. Partial 16s rRNA sequences of 50 reference strains, corresponding to positions 290 to 509,690 to 904, and 1180 to 1379 of the E. culi 16s rRNA sequence (5). Abbreviations for nucleotides used in the alignment: R, purine (A or G); Y, pyrimidine (C or U); M, A or C; W, A or U; S, C or G; K, G or U; D, A, G, or U; H, A, C, or U; V, A, C, or G; B, C, G, or U; N, A, C, G, or U; the dots indicate padding. These abbreviations and symbol are currently under consideration by a commission of the International Union of Pure and Applied Chemistry-International Union of Biochemistry. For abbreviations of organism names, see Table 1. The portions enclosed in boxes are sequences that are common to the members of individual branching groups (Fig. 2).

was added as an RNase inhibitor. When cells which have 926, and primer R1400 covered the sequence from position particularly high levels of RNase activity were prepared, 1392 to position 1406. phenol was added along with the Macoloid solution as an The reverse transcriptase sequencing reaction was additional RNase inhibitor. Nucleic acids were purified by stopped by adding 5 ~l of stop mixture (15 mM EDTA [pH phenol-chloroform extraction and cold isopropanol precipi- 8.0]), and the preparation was dried in vacuo. After a dye tation. DNA was digested with 60 U of RNase-free DNase I mixture (0.05% bromophenol blue and 0.05% xylene cyanol (Takara Shuzo Co., Ltd.). Each RNA preparation was dried in formamide) was added, the reaction mixture was heated and suspended in 10 mM Tris-HC1 buffer (pH 8.5) at a for 3 min at 97°C before it was loaded onto the sequencing concentration of 8 pg/pl. 16s rRNA sequences were determined by using the re- gel. A nongradient 8% polyacrylamide gel (thickness, 0.35 verse transcriptase reaction and oligonucleotide primers mm; length, 50 cm) was used for electrophoresis. specific to the 16s rRNAs. For sequencing we used the Determination of phylogenetic relationships. Sequences protocol described by Lane et al. (18), with some modifica- were initially aligned on the basis of obvious primary struc- tions. Three DNA primers were used in the sequencing ture homology. The evolutionary distance per site (K)was reactions; primer R520 covered the sequence from position calculated as follows: K = -(1/2) In [(l - 2P - Q) (1 - 517 to position 531 (Escherichia coli numbering) (5), primer 2Q)””l (17), where P and Q were the fractions of nucleotide R920 covered the sequence from position 907 to position sites having transition and transversion types of substitu- VOL.43, 1993 PHYLOGENETIC RELATIONSHIPS OF MARINE BACTERIA 11

410 420 430 ECOL GAAGAAGGCC UUCGGGUUGU AAAGUACWU VCHOl VCHOZ VCHOJ VCH04 VCHOS VMIM VMAR vcos VHOL LDAM VFIS VLOG LAN0 VAES VGAZ VMET VORD VORI LPEL VAL0 VCAM VCAR VD IA VFLU WAR VNAT VNER WIG WAR VPRO PPHO PANG PLEI PLSH AHAN ACAV AMED ASMA AVER

ALES ALnA ALNI SPUT MCOM WAG PSAE PSFL PSNA DMAR

tions in two sequences, respectively. A phylogenetic tree Vibrio. The members of the genus Pseudomonas were was constructed by using the neighbor-joining method (27). divided into two clearly diverse branches, and the members Nucleotide sequence accession numbers. The partial 16s of the genus Vibrio occurred in five branches. A total of 30 rRNA sequences reported in this paper have been deposited Vibrio and Listonella strains were examined; 16 Wbrio in the DDBJ (Mishima, Japan), EMBL (Heidelberg, Germa- strains and one Listonella strain constituted a large branch, ny), and GenBank (Mountain View, Calif.) nucleotide se- and 13 strains occurred on branches that were distinct from quence data bases under accession numbers D11169 to the branch that contained the other 17 strains. In particular, D11315 (Table 2). vlibrio marinus was far removed from the other members of the genus Wbrio. The large cluster consisting of 17 strains RESULTS could be further divided into two subgroups. One of these included Vibrio aestuarianus, Vibrio gazogenes, Kbrio Partial sequences consisting of 600 16s rRNA bases, meschnikovii, Wbrio ordalii, Vibrio orientalis, and Lis- covering base positions 290 to 509, 690 to 904, and 1180 to tonella pelagziz; these organisms had similarity values and K (Escherichia coli 1379 numbering) (5), were determined for values ranging from 96.39 to 98.85% and from 1.02 to 3.86, 50 bacterial reference strains. The sequence alignments are respectively. The members of the other subgroup, including shown in Fig. 1. An unrooted phylogenetic tree constructed from the Kvalues is shown in Fig. 2. 11 species belonging to the genus Vibrio, had similarity According to our phylogenetic analysis, 7 of the 10 genera values and K values that ranged from 95.78 to 99.79% and used in this study (the genera Aeromonas, Alteromonas, from 0.22 to 3.81, respectively. When the data for all 17 Deleya , Marinomonas, Photobacteriurn, Plesiomonas, and strains were included, the similarity values and K values Shewanella) represent distinct isolated lines of descent. In were more than 95.32% and less than 5.06, respectively. This contrast, the members of the genus Listonella were divided finding indicates that the 17 strains constitute a fairly homo- among several branches along with species of the genus geneous group. 12 KITA-TSUKAMOTO ET AL. INT. J. SYST.BACTERIOL.

VALG VCAM VCAR VD IA VFLU VHAR VNAT VNER WIG VPAR VPRO

Figure 3 shows the similarity values and K values for the Pseudomonas species were used. Two species, Pseudo- 16s rRNA sequences of the organisms used in this study. monas fluorescens and Pseudomonas aeruginosa, do not High similarity values and low K values were observed require Na+ for growth, while Pseudomonas nautica does. among the species in the separate groups (new genera) of the For the two terrestrial species the similarity and K values genus vibrio. Five strains of K cholerae, including two were 96.15% and 4.02, respectively, indicating a close rela- biotypes and two serotypes, formed one clearly distinguish- tionship. able cluster along with vibrio mimicus; the levels of similar- Plesiomonas shigelloides was far from the other members ity among the strains ranged from 99.06 to 100.00%, and the of the family Vibrionaceae (Fig. 2). The location of this K values ranged from 0.00 to 0.78, indicating that these species was rather close to the locations of family Aeromon- organisms are very closely related. On the other hand, the adaceae and E. coli. distances between the V. cholerae-V. mimicus group and Another outstanding feature of the results of our 16s other vibrio groups were quite large. Three species of the rRNA sequence analysis was that each bacterial genus had genus Photobacterium were members of one branch, which its own specific sequence pattern that was common to the was located near the large compact cluster that included 17 species in that genus. These common sequences which species of the Vibrionaceae. VZbrio jischeri, vibrio logei, characterized each genus are indicated in Fig. 1. For exam- and Listonella anguillara also were on one branch near the ple, all three species of the genus Photobacterium had big cluster but were quite distant from each other. The common sequences which were not identical to base se- similarity value and K value ranges for Listonella species quences of other genera; they were U, G-A- -C, U, C, G, were much greater than the ranges for most genera, showing CA, and A at positions 307, 379 to 384,443,455,459,473 to that this group of organisms is heterogeneous. The similarity 474, and 491, respectively. The genus Aeromonas had a values and K values for the genus Pseudomonas also exhib- specific sequence pattern characterized by G-A- -C, G, C, ited a great deal of variability, ranging from 88.48 to 96.15% A------A--U, C, and UG------U at positions 379 to 384, and from 4.02 to 12.95, respectively. In this study, three 408, 434, 445 to 456, 463, and 478 to 489, respectively. VOL.43, 1993 PHYLOGENETIC RELATIONSHIPS OF MARINE BACTERIA 13

VAES VGAZ VMET VORD VORI LPEL VALG .- VCAM VCAR VDIA VFLU VHAR 1 VNAT .- VNER ,- VNLG .- WAR :- VPRO :- PPHO PANG PLEI PLSH AHAN ACAV AMED ASMA AVER ALES ALHA ALNI SPVT ----

MCOM --a_- MVAG --- #---A- CC-U-G-G-A ----- PSAE ------PSFL ----A- - PSNA _---A--

FIG. 1-Continued.

Likewise, the separate groups (genera) of the genus Vibrio the basis of the results of a 5s rRNA sequence analysis (22), and the genera Alteromonas, Marinomonas, and Pseudo- and in 1983 the genus Marinomonas was distinguished from monas had their own characteristic sequence patterns. If the the genus Alteromonas as a result of data from a nucleic acid vibrio groups were not separated, the common sequence analysis (35). The genus-specific common sequence patterns patterns were less clear; in particular, the group had no (Fig. l), as well as the similarity and K values (Fig. 3), common sequence at base positions 290 to 509, where all of support the separation of these three genera. the other genera had one or more common sequences. This The genus Aeromonas was once placed in the family result confirms the validity of separating the members of the Vibrionaceae. In 1986, Colwell et al. proposed a newly genus Vibrio into several new genera. Also, our results created family Aeromonadaceae on the basis of the results suggest that the region of 16s rRNA from base positions 290 of 5s rRNA sequencing; a member of the genus Aeromonas to 509 is evolutionarily more variable than the other two was the type species of this family (8). Figure 2 shows that regions which we studied. species belonging to the genus Aeromonas form a branch that is clearly distinct from the other species of family DISCUSSION Vibrionaceae. This result supports the proposal of Colwell et a1 . The phylogenetic relationships which we determined on On the other hand, our phylogenetic data also suggest that the basis of a 16s rRNA sequence analysis of 50 reference the current taxonomic system should be revised in many strains conform to the current taxonomy of marine bacteria respects. Our 16s rRNA sequence analysis revealed the in many respects. For example, the genera Marinomonas, clear phylogenetic divergence of the marine bacteria from Alteromonas, and Shewanella are clearly distinguished from their terrestrial counterparts. Three Pseudomonas species each other. The members of these three genera were placed were used in this study, including one marine species,

together in the genus Alteromonas until 1983. The genus Pseudomonas nautica , which requires Na + for growth, Shewanella was separated from the genus Alteromonas on and two terrestrial species, Pseudomonas fluorescens and 14 KITA-TSUKAMOTO ET AL. INT. J. SYST.BACTERIOL.

ECOL VCHOl VCHOZ VCHO3 VCH04 VCHOS VMIM VMAR vcos VHOL LDAU WIS VLOG LANG

LPEL VALG VCAM VCAR VDIA VFLU VHAR VNAT VNER WIG WAR VPRO PPHO PAN0 PLEI PLSH AHAN ACAV AMED ASMA AVER ALES ALHA ALNI SPUT

Pseudomonas aeruginosa, which do not require Na+. Ac- rRNA sequence (21). In 1990, Steven proposed that K cording to our phylogenetic analysis, Pseudomonas nautica marinus should be renamed Moritella marinus on the basis is on a branch that is distinct from the branch containing the of the results of molecular genetic methods, including 5s two terrestrial Pseudomonas species. Deleya marina, which rRNA sequencing and DNA-DNA hybridization (32). Our was classified previously as Pseudomonas marina (24), results based on 16s rRNA sequencing data support this requires Na+ for growth, like Pseudomonas nautica. This proposal. species was also on a branch that was distinct from the The second group comprises V. cholerae and V mimicus. terrestrial Pseudomonas species branch. Although addi- Whereas most species of the genus Vibrio require Na+, tional study is needed, our results suggest that the halophilic, having optimal Na+ concentrations of 2 to 3%, V cholerae, marine pseudomonads should be separated from the genus the type species of the genus Ebrio, is able to grow without Pseudomonas and may be placed in newly created groups. Na+ in the medium (2), although growth is stimulated by a The three species of the genus Pseudomonas had common, small amount of Na+. In addition to the salt requirement, V. genus-specific sequence patterns. However, the two non- cholerae has many characteristics that distinguish it from halophilic strains shared far more and clearer common other vibrio species. In this study, various strains of K sequence patterns than all three species shared. cholerae formed a tight cluster and were clearly separated Subdivision of the family Vibrionaceae. A comparison of from other species of the genus Ebriu. K mimicus was the phylogenetic relationships among 34 strains of the family proposed as an atypical strain of V. cholerae; these two taxa Ebrionaceae suggested that the family should be separated are very closely related both phenotypically and genotypi- into at least seven groups, which correspond to genera or cally (DNA homology) (9). families. The third group is composed of Ebrio costicola, vibrio The first distinct group is V. marinus. MacDonell and hollzkae, and Listonella damsela . The members of this group Colwell have also suggested that K marinus is significantly are different from members of other groups and do not form different from other vibrio species on the basis of its 5s a tight cluster. The similarity values and K values for these VOL.43, 1993 PHYLOGENETIC RELATIONSHIPS OF MARINE BACTERIA 15

1300 1310 1320 1330 1340 1370 ECOL GUCCGGAUUG GAGUCUGCAA CUCGACUCCA UGAAGUCGGA AUCGCUAGUA GGUGAAUACG

VMIM VMAR vcos VHOL LDAM WIS VLOG LANG VAES VGAZ WET VORD VORI LPEL VALG VCAM VCAR MIA WLU VHAR VNAT WER WIG VPAR VPRO PPHO PANG PLEI PLSH AHAN ACAV AMED ASKA AVER ALES ALHA ALNI SPUT MCOM WAG PSAE PSFL PSNA DMAR

species range from 92.95 to 95.08% and from 5.37 to 7.40, the suggestion of MacDonell and Colwell (22) that the respectively. The similarity values are much lower than the genus Plesiomonas should be separated from the family values for species in other branches. We suggest that this Vibrionaceae. It seems that evolutionarily, Plesiomonas group may be divided into two or three genera on the basis shigelloides is closely related to the family Aeromonadaceae of phylogenetic relationships. Dorsch et al. also suggested and the family . that K hollisae should be elevated to genus rank (11). The sixth group comprises three species of the genus The fourth group, which also does not form a compact Photobacterium. The genus Vibrio and the genus Photobac- cluster, consists of three species, V Jischeri, V: logei, and L. terium have many common characteristics, but they can be anguillara. This group should be divided into different separated on the basis of accumulation of poly-P-hydroxy- genera. Dorsch et al. suggested that L. anguillara should be butyrate and utilization of D-mannitol by Photobacterium separated from the other vibrio species (11). Two of these species. These two genera are closely related, and the organisms, V: fischeri and I? logei, have the ability to distinction between them based on phenotypic characteris- luminesce. The other luminescent vibrio species, Vibrio tics has often been a source of confusion. According to our haweyi and V. orientalis, fall into another branch, suggest- data, a fairly clear separation of the two genera occurs on the ing that luminescence is not a key criterion for distinguishing phylogenetic tree (Fig. 2) and in similarity and distance vibrio groups. matrices (Fig. 3). The fifth group consists of the genus Plesiomonas. Like The seventh group includes the remaining species of the the species of the family Aeromonadaceae, the genus Plesi- family Vibrionaceae. Excluding V: cholerae, V mimicus, I/. omonas is placed by itself on a distinct branch. The levels of costicola, K hollisae, L. damsela, V marinus, E fischeri, similarity between the genus Plesiomonas and groups be- K logei, L. anguillara, and Plesiomonas shigelloides, the longing to the family Vibrionaceae are much lower than the other 17 species of the Vibrionaceae form one large cluster. levels of similarity between this genus and members of the This group seems to be the main group of the genus Vibrio. familyAeromonadaceae or even E. coli. Our results support Although the genus Listonella was separated from the genus 16 KITA-TSUKAMOTO ET AL. INT. J. SYST.BACTERIOL.

FIG. 2. Unrooted phylogenetic tree based on 16s rRNA sequences, showing the relationships among the 50 reference strains which we used. For abbreviations of organism names, see Table 1.

vibrio on the basis of 5s rRNA sequences (22), according to Our clustering, including subdivision of the current genus the phylogenetic relationships based on a 16s rRNA se- Vibrio, is in accord with the sequence patterns of the 16s quence analysis the species of the genus Listonella occur on rRNAs. Each separate group on the phylogenetic tree has a three separate branches. A comparison of the common common, characteristic sequence pattern. This is particu- sequence patterns of the genus Vibrio and the genus Lis- larly clear when the grouped and ungrouped members of the tonella showed that species of both genera have almost genus ‘vibrio are compared. Our results indicate that the identical sequence patterns. According to our results, the members of the genus Vibrio should be separated into at genus Listonella is a rather heterogeneous group. Although least five groups at the genus and family levels. From our further study, including a detailed comparison of 5s rRNA observations of levels of interspecies similarity and dis- data, is needed, some species of the genus Listonella may be tances among the genera, we concluded that the similarity combined into genera which include species of the current values and Kvalues which separate different genera are 95 to genus vibrio. 96% and 5.0 to 4.0, respectively. If these values are adopted, Criteria that separate taxa. In this study a phylogenetic the group consisting of V. costicola, V. hollisae, and L. analysis was performed by using 600 16s rRNA nucleotides damsela should be divided into two or three genera. Lower and three universal primers (18) that have been used very similarity values or higher distance values may indicate that frequently. Therefore, the similarity values and K values the species should be separated into different genera, al- should change when they are recalculated with the complete though other characteristics (e.g., phenotypic characteris- nucleotide sequences of the 16s rRNAs. However, by tics) of the species should also be taken into consideration comparing similarity values and K values in this study, we before a final decision is made. arrived at a concept for distinguishing the taxonomic hierar- On the basis of a comparison of the distances among chy on the basis of a 16s rRNA sequence analysis. various families in Fig. 3, the similarity value which distin- Within the seven groups on our phylogenetic tree, the guishes different families is 90 to 91%, and the Kvalue is 9 to similarity values and K values ranged from 92.95 to 99.79% 10. The similarity values and Kvalues among species within and from 0.22 to 7.40, respectively. The lowest similarity the current family Vibrionaceae range from 86.74 to 99.79% value (92.95%) and the highest K value (7.40) were obtained and from 0.22 to 14.71, respectively. This means that the with the species V. costicola, V. hollisae, and L. damsela. species of the Vibrionaceae are too diverse to be included in When the branch containing these three species was ex- a single family. On the basis of our results, three species of cluded, the similarity values among the species of each the fibrionaceae, V. marinus, V. costicola, and Plesiomo- group were more than 95% and the Kvalues were less than nas shigelloides, are distant enough to create new families. 5.00, with one exception. With respect to similarity values and K values at the -BCOL %KO. WK0: %KO: vcIIoa VlIIll%KO:

VllM 88.98 88.58 89.11 86.14 81.31 80.05 89.09 89.06 89.39 vcm 88.76 81.83 67.94 88.40 89.43 VBOLLMll 91.80W.60 91.95 92.46 91.4891.13 91.11 91.03 91.8493.49 93.21 93.26 s2.n ~3.05 wis 93.03 93.33 90.- 91.16 91.50 91.w 92.19 nooull0 89.4391.13 91.54 91.61 90.8791.84 W.64 91.21 91.8993.19 93.37 93.18 91.19 91.81 93.61 93.59 VASS 91.46 91.03 91.10 91.12 93.81 89.16 90.40 94.11 93.11 94.11 95.84 VOAZ 91.37 92.49 91.18 91.19 91.44 91.91 91.99 88.31 W.25 91.99 91.84 93.49 94.33 m 91.30 OJ.~93.56 92.05 91.11 92.03 93.13 8o.u 9o.ia 94.22 oz.9a 94.60 96.41 VORD 91.64 93.53 93.72 91.08 91.96 92.48 93.15 90.07 90.25 93.18 92.41 94.W 94.15 VORILPBL 91.0091.71 93.31 93.72 91.0891.83 9i.m 91.46 93.1593.33 69.25 91.26 94.58 03.31 94.96 05.18 93.34 93.31 91.46 92.00 88.81 Bl.51 94.18 93.01 94.56 05.08

VAL0 91.11 92.13 93.35 88.60 90.78 94.13 93.11 94.98 95.85 96.10 97.89 95.86 VW 91.10 91.62 93.24 88.62 90.40 94.91 94.73 94.97 95.84 90.59 91.11 95.50 VCAR 91.13 91.61 93.26 88.80 90.71 94.94 94.76 94.13 98.23 95.93 91.38 9s.m MIA 91.89 91.40 93.84 89.58 91.41 94.57 03.09 94.13 95.83 96.40 91.20 95.31 WLU 91.74 91.16 93.35 88.34 91.21 95.46 95.30 95.04 96.79 98.35 91.83 W.13 vlwl 91.53 91.03 93.25 88.58 90.76 94.93 94.15 94.23 96.23 95.95 91.38 95.50 mrS 91.12 91.03 93.70 89.58 90.59 94.40 93.47 94.06 94.91 98.W 97.87 96.58 WBR 91.15 92.10 93.40 89.05 91.30 95.18 94.13 95.06 96.23 96.35 91.98 95.91 Xi10 91.12 91.45 92.4292.94 90.07 91.26 94.14 93.47 94.05 94.91 95.46 96.39 96.40 W A R WROWAR 90.75 91.16 87.08 90.51 94.87 94.44 95.04 96.11 96.09 91.95 95.88 91.64 93.20 93.55 91.67 91.31 92.03 93.13 89.40 91.80 95.17 94.19 96.59 96.03 96.7E

PPHO 90.76 93.01 93.17 91.84 91.91 92.21 93.19 89.20 91.40 92.62 93.84 94.4094.59 95.8595.48 94.16 94.74 94.41 94.98 94.65 94.49 93.93 PAN0PLBI 91.00 94.20 94.35 91.61 91.55 93.05 94.21 90.54 90.92 92.82 94.01 94.13 95.41 95.45 L96.01 95.68 95.82 95.50 95.34 94.80 91.30 93.12 93.84 91.41 91.91 93.21 03.52 88.83 90.80 92.94 95.15 B4.12 94.39 94.19 95.47 94.68 95.05 94.88 95.58 95.21 PLSH 92.31 69.65 89.81 88.99 89.21 89.02 89.43 88.09 89.08 91.05 91.64 91.20 90.11 00.97 00.80 00.45 91.12 91.01 91.01 89.81 W.83 91.16 W.51 00.51 91.50 W.13 91.01 91.32 91.53 90.59 91.22 91.19 90.11 90.33 ARAH 90.67 88.87 88.85 87.88 81.50 81.85 88.73 88.95 87.95 89.71 89.81 91.35 80.19 ACAV AllBD ::::: ::::: ::I:: ::::: :;::: ::::: :::% ::::: ::::: AWAVER ::::: ::::: ::::: ::::: 88.19 85.55 85.33 85.38 84.93 85.35 85.36 88.74 85.29 87.39 88.98 89.19 89.86 90.81 89.12 89.35 81.50 87.16 81.45 89.23 89.94 81.90 89.81 90.56 91.58 91.13

ALES 87.57 87.45 67.88 81.05 87.29 88.91 81.88 89.01 81.90 88.43 81.08 118.05 88.55 ALNIALKA 81.52 87.27 111.84 85.90 81.06 88.97 87.83 8n.w 111.33 81.03 ~8.m07.55 81.87 87.24 87.00 87.51 85.71 88.87 86.17 87.62 89.23 81.67 87.83 88.76 87.03 87.29 SPW 88.91 81.66 87.94 87.15 81.80 87.48 88.20 89.15 87.86 88.14 89.13 87.00 85.63 86.81 87.43 87.41 87.10 87.88 87.32 86.81 81.19 87.08 87.31 87.81 87.88 87.30 87.99 87.52 87.85 88.65 18.16 87.M 88.16 88.14 88.37 88.38 81.10 87.88 86.49 87.48 81.28 88.98 88.79 81.40 87.91 87.08 87.29 87.29 88.16 86.49 61.98 88.14 87.74 07.61 88.93 88.18 88.93 89.83 89.49 90.23 89.74 89.49 89.12 89.51 88.11 90.00 88.51 88.74 80.31 85.81 84.52 85.17 84.27 82.05 64.99 88.28 88.26 87.57 84.82 nconMA0 86.73 87.00 88.85 89.50 89.08 69.47 89.33 90.07 88.31 88.51 87.06 86.15 88.51 86.83 87.58 85.48 87.73 88.81 67.09 86.10 87.33 87.82 89.18 89.01 88.89 89.01 89.18 89.87 88.44 88.61 88.71 89.83 88.61 68.69 89.00 89.05 89.88 87.88 89.34 81.80 88.65 69.90 64.85 86.00 85.95 05.02 85.33 85.95 88.21 86.35 88.11 84.42

PMB 85.58 88.51 86.80 85.61 85.30 05.66 86.m 8s.a 82.88 84.76 8s.il 83.27 84.53 84.41 85.14 84.53 85.39 85.11 85.23 84.88 83.44 8s.a~88.~1 85 14 85 11 86.4~88.45 06.01 85.83 83.18 86.33 85.58 86.66 85.84 15 06 85.46 8s 48 85.40 83.56 88.36 as.53 84.01 84.07 85.16 PSPLPSNA 83.89 85.60 86.06 84.85 84.47 84.88 85.81 83.95 82.38 83.74 84.21 81.87 83.40 82.98 84.59 83.45 84.43 64.59 83.63 83.41 84.41 85.26 85.87 84149 65:02 85.51 85.85 85.28 84.89 82.55 85.53 84.80 85.21 64.47 83:50 84.49 84115 84.78 61.66 85.28 85.13 83.42 83.95 84.81 84.47 84.21 84.17 83.88 83.81 84.24 83.92 II3.52 84.37 83.80 84.91 83.01 85.24 63.86 84.94 81.21 84.57 84.81 84.81 84.58 64.61 85.8E 85.40 85.34 86.31 85.16 85.32 85.14 84.23 84.21 85.50 85.47 85.13 18.66 84.73 86.42 18.09 87.57 86.85 87.17 86.14 85.90 85.15 85.18 DluR 83.99 82.14 82.07 82.03 82.01 82.51 82.78 85.19 84.10 84.10 84.51 85.40 82.66 63.79 82.63 83.95 82.88 83.8S 83.87 82.81 83.64 84.18 82.76 83.30 83.85 13.06 63.79 13.93 13.79 81.21 84.49 83.13 84.12 13.04 D5.51 83.94 84.30 84.43 83.26 84.63 83.40 82.32 81 ,45 85.61 83.41 03.06 86.16 85.33 87.41 WIN SPW WA0 PsM pspL psNA DMR I - ECOL VCHOl VCKOZ VCR03 Vm04 %KO5 VXIll VlUR VCOB WOL LDM WIS VIP0 LAN0 VABS VW Vl5I VORD VORI LPRL VALO VCM VCAR MIA WLU WAT WER WIO WAR VPRO PPUO PANO PWI PLSH ABAN ACAV MDASUA AVER ALES UKA ALNI nCm -ECOL VCKOl 10.06 vcno: vcio: VCKOi WIIIVCKO!

MlhR 12.07 12.48 12.10 14.71 13.76 13.28 11.82

vcos 12.31 12.26 11.56 13.33 13.13 12.85 11.61 13.117 WOLLUAn 8.94 8.91 8.31 9.11 8.81 8.75 1.81 10.29 1.53 1.26 8.45 7.76 1.11 7.17 WIS 10.41 1.51 1.35 1.85 1.97 1.86 1.75 nw 11.51 9.13 6.96 9.69 9.16 9.39 8.81 LAN0 6.97 1.49 1.61 0.13 9.60 9.16 1.85 VAES VMZ V7a.T VORD VORlLPEL

7.61 7.15 8.33 VALO 9.53 (1.55 0.21 1.00 11.91 9.91 5.10 6.91 5.19 4.15 4.31 1.43 4.03 3.01 3.55 1.11 3.99 VCM 9.46 6.98 1.26 8.11 8.84 6.75 7.16 12.51 10.58 5.83 5.41 5.51 4.50 3.79 3.00 4.92 3.61 4.49 3.46 4.40 vcm 9.10 7.41 7.15 0.61 9.12 8.19 1.03 12.48 9.89 5.07 5.53 6.12 4.00 4.30 1.56 4.61 3.06 4.04 3.03 3.97 MIA 9.81 7.27 6.65 8.30 8.38 8.14 e.8~11.10 9.20 SAT 1.m 6.14 4.13 3.99 1.0s 1.08 3.10 4.62 3.59 4.55 WLU 9.30 1.81 1.51 8.5s 8.81 8.46 1.26 11.44 9.49 4.94 4.96 5.13 3.24 4.03 2.30 4.15 2.90 3.E3 3.06 1.34 VlUR 8.91 1.58 7.29 8.63 8.93 8.58 1.11 12.44 10.05 5.45 5.10 6.08 4.00 4.41 2.73 4.81 3.38 4.36 2.85 3.94 WAT 9.49 1.21 6.09 8.46 8.w 8.65 6.78 11.31 10.34 6.09 1.14 6.53 5.50 4,34 2.13 3.05 2.90 2.18 1.11 4.18 VNLR 8.m 7.41 1.25 8.37 8.19 8.40 1.19 11.w 0.43 5.21 6.24 5.40 4.05 4.05 1.13 4.16 1.04 3.31 2.44 3.74 vN10 7.- 7.27 1.01 8.95 6.81 8.11 7.12 10.55 9.35 6.08 7.02 5.71 4.49 4.81 3.30 3.28 3.60 3.38 3.40 3.ES W A R WROWAR 1O.W 8.50 6.31 9.38 9.14 9.34 1.91 13.75 10.37 5.01 5.88 4.80 4.08 4.18 2.15 4.19 2.79 3.86 3.01 4.40 9.11 1.46 7.04 9.01 0.07 8.71 6.76 11.35 9.19 5.11 6.43 5.74 4.46 3.78 1.69 3.07 2.54 3.38 1.11 3.03 1 PPKO 10.16 1.66 7.44 8.78 8.52 0.41 7.38 11.81 9.25 8.11 6.87 6.13 4.88 3.68 5.66 5.~3.11 5.51 5.14 8.3s 5.46 6.42 5.95 5.44 5.96 6.11 5.80 5.19 5.48 5.91 5. PLEIPAN0 B.90 6.31 6.09 7.86 7.83 1.54 6.14 10.00 9.41 1.78 6.22 6.13 4.41 4.118 4.03 4.M 4.13 4.59 4.84 5.25 4.31 4.74 4.51 4.16 4.30 4.06 4.51 4.12 4.17 3.95 4. 9.31 6.81 6.M 8.10 7.36 1.30 6.80 11.01 9.94 1.74 5.13 8.48 6.06 8.54 4.64 5.73 4.92 5.24 4.73 4.95 4.91 5.40 5.12 5.37 5.25 5.10 5.16 4.80 4.97 4.88 4. PLSH 1.18 11.13 10.85 12.12 11.18 12.05 11.31 12.18 11.71 9.40 8.83 9.38 10.70 9.43 9.65 1O.W 8.38 9.24 9.14 10.81 9.24 e.18 9.95 9.17 9.38 9.68 9.20 9.12 8.67 10.04 9.21 9.43 9.61 10.21

AHAN ACAV N5D ASMAAVER

ALES ALKAALNI

SPUI 14.68 14.05 13.68 14.43 13.54 13.98 13.29 11.79 13.85 13.25 12.43 14.79 16.58 11.70 14.- 14.33 14.31 13.19 14.42 15.21 14.3. 14.10 14.44 14.01 13.43 14.32 13.E6 14.28 13.49 14.93 13.82 13.66 13.39 13.33 15.38 13.82 14.55 14.17 15.59 14.41 14.03 14.27 14.58 ncon 14.73 14.09 13.33 14.19 13.93 14.18 12.10 15.35 13.29 13.05 13.55 14.79 12.65 14.47 11.55 12.11 11.19 12.97 12.01 11.18 11.37 11.15 11.52 10 53 11 44 11.50 11 08 10.97 11.20 13.10 10.91 12.73 11.85 11.83 15.39 11.16 16.30 18.30 19.95 11.11 13.02 13.02 13.95 17.22 m- 15.26 13.10 14.35 13.44 14.82 14.75 13.80 16.0'7 13.61 14.71 14.40 15.89 14.08 14.36 11.95 11.06 11.03 11.86 11.91 11.00 11.51 12.49 11.29 11:29 12:51 11.48 12:lO 11.88 11.30 13.35 11.41 12.16 11.73 10.18 16.65 15.92 15.92 17.21 lE.43 15.84 13.01 15.33 15.72 17.76

PMB 16.02 15.14 14.13 15.90 18.10 16.08 14.59 15.69 19.6s 11.44 16.98 1s.ze ~~.li18.~0 16.21 17.15 16.11 15.~1 16.81 11.01 m.11 16.i~ 15.10 15.01 1s.1~ 15.11 15.42 15.85 15.44 18.31 15.44 18.24 14.86 15.85 16.20 18.36 i6.az 16.77 16.91 15.33 16.~018.1~ 18.~8 18.69 P 11F L PSUAP11FL 18.03 16.00 15.33 16.54 18.79 16.65 15.41 i8.io 19.9~ i8.w 18.14 11.28 18.82 19.90 i?.a3 19.04 11.42 18.99 ia.61 i8.n 11.21 10.96 16.14 11.30 16.~9 16.~0 16.30 18.74 16.49 19.91 16.33 17.32 i6.or 11.24 18.33 11.45 17.53 17.38 10.55 18.~8 16.51 18.84 18.23 18.78 17.94 18.44 16.28 18.76 13.65 18.43 18.76 19.42 18.08 19.21 11.13 11.48 16.17 19.26 11.44 18.39 11.63 17.18 17.71 11.91 11.31 16.51 16.90 18.81 11.23 17.14 18.82 11.08 11.75 18.01 IE.11 16.81 18.50 15.09 17.44 15.64 15.84 14.11 14.95 14.62 14.91 16.13 11.22 17.00 OW 18.36 20.56 10.06 11.10 20.89 10.58 20.21 16.95 18.16 18.60 18.06 11.11 20.W 18.83 10.36 18.51 19.91 1B.61 18.77 20.30 18.89 18.18 20.20 1g.m 18.31 19.31 18.10 18.94 18.82 20.11 18.08 19.14 18.18 19.96 16.42 18.74 10.08 18.31 19.31 18.03 18.99 20.88 20.39 18.61 19.17 19.81 15.54 18.43 14.26 -zm11 ECOL vcnoi vcnmVCKOJ VCROI VCAOS wm ww vcoa YROL LUM Ypis nw wo VMS mz ~7a.~vom vow LpEL vmo VCM Vera nlA vpLu mu vNAr WBR WIO wm WRO PPRO PANO PLEI PLSR .UIM ACAV -0 rn AV~R ALES urn ALNI SPW won WAO pwa PSPL PSNA 1 -FIG. 3. Matrices of similarity values (upper part) and K values (lower part) for partial 16s rRNA sequences of 50 reference strains belongingto different groups, as determined J by using an unrooted phylogenetictree. For abbreviations of organism names, see Table 1. The values enclosed in boxes are values for members of the same branch (Fig. 2). 8 18 KITA-TSUKAMOTO ET AL. INT. J. SYST.BACTERIOL. species level, multiple strains of only one species, K chol- 9. Davis, B. R., G. R. Fanning, J. M. Madden, A. G. Steigerwalt, wae, were compared in this study. The similarity values and H. B. Bradford, Jr., H. L. Smith, Jr., and D. J. Brenner. 1981. Kvalues for these organisms were 99.23 to 100.00% and 0.00 Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio iio 0.60, respectively. Therefore, we concluded that the 16s mimicus. J. Clin. Microbiol. 14:631-639. rRNA similarity value and K value that define a species are 10. Deming, J. W., L. K. Somers, W. L. Straube, D. G. Swartz, and 99.2% and 0.6, respectively. However, these values are valid M. T. MacDonell. 1988. Isolation of an obligatory barophilic only in this present study, since the values were obtained bacterium and description of a new genus, Colwellia gen. nov. from a limited number of 16s rRNA nucleotides. Also, the Syst. Appl. Microbiol. 10152-160. criteria for distinguishing species may vary in different 11. Dorsch, M., D. Lane, and E. Stackebrandt. 1992. Towards a taxonomic groups. Kawasaki (16a) has set a similarity value phylogeny of the genus Vibrio based on 16s rRNA sequences. of 99.3% as the criterion for separating species on the basis Int. J. Syst. Bacteriol. 42:58-63. of the results of a 16s rRNA sequence analysis for meth- 12. Egidius, E., R. Wiik, K. Andersen, K. A. Hoff, and B. Hjeltnes. 1986. Vibrio salmonicida sp. nov., a new fish pathogen. Int. J. anogens which belong to the archaebacteria. For K mim- Syst. Bacteriol. 36518-520. icus, which was a member of a compact cluster along with 13. Grimes, D. J., J. Stemmler, H. Hada, E. B. May, D. Maneval, five strains of I/. cholerae, the similarity values and Kvalues F. M. Hetrick, R. T. Jones, M. Stoskopf, and R. R. Colwell. 1984. in comparisons with the K cholerae strains ranged from Wbrio species associated with mortality of sharks held in 99.06 to 99.60% and 0.40 to 0.78, respectively. According to captivity. Microb. Ecol. 10:271-282. DNA-DNA relatedness data, K mimicus is not related to K 14. Guerinot, M. L., P. A. West, J. V. Lee, and R. R. Colwell. 1982. cholerae at the species level (9). More data are needed to Wbrio diazotrophicus sp. nov., a marine nitrogen-fixing bacte- determine more accurate values for species differentiation. rium. Int. J. Syst. Bacteriol. 32:350-357. The results of our 16s rRNA sequence analysis showed 15. Hada, H. S., P. A. West, J. V. Stemmler, and R. R. Colwell. 1984. sp. nov., a pathogen of bivalve molusks. that molecular genetic data are very valuable for establishing Int. J. Syst. Bacteriol. 34:1-4. clear-cut definitions of various levels of bacterial taxa. 16. Hickman, F. M., J. J. Farmer 111, D, G. Hollis, G. R. Fanning, However, more detailed studies will be needed to determine A. G. Steigerwalt, R. E. Weaver, and D. J. Brenner. 1982. the exact similarity values or K values which distinguish Identification of Wbrio hollisae sp. nov. from patients with these taxa and to define species, subspecies, and biotypes diarrhea. J. Clin. Microbiol. 15395401. that are very closely related phenetically and phylogeneti- 16a.Kawasaki, H. Personal communication. cally. 17. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120. ACKNOWLEDGMENTS 18. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and We thank Y. Oyaizu-Masuchi for technical advice concerning N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA sequencing of 16s rRNA. sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. This investigation was supported by grants from the Ministry of USA 82:6955-6959. Education of Japan and the Ministry of Agriculture, Forestry and 19. Lawson, P. A., S. E. Gharbia, H. N. Shah, D. R. Clark, and Fisheries of Japan. M. D. Collins. 1991. Intrageneric relationships of members of the genus Fusobacterium as determined by reverse tran- REFERENCES scriptase sequencing of small-subunit rRNA. Int. J. Syst. Bac- 1. Baumann, L., P. Baumann, M. Mendel, and R. D. Allen. 1972. teriol. 41:347-354. Taxonomy of aerobic marine eubacteria. J. Bacteriol. 110402- 20. Love, M., D. Teebken-Fisher, J. E. Hose, J. J. Farmer 111, F. W. 429. Hickman, and G. R. Fanning. 1981. Vibrio damsela, a marine 2. Baumann, P., and R. H. W. Schubert. 1984. Family 11. 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