JOURNAL OF BACTERIOLOGY, JUlY 1973, p. 307-315 Vol. 115, No. 1Copyright 0 1973 American Society for Microbiology Printed in USA.

Molecular Relationships Among theSalmonelleae

J. H. CROSA', D. J. BRENNER, W. H. EWING, AND S. FALKOW

Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20012; Center forDisease Control, Atlanta, Georgia 30333; and Department of Microbiology,

University of Washington, Seattle, Washington 98195

Received for publication 9 April 1973

Polynucleotide sequence relatedness studies were carried out to determine theextent of divergence present in members of the tribe Salmonelleae and betweensalmonellae and other enteric bacteria. Typical Salmon lla were 85 to 100%related. Two groups of biochemically atypical Salmonella showed somewhatlower binding to typical salmonellae and to each other. Arizona were 70 to 80%related to salmonellae. Two groups of Arizona were detected. These groupscorrelated with the presence of monophasic or diphasic flagellar antigens.Salmonella and Arizona were no more related to Citrobacter than to Escherichiacoli (45-55%). Relatedness of Salmonella and Arizona to other ente'robacteriaranged from. 20 to 40% with klebsiellae and shigellae, to 20 to 25% with erwiniae,and to less than 20% with edwardsiellae and Proteus mirabilis.

Bacteria belonging to the family Entero-bacteriaceae share significant segments ofcomplementary polynucleotide sequences (2,3). Previous work in our laboratories (5) re-vealed that substantial divergence may existeven among closely related enterobacteria.Such divergence is evident even within singlespecies. Findings with Escherichia coli, forexample, imply that this species represents acomposite gene pool within which are distinc-tive subgroups that are evolving towards differ-ent degrees of specialization. Moreover, thedistribution of related nucleotide sequences issuch that even bacteria of the genus Shigellamay be considered as specialized forms of E.coli (5).Whereas molecular parameters for E. coli and

other enterobacteria have been examined insome detail during the last few years, this is notthe case for members of the tribe SalmonelleaeBergey, Breed, and Murray. This tribe is com-posed of the genera Salmonella, Arizona, andCitrobacter, whose species are grouped on thebases of biochemical and immunochemical con-siderations (10, 11). In this report Bethesdastrains are considered as part of Citrobacterfreundii.

Available genetic data indicate that themajor gross features of the chromosomes of

I Present address: Department of Microbiology, Universityof Washington, Seattle, Wash. 98195.

Salmonella typhimurium (17, 20) or S. typhi(14, 16), and other enterobacteria such asShigella flexneri (13, 21) and E. coli (22) areessentially identical. Little is known about thegentics of the rest of the tribe Salmonelleae.The present investigation was undertaken todetermine the degree of evolutionary divergenceamong members of this tribe as assessed bynucleotide sequence relationships.

MATERIALS AND METHODS

Organisms and media. All Salmonella andArizona species (unless designated below) were ob-tained from the Center for Disease Control, Atlanta,Ga., and were thoroughly characterized both bio-chemically and antigenically. Citrobacter strains 1C,H310, and 3796 were obtained from S. Schaefler;Enterobacter cloacae 165 was obtained from Vee J.Brenner; Levinea (23) was obtained from V. M.Young. S. typhi 643, S. typhimurium strains 7823 and,LT2, E. coli K12, Serratta marcescens, Jnterobacteraerogenes, and Shigella flexneri 24570 have beendescribed previously (3). Erwiniae were obtained fromthe American Type Culture Collection or from M.Starr.

Cultures of bacteria were maintained on meatextract or nutrient agar slants. Brain-heart-infusionbroth (Difco) was used for routine cultivation oforganisms on a dry air rotary shaker at 37 C. Forlabeling DNA, log-phase cells were suspended in a tris(hydroxymethyl) aminomethane-glucose mediumlacking phosphate salts and containing 0.05% brain-heart-infusion broth. Carrier-free HS32PO4 (10-15

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CROSA ET AL.

mCi; New England Nuclear Corp., Boston, Mass.)was added, and the cultures were incubated at 37 Cwith shaking for 16 to 18 h.

Preparation of deoxyribonucleic acid. Both la-beled and unlabeled deoxyribonucleic acid (DNA)were prepared by a modification of the method ofBerns and Thomas (3). The DNA was sheared in apressure pump at 50,000 lb/in2 to an average single-stranded fragment molecular weight of 1.25 x 106,and filtered afterwards through metricel filter disks(0.45-M pore size, Gelman Instrument Co., Ann Arbor,Mich.). The labeled DNA fragments were then dena-tured by heating, further purified by passage througha hydroxyapatite (HA) column equilibrated with 0.14M phosphate buffer (PB; an equimolar mixture ofNaH2PO, and Na,HPO4, pH 6.8) plus 0.4% sodiumdodecyl sulfate (SDS), and held at 60 C. Material thatbound to the column under these conditions wasdiscarded. This procedure decre6sed the zero timebinding (label bound to HA immediately after theDNA is denatured) to 2% or less. The specific activityof labeled DNA obtained under these conditions is 0.5x 101 to 1.5 x 106 counts per min per ug.DNA reassociation. Thermally denatured, labeled

DNA fragments were incubated at 60 or 75 C with a1,500-fold excess (150 ;g/ml) of denatured, unlabeledDNA fragments in 0.28 M PB, and the reaction wascarried to approximately 100 Cots (DNA concentra-tion x time units; reference 6) for the unlabeled DNAin a 16-h incubation. After incubation the concentra-tion of PB was diluted to 0.14 M, and the preparationwas subjected to chromatography on HA to separatesingle-stranded DNA from duplexed DNA by using abatch procedure (4). HA was equilibrated with 0.14MPB plus 0.4% SDS and held at 60 or 75 C.

Single-stranded DNA does not bind to HA at thissalt concentration, whereas reassociated DNA isbound to HA. Four 15-ml portions of 0.14 M PB plus0.4% SDS at the incubation temperature served toremove unreacted DNA. HA was next washed witheither 4 15-ml portions of 0.4 M PB (from which thesalt concentration of the double-stranded DNA hadbeen removed from the column) or (when thermalstability of the duplexed DNA was of interest) in aseries of 15-ml portions of 0.14 M PB buffer atincreasing 5-C temperature increments up to 100 C.As the incubation temperature exceeds the Tm(e) (thetemperature at which 50% of the DNA bound to HA isdissociated) of various stability classes of DNA, theresulting single-stranded fragments are eluted fromHA, and a thermal elution profile is obtained. The HAwas finally washed with a 15-ml portion of 0.4 M PBto elute any material that remained bound to HA. Alleluates were collected in counting vials and assayedby Cerenkov radiation (7). In control experiments thereassociation of labeled DNA in 0.28 M PB was lessthan 1%. Relative binding values were not correctedfor the backgound reassociation of labeled DNA.

Spectrophotometric determination of molecularweight. The estimation of the genome sizes in testorganisms was accomplished essentially as describedby Gillis et al. (15) with some modifications. Thistechnique is described below in general terms.

Sheared DNA at an absorbancy of 2.000 0.01 in 2

x SSC at 260 nm (SSC = 0.15 M NaCl + 0.015 Msodium citrate) is denatured by heating in a boilingwater bath for 5 min. The DNA is then transferred to10-mm cuvettes in a 70-C chamber of a Gilford 2000spectrophotometer set at a changing optical density of0.25 in the vicinity of 2. The absorbancy of eachsample and the temperature is recorded automati-cally at 40-s intervals on a Minneapolis Honeywellrecorder for 60 min. The decrease in absorbencyduring 30 min, beginning 10 min after initiation of therenaturation process, is employed in the estimation ofthe molecular complexity of the DNA.

RESULTSNucleic acid relatedness among Salmo-

nelieae. The reassociation data obtained when32PO0-labeled DNA fragments from S. typhi-murium LT2 are reacted with unlabeled DNAfragments from other organisms at two in-cubation temperatures are compiled in Table1. The concentration of labeled fragments (0.1,g/ml) is sufficiently small, compared to that ofunlabeled DNA (150 ,g/ml), to preclude reac-tions of labeled fragments with one another.The values of reassociation for the homolo-

gous reaction (labeled and unlabeled S.typhimurium DNA) were normally between 80and 90% (Table 2), whereas the reaction wasless than 1% in labeled preparations incubatedin the absence of unlabeled DNA. The lowertemperature of incubation (60 C) allows reas-sociation of partially complementary sequences,implying the formation of hybrids betweensequences which could differ by as much as onebase in five. The higher temperature of incuba-tion (75 C) is more stringent, permitting onlythe reassociation of sequences with a smallerdegree of mismatching (eight base pairs or lessper hundred). As a consequence, the closer thebinding values obtained at both temperatures,the more precise the degree of relatednessbetween the sequences of the reassociatedstrands.The DNA duplex data in Table 1, shows that

the bacteria grouped as Salmonelleae presentthe following general features. The Salmonellatested show a high degree of relatedness witheach other. With few exceptions, the interspe-cies variation is no greater than that observedbetween different strains of S. typhimurium.A few of the atypical biotypes of Salmonella

were tested. They are included in Salmonellabecause of overall biochemical similarity (10)and they appear to show significantly moredivergence from S. typhimurium than is seen inthe strains of typical Salmonella tested (Table1).The genus Arizona which presents a similar

pattern of pathogenicity to that seen in

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TABLE 1. Relative binding of enterobacteria to Salmonella reference strains

Source of labeled DNA Source of labeled DNA

S. S. S .Source of S. typhimurium LT2 b7z phoe- Source of S. typhimurium LT2 binza phoe-

unlabeled DNA nix unlabeled DNA nixBind- Bind- Bind- Bind- Bind- Ta Bind- Bind- Bind-ing at Tm(e, ing at ing at ing at ing at m(e) ing at ing at ing at60C at 75C 60C 60C 60C at 75C 60C 60C(%) 60 C ( ( (%) 60 750(%C 60

.~~~~~~~~~~~~~% 1100

93988989808587

8784807494939393

94

92

9194

91

95

91878788769389929185

878496918985

881038292

7472747278

0.60.6

3.2

3.4

1.5

1.7

3.3

1.6

1.63.71.7

1.6

1.10.8

4.9

5.0

3.8

100

8897

82

71

9190

90

91

8891

91

88

70

83

857991

86

8498

6265676568

86

8992

10091

89808682

92

89869381

939086

87

89

93

9090

75

81

78

7879797778

838679788682

82

81

7883787880

807780

100

76

80

7777

80

82

Arizona 85Arizona 91Arizona 111Arizona 118Arizona 143Bethesda 1ABethesda 2ABethesda 3ABethesda 4ABethesda 6Bethesda 7Bethesda 8ABethesda 9ABethesda 29ACitrobacter freundii460-61

C. freundii 876-58C. freundii 3796C. freundii 4675-65C. freundii 1 CC. freundii H 310C. diversus 1381-70C. diversus 3616-63Levinea amalonatica

25405L malonatica 25408Escherichia coli K-12Shigella flexneri 2a24570

S. flexneri 6 (Newcas-tle)

Edwardisella tarda1126-64

E. tarda 1795-62E. tarda 3592-64E. tarda 3888-64E. tarda 6243-61Enterobacter

aerogenes 1627-66E. cloacae 165E. cloacae 1347-71E. hafniae 4360-67Erwina amylovora7400

E. carnegieana 13452E. carotovora 495E. dissolvens ED 105E. dissolvens ED114E. herbicola 2552E. nimipressuralis EN

1E. tracheiphila 27004Klebsiellapneumoniae 2

Serratia marcescensSM6 W2

Proteus mirabilis 1

717769807746494942504646475850

474750

505248

484639

38

19

1810181836

32392022

282036331940

1640

25

7

3.83.5

3.713.513.113.212.712.813.413.012.813.6

14.214.8

13.713.812.9

11.0

14.0

18.7

15.918.517.518.416.0

15.913.218.916.5

14.617.413.513.715.714.1

16.815.3

14.6

697267747417202119221918222523

222423

252717

231415

13

1

5122

17

141623

11313121

15

718

5

2

81

58

60

5756

59

54

29

36

82

51

55

5655

58

54

27

36

Salmonellatyphimurium LT2

S. typhimurium 4066S. typhimurium 7823S. aberdeen 687-72S. anatum 78S. argentina 1341°S. binza 1927-72S. bonariensis

156-C12S. brazil 298-1181S. canastel 192°S. carrau 2253-72S. chameleon 1110°S. cholera-suis 34S. cholera-suis 686-72S. cholera-suis 789-63S. cholera-suis

3183-62S. cholera-suis

3326-54S. cholera-suis

4414-63S. cholera-suis 36S. cholera-suis

4499-63S. cholera-suis

6033-61S. enteritidis 64

[9, 12: g, m: -]S. gallinarum 74S. gaminara 3296-71S. give 2176-72S. illinois 121 38260S. marinus 1112kS. mendoza 294S. montevideoS. muenchen 599-72S. newington 84S. onderstepoort

94-282S. paratyphi AlS. phoenix 791bS. pullorum 75S. rubislaw 102-193S. senftenberg 1381S. thomasville

1132-72S. typhi 643S. typhi-suis 38S. wichita 105-9S. worthington

245-72Arizona 1Arizona 14Arizona 15Arizona 20Arizona 62

'Tm,., thermal elution midpoint; that temperature at which 50% of the DNA bound to HA at the 60 or 75 C incubationtemperature is eluted. ATm, , is the decrease in Tme, between heterologous DNA reactions and the homologous SalmonellaDNA reactions.

'Biochemically atypical Salmonella.

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TABLE 2. Homologous DNA reassociation reactionsa

Binding (%)Source ofDNA

60C 75C

Salmonella typhimurium LT2 86.6 88.7S. phoenix 791 82.5 -

S. binza 1927-72 83.8 -

Arizona 1 88.0 84.3Arizona 62 84.0 83.4

a In all reactions 0.1 gg of sheared 32PO4-labeledDNA per ml together with 150 ig of sheared DNA perml from the same strain were denatured and in-cubated for 16 h at the indicated temperature in 0.28M PB. The averages shown represent 8 to 36 reac-tions.

Salmonella has about 70 to 80% nucleotidesequences in common with S. typhimurium.Species of Citrobacter and organisms includedin the Bethesda-Ballerup group share about 45to 50% nucleotide sequences with S. typhimu-rium, and seem to be no more related to S.typhimurium than are E. coli, Shigella orLeuinea. Relatedness between S. typhimuriumand strains of Klebsiella pneumoniae, En-terobacter cloacae and E. aerogenes is between30 and 40%. A similar extent of reaction isseen with Erwinia dissolvens and Erwinianimipressuralis. These organisms are in factmore properly placed with E. cloacae than withErwinia (Brenner, unpublished observations).The nucleotide sequences of Salmonella held incommon with other enterobacterial DNAs, suchas Edwardsiella tarda, species of Erwinia, S.marcescens, Enterobacter hafniae and, Proteusmirabilis, are of a lower order of magnitude(7-28%).Sequences held in common between different

kinds of Salmonella are quite precise as judgedby the criteria of reassociation at the highertemperature. The same observation holds forsequences shared by Salmonella and bacteria ofthe genus Arizona. In contrast, the sequencesheld in common between S. typhimurium andstrains of Citrobacter, E. coli, and all otherenteric bacteria are quite divergent by thiscriterion.Table 1 also shows values of reassociation at

60-C incubation temperature obtained by using32PO -labeled DNA from two additionalSalmonella. Results obtained with S. binza, abiochemically typical Salmonella, are similar tothose obtained with S. typhimurium. When32PO0-labeled DNA from the atypical strain S.phoenix is used, reassociation values fall to anaverage of 80% with arizonae, as well as withmost of the typical and atypical salmonellae.

Only one of the other atypical strains, S.canastel, appears to present a higher value ofrelatedness to S. phoenix than to S.typhimurium or S. binza. S. canastel and S.phoenix are classified as Salmonella subgenusII by Kauffman (18), whereas the other threeatypical salmonellae are members of subgenusIV.Thermal stability of interspecies DNA du-

plexes among the Salmonelleae. The thermalstability of interspecies enteric DNA duplexesis studied in 60-C reactions by measuring thethermal release of reassociated DNA fragmentsbound to hydroxyapatite. The thermal elutionprocedure generates a profile from which onecan approximate the percentage of unpairedbases in a DNA heteroduplex (1.0% unpairedbases per 1.0 C decrease in thermal stability;references 1, 19).

Figure 1 shows the thermal elution profiles ofsome S. typhimurium-enterobacterial DNA du-plexes for representatives of the Salmonelleae.

100-

w

< 80-

0 -

cr

I 60-

0

cr0

F 40--Jw

za

20-

0

*11.0

_ .

.I

0

A/

0A---A---"00OAM PB

'II**,1

.

60 70 80 90 100TEMPERATURE, °C

FIG. 1. Thermal elution profiles of reassociatedenterobacterial DNA duplexes. An 0.1-Ag amount oflabeled S. typhimurium DNA fragments was in-cubated with 150 gg of unlabeled DNA fragmentsfrom the indicated organisms in 1 ml of 0.28M PB for16 h at 60 C. The samples were then subjected tothermal chromatography on HA and assayed asdescribed in Materials and Methods. Symbols: (0) S.typhimurium LT2 DNA homoduplex, (x) S. ty-phimurium LT2-S. typhi 643, (A) S. typhimuriumLT2-S. marinus (-) S. typhimurium LT2-Arizona 62,(0) S. typhimurium LT2-C. freundii 4675-65.

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RELATIONSHIPS OF SALMONELLEAE

In Table 1 are shown the ATm(e) values (Tm(e)homoduplex minus Tm(e,) heteroduplex) for mostof the S. typhimurium heteroduplexes.

Heteroduplexes formed with DNA from S.typhimurium and typical salmonellae have avery low degree of mismatching (0.5-1.5% un-paired bases). The degree of unpaired basespresent in DNA duplexes formed between S.typhimurium and atypical salmonellae or arizo-nae is between 3 and 5%. In the case of S.typhimurium-Citrobacter DNA heterodu-plexes, the degree of mismatching reaches val-ues as high as 15%. Between 15 and 20%unpaired bases are present in DNA duplexesformed from S. typhimurium and members ofother tribes of enterobacteria.Frequency distribution of relatedness of

Salmonella, Arizona and Citrobacter. Thedegree of heteroduplex formation between S.typhimurium DNA and DNA from 66 Sal-monella, Arizona, and Citrobacter cultures (in-cluding Bethesda-Ballerup) was plotted asnumber of strains versus percent relatedness(Fig. 2). The mode for the S. typhimuriumreaction with typical salmonellae was 91 to 95%.Relatedness values were symmetrically dis-tributed around this mode. The distribution ofrelatedness graphs for the atypical salmonellae,members of the genus Arizona, and Citrobacterare also shown in Fig. 2. The relative related-ness data for these bacteria also approximate asymmetrical distribution around their modes,despite the relatively small number of strainsassayed in some of them (especially the atypicalsalmonellae). The mode for Arizona is shiftedtowards lower relatedness (71-75%), whereasthe atypical salmonellae show a relatednesscurve which falls midway between the distribu-tion of Arizona and typical salmonellae. The

16 -

142

zuD0

w(r 8

Citrobocterstrains

Il

typical Salmonelloa/strainls

I1541jj~55jf

/ I16

Arizono

atypical Salmonsill\> /\ ~~~~~stroins

_ *141-45 46-50 51-55 56-60 61-65 66-70 71-75 76-SO SI-tS1 96-90 51.55 96-100

% RELATIVE BINDING

FIG. 2. Frequency distribution of relatedness ofstrains to S. typhimurium LT2. Values shown wereobtained from 60-C reassociation reactions in whichS. typhimurium L72 was the reference strain.

distribution of relatedness of Citrobacter to S.typhimurium is substantially lower than that ofthe other Salmonelleae (mode = 46-50%).

Differences in genome size among theSalmonelieae. DNA reassociation is a collision-dependent, second order reaction (6). Gillis etal. (15) described a procedure for measuring thegenome size of a strain based on the spectropho-tometric differences in initial rates of DNAreassociation. These differences in initial ratesof renaturation are proportional to the molecu-lar weight.We used this method, as well as reciprocal

binding values, to approximate the relativegenome size of a number of enteric bacteria.The values obtained indicate that Arizona 62and Arizona 91 have the largest genome sizes,about 35% larger than that of E. coli K-12 (E.coli K-12 DNA is approximately 2.5 x 109daltons). In general, the genome size in arizonaeis 20 to 35% larger than that of E. coli K-12.Salmonella genomes were mainly 10 to 20%larger than E. coli K-12. The Citrobacter andBethesda genomes ranged from 15% larger thanE. coli K-12 to 15% smaller than E. coli K-12.Molecular parameters for bacteria of the

genus Arizona. In preliminary hybridizationexperiments with the spectrophotometricmethod described by De Ley et al. (9), thearizonae could be separated into two subgroups.This method, in our hands, gave only an ap-proximation of quantitative nucleic acid re-latedness. It seemed worthwhile to label DNAfrom Arizona serotypes to further investigatethis inference of two distinct subgroups ofarizonae and to determine the extent of diver-gence within related sequences.

Relatedness among arizonae was assayed byusing 32PO0-labeled DNA from Arizona 1 andArizona 62 (Table 3). There is indeed a differen-tiation of strains into two subgroups based onrelatedness to either Arizona 1 or Arizona 62.This difference was further emphasized by theincreased ATm(e) values at 60 C and by thedecrease in reaction at 75 C, when the heterodu-plexes are formed between labeled DNA fromstrains belonging to the other subgroup. Theonly exception to these groups is Arizona 118which exhibits below 80% relatedness to bothreference strains of Arizona. A biochemicalreexamination of this strain indicated that it is,in fact, an atypical Salmonella.The strains more highly related to Arizona 1

exhibit 90% or higher relative binding, whereasthose strains more closely related to Arizona 62exhibit binding between 85 and 89%. Thesedifferences are probably the result of the largergenome size of Arizona 62 as compared to the

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TABLE 3. Relative binding of enterobacteria to Arizona strains

Source of labeled DNA

Source of unlabeled DNA Arizona 62 Arizona 1

Binding at ATm(*, Binding at Binding at ATml() Binding at60 C (%) at 60 C 75 C (%) 60 C (%) at 60 C 75 C (%)

Arizona 1 74 4.3 68 100 _ 100Arizona 14 73 4.9 70 91 0.7 90Arizona 15 76 4.3 69 92 0.3 90Arizona 20 77 5.1 68 93 0.7 89Arizona 62 100 - 100 81 4.4 73Arizona 66 89 0.3 89 82 4.2 73Arizona 85 89 0.3 87 80 4.3 71Arizona 91 85 0.5 85 81 4.3 75Arizona 111 88 0.5 86 73 4.2 66Arizona 118 75 4.0 69 78 4.0 70Arizona 143 87 0.5 85 82 4.4 73Arizona 345-71 71 4.3 65 92 0.5 90Arizona 1485-72 88 1.0 84 81 4.2 70Arizona 1900-72 84 0.9 85 82 5.0 70Arizona 1986-72 92 0.4 93 81 4.2 71Arizona 2175-72 89 0.1 89 82 4.3 73Arizona 2238-72 76 5.1 70 93 0.3 92Arizona 3246-71 91 0.1 86 80 4.3 70Salmonella typhimurium LT2 73 4.2 68 75 4.3 64S. anatum 78 71 3.6 - - - -

S. cholera-suis 36 73 3.7 67 78 4.7 64S. brazil 298-1181 77 3.5 67 79 4.4 69S. bonariensis 156-C12 73 3.3 71 - - -

S. canastel 192 73 2.7 75 79 4.4 69S. chameleon 1110 74 2.9 - 77 4.7 67S. marinus 1112 74 3.2 _ 79 4.8 69S. muenchen 599-72 72 2.3 - - - -

S. phoenix 791 75 2.9 74 79 4.8 70Citrobacterfreundii 5118-60 - -__ 56 - -

Bethesda 6 51 13.0 25 54 13.1 21Bethesda 9A - - - 53 - -

Bethesda 29A 49 12.5 29 - - -

Citrobacter diversus 1381-70 - - - 55 13.5 28Levinea amalonatica 25405 _ - _ 54 11.9 23L. malonatica 25408 - - - 52 13.3 27Escherichia coli K-12 44 13.3 20 48 14.2 18Shigella flexneri 24570 - - - 43 15.3 15Enterobacter aerogenes 1627-66 - - - 40 12.9 16E. cloacae 1341-71 - - - 41 15.4 17E. hafniae 4360-67 - - - 22 19.5 3K. pneumoniae 2 - - - 41 14.0 18Erwinia carotovora 495 - - - 22 15.7 4E. camegieana 13452 - - - 35 12.3 15E. herbicola 2553 - - - 24 15.2 5E. dissolvens ED 114 - - - 39 13.6 14E. amylovora EA 169 - - - 25 16.1 6E. nimipressuralis EN 1 - - - 42 11.9 17E. tarda 1126-64 - - - 19 18.5 2Serratia marcescens SM6 W2 - - - 26 16.1 7Proteus mirabilis 1 - - - 9 16.3 3

rest of the arizonae. The smaller percentage of When relative reassociation results for la-reassociation of other enterobacterial DNAs to beled DNA from Arizona 1 are plotted, theArizona 62 DNA than to labeled DNA from values fall on two curves (Fig. 3A). The modeArizona 1 supports this assumption (Table 3). for the arizonae of the Arizona 1 group is 91 to

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RELATIONSHIPS OF SALMONELLEAE

atypicol Salmonellastrains

I

Arizona ,

/ group ,

I %\ Arizona,' \ group

66-70 71-75 76-80 81-85 86-90 91-95 5

4). All diphasic strains are preferentially relatedA to the diphasic Arizona 62.

The frequency distribution of relatednesscurves for the heterologous subgroup overlapswith that of Salmonella for both Arizona 1 andfor Arizona 62. Relatedness of both arizonae to

62 other enterobacteria is shown in Table 3. theyshare 70 to 80% of their nucleotide sequenceswith salmonellae and 50 to 55% with strains ofCitrobacter. In general, relatedness of arizonaeto other groups of enterobacteria closely paral-lels the results obtained by using Salmonellareference DNA.

Arizona 62 group'I

I %B

otypical aSalmonella. '.

strains. aIJ

..

,. 9-81-85 86-90 91-95 96-100

% RELATIVE BINDING

FIG. 3. Frequency distribution of relatedness ofstrains to arizonae. (A) Frequency distribution ofrelatedness to Arizona 62, (B) Frequency distributionof relatedness to Arizona 1. Values shown were ob-tained from 60-C reassociation reactions in whichArizona 62 and Arizona 1 were the reference strains.The graph for Arizona 62 is shifted one interval to theright to compensate for genome size differences inArizona 62 and Arizona 1.

95%, and for the Arizona 62 group the mode isshifted to the 81 to 85% interval. There is no

overlap between the two frequency distribu-tions. When Arizona 62 is used as the referencestrain, the mode values are 86 to 90% for theArizona 62 group and 71 to 75% for the Arizona 1group (Fig. 3B). The lower binding of bothgroups to Arizona 62 can be ascribed to the largegenome size of Arizona 62. To directly comparethe distribution curves obtained with both ari-zonae, we have shifted the graph for Arizona 62one interval to the right to compensate for thegenome size differences.Many strains of Arizona exhibit reversible

phase variation in flagellar or H antigens (10).An examination of the monophasic or diphasicH antigen behavior of the arizonae used in thisstudy revealed that all diphasic strains were

rapid lactose fermenters, whereas all mono-

phasic strains exhibited delayed fermentationof lactose. These characteristics coincided per-fectly with the DNA relatedness data. All mon-ophasic strains tested are preferentially relatedto Arizona 1, which is itself monophasic (Table

DISCUSSIONIt has been assumed, primarily from bio-

chemical and serological findings, that the en-teric bacteria form a spectrum of overlappingspecies. We, in fact, do find that Salmonellaand Arizona show considerable overlapping,almost as much as E. coli and shigellae (5).Nonetheless, there is no clear-cut evidence for acontinuum of species at this point in the evolu-tion of the Enterobacteriaceae. For example, wehave found that members of the genus Citrobac-ter, which is placed in the tribe Salmonelleae onthe basic of biochemical similarity, are notsignificantly more related to S. typhimuriumthan to E. coli. with other enterobacteria, thefrequencies of common sequences are still lower(2, 3).Although all the cultures of Citrobacter, Be-

TABLE 4. Grouping of Arizona strains based onantigenicity and nucleotide sequence relatedness

DNAa HStrain DNAg g

antigenhomology group type

Arizona 1 1 MonophasicArizona 14 1 MonophasicArizona 15 1 MonophasicArizona 20 1 MonophasicArizona 345-71 1 MonophasicArizona 2238-72 1 MonophasicArizona 62 62 DiphasicArizona 66 62 DiphasicArizona 85 62 DiphasicArizona 91 62 DiphasicArizona 111 62 DiphasicArizona 143 62 DiphasicArizona 1485-72 62 DiphasicArizona 1900-72 62 DiphasicArizona 1986-72 62 DiphasicArizona 2175-72 62 DiphasicArizona 3246-71 62 Diphasic

a Group 1 and Group 62 merely refer to the refer-ence organism to which the strain exhibited greaterrelatedness.

10

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6-

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66-70 71-75 76-80

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CROSA ET AL.

thesda and Levinea show about 50% relatednessto salmonellae and arizonae, these organismsare far from a homologous group. Data obtainedwith these organisms will be presented sepa-rately (J. H. Crosa, D. J. trenner, and W. H.Ewing, manuscript in preparation).When polynucleotide sequence relationships

among the various Salmonella are examined, itbecomes evident that regardless of the sero-group to which each representative belongs, allshow a high degree of relatedness with eachother, even under the most stringent conditions(i.e., 75 C incubation temperature). For exam-ple, S. paratyphi (serogroup A), S. typhimurium(serogroup B), S. bonariensis (serogroup C2), S.mendoza (serogroup D,), S. typhi (serogroupD1), S. anatum (serogroup El), S. rubislaw(serogroup F), S. wichita (serogroup G2), S.onderstepoort (serogroup H), and S. gaminara(serogroup I) show a high degree of relatednessto S. typhimurium LT2. Although the degree ofrelatedness is not exactly the same, the varia-tion is not greater than that determined be-tween different strains of S. typhimurium. Onthe other hand, it appears that the so-calledatypical salmonellae possess nucleic acid se-quences that have diverged significantly fromthe average typical Salmonella. They appar-ently fall into a class midway between thetypical salmonellae and the arizonae.The closely related atypical strains S. canas-

tel and S. phoenix belong to Kauffmann'sSalmonella subgenus II. The three other atypi-cal strains tested, S. argentina, S. chameleon,and S. marinus, are members of Kauffmann'ssubgenus IV. We have not determined related-ness among the subgenus IV strains. In fact, wehave not tested enough atypical strains to reachany definite conclusions about these organisms.

Arizonae share about 70 to 80% of theirgenome with salmonellae. On the basis of anti-genic characteristics, the genus Arizona can besubdivided into two subgroups. These sub-groups differ by an average of about 10% nucleo-tide similarity in intergroup DNA reassociationreactions. These results imply that the phasevariation in H antigens is responsible for only apart of the chromosomal difference betweenthese groups. The difference in rapidity oflactose fermentation probably also accounts forsome of the nucleotide sequence differencesevident in the two groups of arizonae.

Previous studies of specific proteins (for ex-ample, alkaline phosphatase, 8) implied greaterrelatedness between E. coli, Shigella, Salmo-nella, Arizona, and Citrobacter, and it was con-cluded that all of these bacteria comprised aunique tribe. We believe that, although some

specific cistrons may be preferentially con-served to varying degrees during bacterial evo-lution (2), overall relatedness does change. Weassume that the enteric species were initiallyderived from only one or a few ancestral types,but that divergence into different specializedforms of enteric bacteria may be quite ancientin the evolutionary sense. The only evolutionaryvestige of an enteric archetype strain, we feel, isfound in a pool ofcommon nucleotide sequencesthat are found in virtually all enteric bacteriaand which comprise about 20% of the totalgenome. We are currently attempting to isolateand identify such common core sequences.A 1963 recommendation (11) includes all

arizonae in Arizona hinshawii (Ewing and Fife)and recognizes three species of Salmonella, S.cholerae-suis (Smith) Weldin, S. typhi (Schro-eter) Warren and Scott, and S. enteritidis(Gaertner) Castellani and Chalmers. All sal-monellae other than S. cholerae-suis and S.typhi are designated as serotypes and bi-oserotypes of S. enteritidis.DNA reassociation data indicate that strains

of Salmonella and Arizona are highly interre-lated but separable. The arizonae examinedthus far are consistent with one species designa-tion, although they form two nonoverlappingpopulations that correlate with manophasic anddiphasic H antigens, as well as with rapidity oflactose fermentation. There is little doubt thatall typical Salmonella form one species from themolecular point of view and that the atypicalstrains are separable into at least two groups,both of which are distinct from the typicalsalmonellae and arizonae.At present few laboratories are equipped to

separate arizonae on the basis of flagellar anti-gen variation. Therefore, any attempt to sepa-rate arizonae into the two groups observed hereis clearly both unnecessary and unfeasible atthis time.From the genetic standpoint, the results of

this investigation indicate that virtually allserotypes of Salmonella are highly related andmight be considered a single species. Currently,the salmonellae are considered to be repre-sented by three species, S. cholera-suis, S.typhi, and S. enteriditis. In this system allsalmonellae other than S. typhi and S. cholera-suis are considered to be serotypes or bi-oserotypes of S. enteriditis (12). Although wecannot differentiate between the three specieson the basis of DNA-DNA duplex formation,the differentiation of these organisms is ofconsiderable importance in the diagnostic labo-ratory. Although our results could be inter-preted to mean that all salmonellae should be

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RELATIONSHIPS OF SALMONELLEAE

considered one species (in which event the namewould be S. cholera-suis, see reference 12), wefeel that any change in nomenclature would beundesirable and impractical. We support thethree nomenspecies idea as the best availablesolution of an historic problem. The threenomenspecies system is nomenclaturally correctand it recognizes common usage and traditionto the extent that these should be recognized. Itshould be emphasized, however, even in thelight of acceptance of this nomenclatural sys-tem, that the differences between salmonellae inbiochemical and serological properties, as wellas differences in pathogenicity and host adapta-tion, probably reflect relatively small changesin a limited number of gene clusters.

ACKNOWLEDGMENTSWe are extremely indebted to G. R. Fanning, G. V. Miklos,

and A. G. Steigerwalt for indispensable assistance throughout'this study.

J. H. Crosa is a fellow of the World Health Organizationand is on leave of absence from Centro de Investigaciones,Microbiologicas, Facultad de Ciencias Exactas, University ofBuenos Aires, Argentina. The experiments performed inSeattle were supported in part by grants from the NationalScience Foundation and the Commission on Enteric Diseasesof the Armed Forces Epidemiology Board.

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