JOURNAL OF BACTERIOLOGY, Dec. 1969, p. 1248-1255Copyright 0 1969 American Society for Microbiology

Vol. 100, No. 3Printed in U.S.A.

Characterization and Relatedness of Marine VibriosPathogenic to Fish: Deoxyribonucleic Acid

Homology and Base CompositionE. D. KIEHN1 AND R. E. PACHA2

Department ofMicrobiology, University of Washington, Seattle, Washington 98105, andDepartment of Microbiology, Oregon State University, Corvallis, Oregon 97331

Received for publication 23 July 1969

Deoxyribonucleic acid (DNA) isolated from several pathogenic marine vibrioswas utilized in studies of base composition and nucleotide sequence relationships.Patterns of relatedness inferred from DNA criteria were found to correlate closelywith those inferred from morphological, physiological, and serological analysis ofthe same organisms. The utility of the phenotypic and genotypic approaches to thedetermination of relatedness is discussed.

In recent years, a great deal of attention hasbeen directed to the problem of microbial classi-fication. The establishment of a taxonomy basedon natural, phylogenetic relationships is impededby the lack of evidence regarding evolutionaryrelatedness (2). Extensive structural differencesand an abundance of developmental and paleon-tological evidence have enabled the classificationof higher plants and animals to proceed withrelative ease. The absence of such informationconcerning microorganisms has resulted inessentially artificial, arbitrary taxonomic systemsfor their classification.The accepted criteria for classification of

microorganisms are mostly morphological, physi-ological, biochemical, and serological. Thesephenotypic criteria represent the combined ex-pression of many genetic loci, and it would beexpected that the greater the number of charac-ters considered, the closer the approximation toknowledge of genetic relatedness would be. Thisis the working hypothesis behind numericaltaxonomy, but it involves possible weaknesses.Account is not taken of the origins and rate ofdevelopment of phenotypic characters. In addi-tion, weighting characters either equally orunequally is not justified by knowledge; yet,certainly characters do differ in their evolutionarysignificance (19, 20).Another difficulty with phenotypic analysis is

that, although two organisms may share the same1 Present address: Salt Institute for Biological Studies, La Jolla,

Calif.2 Present address: Department of Biological Sciences, Central

Washington State College, Ellensburg, Wash.

character, suggesting relatedness, the enzymepathways responsible may not be the same.Indeed, enzymes catalyzing the same reactionsmay be structurally quite different in differentorganisms. Thus, the occurrence of evolutionaryconvergence could mistakenly be interpreted asevidence of actual genetic homology.

Recently, however, new techniques have beendeveloped which enable taxonomic studies to bemade at the molecular level (10, 14). It is nowpossible to determine genetic homology directlyvia physicochemical studies of the deoxyribo-nucleic acid (DNA) molecule itself, in which thetotal genetic potential of an organism is stored.By use of DNA-agar (10, 16) and other methods(5, 8, 14), direct analysis of the degree of commonpolynucleotide sequences in nucleic acids fromdifferent sources is made by studying the forma-tion of molecular hybrids.The organisms used in this study were strains

of marine bacteria considered to be vibrioswhich had been isolated during the course ofepizootics. These pathogens were isolated fromfish in the waters of the Pacific Northwest and ofEurope and are described in detail in the preced-ing paper (17). Representative pseudomonadswere also included, as there is some confusion inseparation of vibrios from the several groups ofpseudomonads owing to inadequate criteria ofdifferentiation.The vibrios and pseudomonads share many

morphological, ecological, and physiologicalcharacteristics, and differentiation is usuallybased on vibrios having curved cells, havingfrequent fermentative potential, and lacking

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pigments. However, the criterion of cell curvatureis extremely variable and may occur in bothgroups, often being dependent on culture ageand growth medium. In addition, some or-ganisms classified as vibrios are not fermentative,and many pseudomonads do not possess dis-tinctive pigments. Definition of the groups (3)also involves other characteristics, includingresponse to vibriostatic agents (18).The experiments described below illustrate the

usefulness of DNA duplex formation and basecomposition studies in the classification anddetermination of relatedness of bacteria. Corre-lations are made with physiological, cultural,and serological analyses of the same organisms(17). The results emphasize the strong compati-bility of approaches based on genotypic andphenotypic characteristics.

MATERIALS AND METHODS

Strains and media. Vibrio strains were maintainedin semisolid Brain Heart Infusion deeps (0.05%Difco Brain Heart Infusion, 2% yeast extract, 0.4%agar, 80% seawater, pH 7.7). Pseudomonad strainswere carried on 1% Fleischmann's yeast extract-I%glucose slants.Zymomonas mobilis was grown in a medium

containing 0.5% Difco yeast extract, 0.5% tryptone,1% glucose (pH 7.3) in large carboys without aerationat 30 C. All other organisms were grown in carboysof Difco Brain Heart Infusion (37 g/liter)-1% NaClmedium (pH 7.3), and were incubated with aerationat room temperature.

For the isolation of radiolabeled DNA, strainswere grown in 200-ml cultures of Brain Heart Infusion(9 g/liter)-1% NaCl (pH 7.3) with 20 jAc each of54C-adenine and '4C-uracil (5 mc/mmole; NewEngland Nuclear Corp., Boston, Mass.).The organisms used in this investigation are listed

in Table 1. The vibrio strains include members ofeach of the three serotypes reported in our previousstudy (17). Two strains of serotype 1 were selectedbecause these organisms wete isolated during differentoutbreaks of disease. Strain 2.1, one of the firstvibrios isolated, was obtained from chinook salmonin seawater, whereas strain 30, though a marinevibrio, was isolated from steelhead trout in freshwater.Two strains of serotype 3 which differed slightly intheir antigenic composition also were studied.DNA isolation. DNA was extracted by use of a

modification of the technique of Marmur (12) de-scribed by Berns et al. (1). Cells were washed andsuspended in water. An equal volume of 4 X SSC[SSC (standard saline-citrate) is 0.15 M NaCl, 0.015 MNaCl, 0.015 M trisodium citrate, pH 7.01 containing54% sucrose and 0.4 sodium lauryl sulfate (SLS) wasadded to the mixture, and the mixture was heatedto 60 to 72 C for 15 min with the addition, if necessary,of additional 25% SLS.

Cell lysates were then incubated at 37 C withPronase for at least 1 hr and dialyzed overnight

TABLE 1. Strains of bacteria

Strain Fish host Source

Northwest U.S. vibrios2.1 .................. Chinook salmon E. J. Ordal30.................... Steelhead trout E. J. Ordal53.................... Chinook salmon E. J. Ordal56.................... Pacific herring E. J. Ordal60.................... Pacific herring E. J. Ordal

European vibrios (V.anguillarum)

NCMB6 (type A) ...... Cod J. M. ShewanV-2911 (typeC).Finnock I. W. SmithV-2916 (type C)........ Finnock I. W. Smith

PseudomonadsAeromonas hydrophila

(A. H. X.).. Rainbow trout E. J. OrdalZymomonas mobilis(ZMC-1) E. J. Ordal

Pseudomonas aeruginosa. J. PateOther bacteria

Cytophaga succinicansstrain 8 E. J. Ordal

against 2 X SSC. The lysates were then phenol-extracted at 60 C and chilled, after which the aqueousphase precipitated with two volumes of ethyl alcohol.DNA was collected on stirring rods and dissolved in0.1 X SSC.

Nucleic acid contents were measured in terms ofultraviolet (UV) absorbance at 260 nm on a BeckmanDU spectrophotometer. Boiled ribonuclease wasadded at a 1:100 (w/w) ratio to nucleic acid. Themixture was incubated at 37 C for at least 45 min, andphenol extraction was performed at room tempera-ture; DNA was precipitated with ethyl alcohol anddissolved in 0.1 X SSC. Storage was at 4 C overCHCl3.DNA duplex formation. Duplex formation experi-

ments were conducted by the method of Hoyer et al.(10). DNA fragments of approximately 0.5 X 101molecular weight were prepared by shearing 1 mg/mlsolutions of DNA at 10,000 psi in a French pressurecell (16).DNA-agar was prepared according to the following

method (16). DNA at a concentration of 200 gg/mlin 0.1 X SSC was denatured by boiling for 5 min,chilled, and concentrated. This solution was boiledfor an additional 2 min and added to an equal volumeof molten 8% Oxoid lonagar in 2 X SSC. Thesecomponents were mixed thoroughly and rapidlysolidified by ice-chilling. The resulting DNA-agarwas granulated by forcing it through a screen twice,and was then washed in three changes of 2 X SSCovernight at 60 C. DNA-agar preparations werestored over CHC13 in tightly sealed bottles at 4 C.The DNA content in the agar preparations was

determined by measuring the UV absorbance at 260nm after dissolving samples in boiling 5 M NaCl04.DNA content was expressed in micrograms of DNAper gram (wet weight).

Competitive DNA-binding experiments were con-ducted by incubating radiolabeled DNA fragments

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with homologous DNA-agar in the presence of"cold" fragments of competitor DNA. Reactionmixtures contained 4 ,ug of labeled DNA, variousamoun:s of unlabeled DNA, 0.1 ml of 20 X SSC,and distilled water to 1 ml total volurre. DNAfragments were denatured by boiling and then wererapidly chilled prior to the preparation of reactionmixtures. The reaction mixtures were again boiled andchilled just before use. Portions (0.5 ml) of reactionmixtures were incubated with 0.5-g amounts ofDNA-agar in silicone coated glass vials at 60 C for atleast 15 hr.

Unreacted DNA was washed out of the DNA-agarsamples with 10 tubes each containing 15 ml of2 X SSC at 60 C. In this procedure, the DNA-agarsample was held in a glass tube by a nylon screen (10)and moved from tube to tube at intervals of 20, 15,and theteafter 10 min in each tube. To elute the DNAwhich had formed stable duplexes at 60 C, the tem-perature was raised to 72 C and the ionic strength waslowered to 0.01 X SSC, conditions strongly favoringstrand separation. This was accomplished in a mannersimilar to the washing, with seven tubes and intervalsof 20, 20, 15, 15, 10, 10, and 10 min in each tube.The radioactivity of each reaction mixture was

measured before incubation, and samples of thewash buffer and eluant were measured, as a check onfull recovery of radioactivity. Radioactivity wasdetermined by cold precipitation in 5% trichloro-acetic acid in the presence of carrier DNA to a totalof 100 jAg of DNA per sample. Precipitated DNA wascollected on membrane filters, dried, and countedin an automatic scintillation spectrometer.

Determination of mole per cent guanine plus cytosine(GC) in DNA. Mole per cent GC in DNA sampleswas determined by measuring the characteristicdenaturation temperatures (Tm) in 0.2 M Na+ andapplying the equation, %GC = (Tm- 69.3) (100)/0.41(13). UV absorbance at 260 nm was monitored on aBeckman DU spectrophotometer with automatictemperature control and absorbance recorder. Themidpoint in the hyperchromic rise of the UV at 260nw was taken as the Tm. Three determinations weremade on each DNA sample, and the standard devia-tion was always less than ±0.4 C. Thus, the averagevalues for per cent GC content are within ±1 mole% GC.

RESULTSTable 2 describes the characteristics of the

DNA-agar and labeled DNA preparations. Itshould be remarked that, although there wasday-to-day variation in the absolute binding ofhomologous, labeled DNA, on a given day it wasconsistently reproducible. Thus, in these studies,duplicate controls were included in every experi-ment. All data from each experiment were cal-culated relative to the controls of the same day.

Levels of binding depression observed incompetition experiments were independent of theparticular batch of DNA-agar used, despitedifferences in the concentration of DNA in the

agar. Binding depression was solely dependenton the sources of the interacting DNA molecules.

Table 3 gives the measured Tm and per cent GCvalues for the organisms studied. It is notablethat all of the vibrios exhibit values in the 41 to46% GC range except for strain 53, which hasthe very high value of 59% GC. On the otherhand, Zymomonas mobilis (Pseudomonas linderi)with 49% GC is notably low for classificationwith the pseudomonads.

Equilibrium centrifugation and chemical baseanalyses of the P. aeruginosa DNA preparation(J. Pate, unpublished data) both indicated 66%GC, which agrees very well with the value asdetermined in this study by thermal denaturationand is the published value for the bacterium (9).

Values for per cent GC have also been pub-lished for A. hydrophila and V. anguillarumNCMB6, and the published values agree with thefindings presented here (9). The value for C.

TABLE 2. Characteristics of labeled DNA andDNA-agar preparations

Specific Range of BindingSpctificy Wet wt binding of ofaciiy(jg of homol- labeled

Source of DNA (counts DNA/gWog DNA toper min of DNA- labelesd pure

of DNA) agar) DMNA agar"

Vibrio strain 567 136 35-45 1.730 ..164 25-30

V. antguillarumNCMB6 ....... 511 250 50-60 1.6

352 45-60

a Labeled DNA (2 ,ug) incubated with 0.5 g ofDNA-agar or agar alone.

TABLE 3. DNA thermal denaturationz temperatureand mole per cent GC

Strain Tm (C) Mole%

VibriosV-2916.86.1 41.060.86.2 41.2V-2911.87.0 43.22.1.87.2 43.730.87.2 43.756.87.9 45.4NCMB6.88.0 45.653.93.4 58.8

PseudomonadsZ. mobilis (ZMC-1).89.2 48.6A. hydrophila (A. H. X.)..... 93.1 58.1P. aeruginosa ................ 96.2 65.7

Other bacteriaC. succinicans ............... 85.0 38.0

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succinicans is within the range observed for otherCytophaga species (9).

Tables 4 and 5 demonstrate DNA interactionsin which heterologous DNA fragments competewith labeled, homologous DNA fragments forbinding sites in agar-fixed, homologous DNA.With vibrio strains NCMB6 and 30, DNA frommost of the vibrios competed strongly. Strains 56and 60 showed little competition, and strain 53,the pseudomonads, and C. succinicans did notcompete at all with the homologous reactions.The ability of heterologous DNA to compete

with the formation of labeled homologous DNAduplexes relative to homologous competitionshould give an indication of the similarity of thetwo types of DNA. Thus, these data suggest thatmost of the vibrios are quite similar, that strains56 and 60 are much less similar, and that strain53, the pseudomonads, and C. succinicans arenot at all related to the reference vibrios.

It is also possible to determine whether thesimilarities two or more organisms show to thereference organism represent a common genetichomology, by adding the DNA from the or-ganisms in question to the same competitionreaction (10). The extent of additive competitionshould reflect the degree of nonoverlapping ofthe genetic homologies. Attempts to discernwhether additive competition existed in mixturesof the DNA competitors used in this study werenot conclusive, but suggest that, in the casesexamined, there were probably no additiveeffects. This implies that common genetic ho-mologies exist among these organisms.The average values given in Table 5 are based

on a minimum of at least three individual experi-ments. Individual experiments were included inTable 4 to suggest the reproducibility of themethod. The present resolution meets well therequirements of this study.

Table 6 represents the same data described inTables 4 and 5 as a percentage depression of thebinding of labeled DNA by unlabeled competitorDNA. In this manner, the relative abilities ofheterologous DNA molecules to bind to thereference DNA-agar is demonstrated. Forexample, strain 2.1 DNA depressed strain 30binding to about the same degree as strain 30DNA itself. This implies a very high degree ofhomology among the polynucleotide sequencesof these two organisms.

If it is assumed that two organisms have thesame complement of DNA, then each shoulddepress the binding reaction of the other to thesame relative degree. Indeed, this is the case here.Strain NCMB6 depressed the reaction of strain30 to a relative value of 78%, and the reciprocalinteraction showed strain 30 depressing the

TABLE 4. Competitive interactions with Vibriostrain 30 labeled DNA for homologous DNA-

agar binding sites

Relative percentage of labeledDNA bounda

Source of 250 pg of com- 500 pg of com-petitor DNA petitor DNA

Trials Avg Trials Avg

Vibrio strains30............ 15,19,18, 17 14,12,11, 11

18, 17, 11, 10,16, 16, 14, 7,14, 19 9

NCMB6 ...... 38, 35, 33 35 31, 29, 26, 3136

2.1 ........... 21,17,19 19 13, 13 13V-2911. 34, 39 37 35, 38 37V-2916. 43,43 43 35,42 3956............ 81 81 81 8160............ 89,92,92 91 99,96 9756 + 60b....... 74,79,84, 82 72,72,98, 83

90 90NCMB6 + 60b 33,31,43 36 30,29,43 3453............ 98 98 104 104

Cytophaga suc-cinticans ...... 107 107 119 119

a Percentages are relative to control reactionmixtures which contained no competitor DNA.

b The indicated amount of DNA is the amounof each competitor DNA in the reaction mixture

TABLE 5. Competitive interactions with V. anguil-larum NCMB6 labeled DNA for homologous

DNA-agar binding sites

Avg relative percentage oflabeled DNA bounda

Source of competitor DNA250 pg of com- 500 pg of com-petitor DNA petitor DNA

Vibrio strainsNCMB6............... 16 10V-2911 ................ 23 21V-2916.22 2330.31 282.1.29 2756.79 7860....... 92 9156 + 60b 8053.111 108

PseudomonadsZMC-1.... 97 104A. H. X.88 100

a Percentages are relative to control reactionmixtures which contained no competitor DNA.

I The amount of DNA indicated is the amountof each competitor DNA in the reaction mixture.

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TABLE 6. Binding depression of labeled DNA tohomologous DNA-agar by unlabeled competitor

DNAa

BindingBind- depression

Source of labeled Source of com- ing relative toDNA and DNA-agar petitor DNA depress- homol-

ion ogousdepression

% %Vibrio strain 30 Vibrio strains

30 89 1002.1 87 98NCMB6 70 78V-2911 63 71V-2916 61 6956 19 2260 3 353 -4 0

C. succinicans -19 0

V. anguillarum Vibrio strainsNCMB6 NCMB6 90 100

30 72 802.1 73 81V-2911 80 88V-2916 77 8556 22 2560 9 1053 -8 0

PseudomonadsZMC-1 -4 0A. H. X. 0 0

a Data are based on the average values of percent labeled DNA bound in competition experi-ments (Tables 4 and 5) at the 500-,ug level of com-petitor DNA. These values were subtracted from100% to reflect the extent of binding depression inthese experiments.

reaction of strain NCMB6 to a relative value of80%. In a direct experiment, without competitorDNA, the binding of labeled DNA from strains30 and NCMB6 to strain NCMB6 DNA-agarwas compared. It was found that the degree towhich strain 30 DNA bound to strain NCMB6DNA-agar was 87% as great as that of homolo-gous strain NCMB6 DNA. Thus, the relativeextent of binding to a heterologous DNA sug-gests the same magnitude of relatedness as canbe inferred from competition experiments.

Figure 1 illustrates graphically the data fromTable 4. Represented are typical curves of com-petition in DNA-binding reactions. Depressionof the binding of labeled DNA to homologousDNA-agar is seen to be greatest when unlabeledcompetitor DNA from the homologous strainwas used, as would be predicted. In each case,500 ,ug of homologous competitorDNA depressedthe binding reaction about 90%.

DISCUSSIONTo draw conclusions from DNA-binding ex-

periments, it is essential to understand the natureof the binding reaction. Recent studies havedemonstrated that the temperature of duplexformation, relative to the natural Tm of theinteracting DNA molecules, is a critical parameterin the formation of specific complementary doublestrands (11, 15). It was determined that the reac-tion products formed at progressively lower tem-peratures contain a rapidly increasing proportionof unstable, nonspecific products. Thus, at Tm25 C duplexes were formed which had essentiallythe same thermal stability as native DNA, but atTm 40 C the predominant products were unstableand did not represent duplexes of homologousnucleotide sequences (11). By varying the reac-tion temperature, there was formed a series ofintermediate products representing various de-grees of partially redundant nucleotide sequences(15).In the determination of sequence homology in

;jg Compet itor DNAFIG. 1. Competitive effect of nonradiolabeled DNA

in the binding of radiolabeled strain 30 DNA to strain30 DNA-agar. The experimental values are the averagevalues listed in Table 4. The sources of the nonradio-labeled DNA are as follows: strain 30 (A), strain 2.1(B), strain NCMB6 (C), strain V-2911 (D), strainV-2916 (E), strain 56 (F), strain 60 (G), strain 53 (H),and C. succinicans (I).

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DNA from different organisms of similar per centGC, the results of low temperature in binding re-actions can suggest considerable homology wherelittle or none exists (11). Theoretically, with in-creasingly stringent reaction conditions, one se-lects against even slight imperfections of comple-mentarity and can approach determinations ofabsolute values of homology. The conditions em-ployed in the present study meet the require-ments necessary to essentially quantitate the de-gree of relatedness of the experimental organisms(11). This is verified by the fact that two DNApreparations from totally unrelated organismswith GC values similar to those of vibrios, themyxobacterium C: succinicans and Z. mobilis, didnot compete at all in the binding reactions ofvibrio strains 2.1 and NCMB6, respectively. Theabsence of nonspecific reaction products is alsosuggested by the very low levels of competitionfrom the DNA of vibrio strains 56 and 60 in thebinding reactions of vibrio strains 2.1 andNCMB6, even though the organisms are probablyall in the same genus, have many common pheno-typical characteristics, and exhibit the same GCcomposition.Two other sources of nonspecific interaction in

the DNA-binding reaction should be discussed.There exists the possibility of retention of labeledDNA by the agar-matrix alone. In the presentstudies it was determined that less than 2% ofinput labeled DNA is agar-bound, a negligibleamount for the purposes of these experiments.

In addition, Fig. 1 demonstrates a nonspecificenhancement of binding with increasing amountsof nonradioactive, heterologous DNA. This isprobably a result of protein or polysaccharide, orboth, in the DNA preparations (8). Whether thepresence of contaminating macromolecules stimu-lated DNA-DNA interactions, or somehow in-creased the retention of unbound DNA in theDNA-agar matrix, if the effect were relativelysmall in comparison with actual specific DNAbinding, the rising slopes seen in the competitioncurves would only occur with relatively heterolo-gous DNA preparations. Figure 1 would seem tobear out this prediction. Thus, the actual potentialsofDNA molecules as competitors could be slightlygreater than indicated.The significance of GC content in DNA to

classification and determination of relatedness isthe following: if organisms are related to a highdegree, their GC contents should be very similar.However, evidence of similar GC content does notimply that organisms are closely related, but onlyallows that the possibility exists. It should also benoted that, although large differences in GC con-tent indicate great overall differences in DNA

molecules, small regions may be homologous, andmay represent genes that evolved at much slowerrates than the bulk of the genome (10, 15). Theseconserved genes could have great significance asto the possible common ancestry of highly di-vergent organisms or groups. For example, it wasfound (6, 7) that in the genus Bacillus, whosevarious members differ in GC by as much as 20%,there was very little overall genetic homologybetween any individual species, but that certaincistrons, such as ribosomal ribonucleic acid(RNA), soluble RNA, and antibiotic resistanceloci, were highly conserved in all species tested. Itmay be noted that these cistrons were not repre-sented in organisms outside of the genus, stronglysupporting the validity of the present taxonomicdefinition of this group. Thus, phenotypic unique-ness was ramified by evidence of a small core ofcommon genes, even though individual specieshave diverged widely from each other.The current problem of classification of the

pseudomonads (3, 4) and vibrios (T. E. Lovelaceand R. R. Colwell, Bacteriol. Proc., p. 18, 1968)exists because of strong similarities, weak andvariable differences, and intermediate organismsthat are usually arbitrarily placed in one or theother "genus." It would indeed be interesting toelucidate the relationships of the intermediateorganisms to the two ends of the spectrum, and tosearch for possible conserved genes betweenthese groups. The possibilities exist that thesegroups may have evolved from a common an-cestor, and that certain genetic continuities mayhave been retained between them.On the sole basis of the serological data pro-

vided in the preceding paper (17), the followingrelationships seem to exist among the vibriosstudied. Members of the Northwest salmonidvibrios (strains 2.1, 4.3, 8, 14, 16, 17, 30, 78, 83,and 61-1) are indistinguishable, are fairly closelyrelated to the European vibrios (strains NCMB6,V-2911, and V-2916), and are more distantly re-lated to the Pacific herring vibrios (strains 56 and60). The European vibrios also represent a homo-geneous group, are related to the Northwest sal-monid vibrios, and are more distantly related tothe Pacific herring vibrios. The Pacific herringvibrios, while distantly related, have an antigen(s)in common with both the Northwest and Euro-pean vibrios. Strain 53 has an antigen in commonwith strain 2.1 but appears very unrelated. Itexhibits no relationship with the European vibrios.

Consideration of the cultural and physiologicaldata given in the preceding paper (17) yields aninterpretation similar to that derived from analysisof antigenic characteristics. With regard to 51cultural and physiological properties, the marine

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vibrios in this study are relatively homogeneous,and all are facultative fermenters of carbohydrates,the sole exception being strain 53. Because of thishomogeneity it is relatively straightforward toassign a cultural-physiological archtype, i.e., ineach of the 51 tests a clear majority of the or-ganisms responded in the same way and this re-sponse will be regarded as typical. By this cri-terion, Northwest salmonid vibrio strains 30 and14 and European vibrio (V. anguillarum) NCMB6are archetypical.

Seven of the eight remaining Northwest sal-monid vibrios are different in only one or twocharacters, and the remaining strain varies in fourcharacters. The other two European vibrios areeach different in three properties with two incommon. The Pacific herring vibrios differ consid-erably; strain 56 varies in 6 characters and strain 60differs in 11, with 4 in common. Of all the vibrios,strain 53, with 21 differing properties, is the mostdisparate. Although it has slightly curved cells,considered typical of vibrios, strain 53 uniquelyproduces a water-soluble pigment and is strictlyoxidative in carbohydrate utilization, suggestingrelationship with the pseudomonads. In sum-mary, cultural and physiological analysis did notallow discrimination between the Northwestsalmonid vibrios and the European vibrios but didsuggest the more distant relationship of thePacific herring vibrios, and the probable totalunrelatedness of strain 53.DNA competition experiments confirm and em-

phasize the interpretations from serology that thevibrios studied can be divided into three groups,that the Northwest salmonid groups and theEuropean groups are quite closely related, andthat the Pacific herring group is much more dis-tantly related. Although competition experimentsenable much more strain differentiation than wasattained in the reported serological experiments(17), other serological techniques clearly discrim-inated between members of the various groups(unpublished data), including the European vi-brios.DNA competition experiments showed that,

among the European vibrios, strains V-2911 andV-2916 are relatively more distantly related thanstrain NCMB6 to the Northwest salmonidvibrios. This conclusion is strongly supported bycultural, physiological, and serological data (17).Thus, V. anguillarum appears to be the pivotalorganism. Of the members of the Northwest sal-monid and the European vibrios that were tested,the least related to strain NCMB6 is strain 30,yet it is still 80% homologous. However, exhibit-ing 69% DNA homology, even strain V-2916 isquite closely related to Northwest vibrio strain 30.

Another notable strain differentiation achievedby DNA competition experiments is the findingthat, among the Pacific herring vibrios, strain 60is much more distantly related to the Europeanvibrios (10% DNA homology) and to the North-west salmonid vibrios (3 % DNA homology) thanis strain 56 (respectively, 25 and 22% DNAhomology). This conclusion is strongly supportedby the cultural and physiological data, and im-plied by the serological experiments. The regionsof genetic homology in strains 56 and 60 relativeto strains NCMB6 and 30 appear to be over-lapping; i.e., they are related in the same way tostrain NCMB6 and 30. The same holds true withhomologous polynucleotide sequences in strainsNCMB6 and 60 relative to strain 30.Although strain 53 has a common antigen with

strain 2.1 and has slightly curved cells, DNAcompetition experiments demonstrate no detect-able homology with the vibrios. Its cultural-physiological characteristics and its high GCcontent suggest that it is a pseudomonad.

Values of GC composition paralleled the DNAcompetition experiments in that no two vibriostrains appeared identical except Northwest sal-monid vibrio strains 2.1 and 30. These strains hadidentical GC content, were indistinguishable inDNA competition experiments, had the sameserotype, and deviated in only 1 of 51 culturaland physiological characteristics. It is noteworthythat strains 2.1 and 30 were isolated at widelyseparated times and locations, strain 2.1 from asalmon in seawater and strain 30 from a trout infreshwater.

It has been demonstrated that strong correla-tions exist between phenotypic and genotypicanalysis of a group of bacteria. Detailed serolog-ical, cultural, physiological, and DNA nucleotidesequence comparisons all suggested the sameoverall patterns of relatedness, but each approachramified and expanded upon the data obtainedfrom the others. In addition, it has been shownthat recently developed techniques in molecularbiology represent powerful contributions to thearray of classical methods available to the taxono-mist.

ACKNOWLEDGMENTS

We thank E. J. Ordal, J. L. Johnson, and B. J. McCarthy formany informative discussions and technical insights. Dr. Ordat'sand Dr. McCarthy's help in preparing this manuscript is deeplyappreciated.

This investigation was supported by a contract (NR137-487)with the Microbiology Branch of the Office of Naval Research,U.S. Navy.

LITERATURE CITED

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2. Bisset,' K. A. 1962. The phylogenetic concept in bacterial tax-onomy. Symp. Soc. Gen. Microbiol. 12:361-386.

3. DeLey, J. 1964. Pseudomonas and related genera. Ann. Rev.Microbiol. 18:17-46.

4. DeLey, J., I. W. Park, R. Tijtgat, and J. Van Ermengem. 1966.DNA homology and taxonomy of Pseudomonas and Xan-thomonas. J. Gen. Microbiol. 42:43-56.

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