JOURNAL OF BACTERIOLOGY, June 1972, p. 793-802Copyright 0 1972 American Society for Microbiology

Vol. 110, No. 3Printed in U.S.A.

Enzyme Evolution in the EnterobacteriaceaelGARY T. COCKS2 AND ALLAN C. WILSON

Department of Biochemistry, University of California, Berkeley, California 94720

Received for publication 6 March 1972

An immunological approach has been used for the study of alkaline phospha-tase evolution in bacteria of the family Enterobacteriaceae. Antisera were pre-pared against alkaline phosphatase from Escherichia coli and Klebsiella aerog-enes and tested against the unpurified alkaline phosphatases of 32 strains ofenterobacteria by double diffusion and quantitative micro-complement fixa-tion. The immunological relationships detected among the alkaline phospha-tases of enterobacteria agree approximately with those reported for five otherenzymes, as well as with the tryptic peptide pattern similarities found for twoother enzymes, and with the relationships detected by interspecific deoxyribo-nucleic acid hybridization tests.

Although bacterial evolution is being studiedincreasingly at the molecular level, work alongthese lines has lagged behind that on verte-brates. Comparative studies of proteins fromvarious vertebrate species, especially thosewith a good fossil record, have been intensive.Such studies are valuable not only becausethey provide a way of measuring the geneticand phylogenetic relatedness of species to eachother but also because they provide insightsinto the mechanism of evolution at the molec-ular level (1, 11, 12, 20, 21, 30, 36-38, 47, 50).Bacterial proteins have not yet been examinedintensively by the comparative biochemicalmethods employed in the studies cited above.However, several deoxyribonucleic acid (DNA)hybridization surveys have been conducted(25), and the percentage of guanine and cyto-sine in DNA has been measured in many bac-terial strains and species. Unfortunately, thelatter measurement gives little informationconcerning degree of sequence similarity (41).The family Enterobacteriaceae has been ofparticular interest to applied and molecularbiologists but even in this case few interspe-cific comparisons have been made at the mo-lecular level. We have carried out a quantita-tive immunological and electrophoretic surveyof alkaline phosphatases (EC 3.1.3.1) from en-terobacteria both to clarify relationshipsamong these organisms and to learn more

I Taken from a thesis submitted by G.T.C to the Univer-sity of California, Berkeley, in partial satisfaction of therequirements for the doctor of philosophy degree in bio-chemistry.

2Present address: Department of Microbiology and Mo-lecular Genetics, Harvard Medical School, Boston, Mass.102115.

about the process of evolution at the molecularlevel in bacteria. A preliminary survey of alka-line phosphatase relationships among entero-bacteria was made by Cordes and Levine (48).

MATERIALS AND METHODSStrains of bacteria. The strains of bacteria used

and the sources from which they were obtained aregiven in Table 1.

Alkaline phosphatase induction and extrac-tion. Alkaline phosphatase synthesis was dere-pressed by growth in low-phosphate liquid culturesor on low-phosphate agar plates (46). Bacteria wereharvested and alkaline phosphatase was released bysonic treatment. After clarification by centrifuga-tion, the extracts were suitable for immunologicaland electrophoretic analyses.Enzyme assays. Alkaline phosphatase was as-

sayed with p-nitrophenyl phosphate as describedelsewhere (2). Acid phosphatase (EC 3.1.3.2) wasdetermined by the procedure of Dvorak et al. (14).

Starch gel electrophoresis. Horizontal starchgels were prepared (15) with either hydrolyzed starchfrom Connaught Laboratories or Electrostarch fromthe Electrostarch Co. since they appeared to giveequal resolution. The pH 7 buffer system consistedof 4 mm citric acid and 32 mm Na2HPO4 in the elec-trode wells and 1.4 mm citric acid, 8.6 mM Na2HPO4in the gel itself. Poulik's discontinuous buffer system(at pH 8.6) was also used (31). Electrophoresis wascarried out in a cold room at 200 or 250 v, for 18 to24 hr.

Alkaline phosphatase activity was made visiblewith a solution of 0.5 mg of a-naphthyl phosphateand 1.5 mg of tetraazotized o-dianisidine per ml in0.06% Na2 B407 10 H20 (18). Mobilities were mea-sured relative to Escherichia coli enzyme which wasincluded in each gel.

Immunodiffusion. Ouchterlony double-diffusiontests were carried out in agar plates (44) at room

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TABLE 1. Bacterial strains

Strain Sourcea

Aeromonas sp.Arizona arizonae 13314bCitrobacter freundi 8090bCitrobacter sp.Enterobacter cloacae 13047bE. liquefaciens 14460bErwinia amylovora 15580bE. amylovora FB-1E. amylovora FB-9E. carotovora 495bE. carotovora EC-1E. herbicola (?) 2E. quercina 1Escherichia coli C9OF 1E. coli B/41E. coli JC4583

SMCATCCATCCSMCATCCATCCATCCHSHSATCCHSHSHSAGSLSL

Strain

E. coli CrookesE. coli W 208E. coli Ymel A3Klebsiella aerogenes 1033cProteus mirabilisP. morganiP. vulgaris FC-1-1Providencia alcalifaciens 9886bProvidencia sp.Pseudomonas maltophiliaSalmonella typhimurium LT-2Serratia marcescens 274S. marcescens SS. marcescens pinkS. marcescens whiteShigella boydi 9207

Sourcea

RVCWCYBMSMCSMCBBIATCCSMCSMCBACwMNSMCSMCATCC

aSource abbreviations: AG, Alan Garen, Yale; ATCC, American Type Culture Collection; BA, BruceAmes, Berkeley; BBI, Department of Bacteriology and Immunology, Berkeley; BM, Boris Magasanik, MIT;CW, Clyde Wilson, Berkeley; CY, Charles Yanofsky, Stanford; HS, Donald Hildebrand and Milton Schroth,Berkeley; MN, Marion Nestle, Berkeley; RV, Raymond Valentine, Berkeley; SMC, Stanford Medical Center,Clinical Laboratories; SL, Stuart Linn, Berkeley.

bType or proposed neotype strain.c Previously referred to as Aerobacter aerogenes 1033.

temperature for 48 hr. The wells contained 10 glitersof liquid. The distance between wells was approxi-mately 5 mm, and the concentrations of antiserumand antigen were adjusted to give sharp precipitinlines. Two antisera were used for immunodiffusionexperiments: an anti-E. coli alkaline phosphataseobtained from rabbit 001 (seventh bleeding, obtainedafter 19 weeks of immunization) and an anti-Kleb-siella aerogenes alkaline phosphatase from rabbit2095 (sixth bleeding, obtained after 11 weeks ofimmunization).Micro-complement fixation. Micro-complement

fixation was done by the procedure of Levine andVan Vunakis (24) except that no protein was used inthe diluent buffer. Incubations were for 18 hr at 4 C.The results of all cross-reaction experiments are

given as immunological distances (32) rather than as

indexes of dissimilarity (49). The index of dissimi-larity is the experimentally determined factor bywhich the antiserum concentration must be raised inorder for a heterologous phosphatase to produce a

complement fixation curve whose peak is equal inheight to that produced by the homologous phospha-tase. The immunological distance is defined as 100times the log of the index of dissimilarity (32). Thereason for expressing our results as immunologicaldistances is that this quantity appears to be propor-tional to the degree of sequence difference betweenhomologous and heterologous protein antigens (32,33). The equation relating immunological distance(y) to percentage difference in amino acid sequence(x) is y 5x, for lysozyme (32). For other proteins a

similar relationship probably exists (33).Alkaline phosphatase purification. The purifi-

cation of the enzyme from E. coli C9OF 1 has beendescribed previously (8). In purifying alkaline phos-

phatase from K. aerogenes 1033 (previously knownas Aerobacter aerogenes 1033), we found that heatingthe cells at 80 C for 15 min in the presence of 2%(w/w) 2-phenoxyethanol gave the highest yield ofsoluble enzyme. The yield was 80%, based on en-zyme assays of whole cells. Alkaline phosphatase wasconcentrated and purified by collecting the precipi-tate formed between 60 and 80% saturation withammonium sulfate. The enzyme was further purifiedby diethylaminoethyl cellulose chromatography andSephadex G-200 gel filtration. For final purification,the enzyme was subjected to starch gel electropho-resis. The enzyme was localized by spraying withnitrophenyl phosphate (46), and the portion of thegel containing alkaline phosphatase was cut out.Antiserum production. The production of anti-

sera against E. coli C9OF 1 alkaline phosphatase hasbeen described (8). For the present micro-comple-ment fixation work we used a mixture of antiseraobtained from rabbits (1, 2, 001, and 002) immunizedfor 15 to 19 weeks with E. coli enzyme. To prepareantisera against K. aerogenes 1033 phosphatase, apiece of starch gel containing the purified enzymewas ground up in Freund's complete adjuvant andinjected intradermally at four sites on the back oftwo male New Zealand White rabbits (2094 and2095). If the specific activities of E. coli and K. aero-genes alkaline phosphatases are the same, 183 unitsas defined here per milligram of protein, each rabbitinitially received 5 ug of protein. During the tenthweek another 130 ,ug of enzyme was injected intrave-nously into each rabbit; the enzyme was eluted fromstarch gels and administered in three injections onalternate days. During the twentieth week eachrabbit received another 250 pg of enzyme adminis-tered as a series of three intravenous injections on

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ALKALINE PHOSPHATASE: ENZYME EVOLUTION

alternate days. Sera were collected at intervalsthroughout the immunization period.

As Fig. 1 shows, the micro-complement fixationtiter rose slowly after the initial injection and rap-idly after each series of booster injections. By con-trast, the cross-reaction specificity of the antisera, asmeasured by the immunological distance betweenthe Klebsiella and Escherichia alkaline phosphates,declined slowly and reached a constant value of ap-proximately 55 after 10 weeks of immunization. Inconsequence, all further micro-complement fixationexperiments described in this article were done withantisera from rabbits immunized for at least 10weeks.Antiserum purity. The antisera were tested

against both unfractionated extracts of cells and pu-rified alkaline phosphatase by immunodiffusion,immunoelectrophoresis, and micro-complement fixa-tion. As noted in reference 8, only rabbit 2 produceddetectable amounts of antibodies to macromole-cule other than alkaline phosphatase. The concentra-tion of these antibodies was too low to interfere withour micro-complement fixation tests.

RESULTSImmunodiffusion. The alkaline phospha-

tases of all strains tested except Erwinia caro-tovora, Aeromonas sp., and Pseudomonas mal-tophilia produced a precipitin line with anti-sera to the alkaline phosphatases of Esche-richia or Klebsiella. The lines produced bySerratia, Providencia, Proteus, and Erwiniaspecies (except Erwinia carotovora) were gen-erally quite faint in comparison with the in-tense lines produced by Escherichia, Klebsi-ella, and Citrobacter species.

Tests for spur formation were carried out inessentially the manner described by Gasserand Gasser (17). Representative results aregiven in Fig. 2 and 3. With the E. coli antise-rum, all strains of E. coli tested (see Table 1)reacted identically. However, as Fig. 2 shows,the precipitin line produced by the homolo-gous E. coli phosphatase formed a spur overthe line given by the phosphatase of any otherbacterial species tested. The simplest interpre-tation of this observation is that E. coli phos-phatase bears several antigenic sites, onlysome of which are shared with the phospha-tases of other species. The lines produced byKlebsiella and Citrobacter phosphatases, inturn, spurred over the lines produced by thephosphatases of the other bacteria listed inFig. 2. Thus it is likely that Klebsiella andCitrobacter phosphatases share several anti-genic determinants with the E. coli phospha-tase that are not possessed by the phospha-tases of other bacteria listed in Fig. 2. Similarresults were obtained with the Klebsiella anti-serum as Fig. 3 shows. It thus appears that on

6000ka,

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2000

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00

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80

60F

40F

20

o10 20 30

Weeks

FIG. 1. Dependence of titer and specificity of an-tisera to Klebsiella aerogenes 1033 alkaline phospha-tase upon length of the immunization period. Theproperties of antisera from two rabbits were meas-

ured as a function of immunization time and aver-

aged. The rabbits received their first injection at 0

weeks and booster injections at the times indicatedby arrows. The titer is defined as the antiserum dilu-tion which results in 50% complement fixation withthe homologous antigen. The immunological dis-tance is that between K. aerogenes and Escherichiacoli alkaline phosphatases.

the basis of immunodiffusion tests the phos-phatases of Klebsiella, Citrobacter, and Esche-richia are more closely related to each otherthan to the phosphatases of Serratia, Proteus,Erwinia, Providencia, and Enterobacter lique-faciens.Micro-complement fixation. Essentially

similar results were obtained with the micro-complement fixation test. As Table 2 shows,antiserum to E. coli phosphatase reacts betterwith E. coli phosphatase than with other phos-phatases, and the heterologous phosphatasescan be clearly divided into two groups on thebasis of reactivity with this antiserum. Thesame two groups are evident from the resultsobtained with the antiserum to Klebsiellaphosphatase.

All naturally occurring strains tested withinthe species E. coli behaved nearly identicallyin micro-complement fixation tests (58; G. T.Cocks, Ph.D. thesis, Univ. of Calif., Berkeley,1971). Thi indicates that the strains probablydiffer froir each other by no more than one ortwo aminb) acid substitutions (8). Likewise,many Klebsiella strains were indistinguishable

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Escherichia

Enterobacter1 iquifaciens

-v 1Serratia

T1 Proteus

Erwiniaamylovora

I Erwiniaherbicola (?)

anti L.scherichia coli

FIG. 2. Cross-reactions detected by the Ouchterlony immunodiffusion technique with antiserum to Esche-richia coli alkaline phosphatase and the phosphatases of various enterobacteria. The symbols -7, -T-, and +refer to the results obtained when two phosphatases are placed in adjacent wells that are equidistant from athird well which contains, the antiserum. When the two resulting precipitin lines meet they may either mergewithout spur formation (i ), or one line may form a spur over the other (-r), or the two lines may cross eachother giving rise to a double spur (+). For example, in the top left part of the figure we see that E. coli phos-phatase forms a precipitin line that spurs over the line produced by Klebsiella phosphatase.

from the reference strain (K. aerogenes 1033) bythis test (43). Other species of Klebsiella, suchas K. pneumoniae, K. mobilis, and K. rubi-acearum, produced phosphatases whose av-erage immunological distance from the refer-ence phosphatase was 30. For a full presenta-tion of strain and species variation in theKlebsiella group see reference 43.

Electrophoresis. The immunological differ-ences among the phosphatases listed in Table2 are accompanied by electrophoretic differ-ences. Bacterial extracts were subjected toelectrophoresis in starch gel at two pH values,and the position of the phosphatases on thegels was determined by a specific staining pro-cedure. Results of electrophoresis at pH 7 areshown diagrammatically in Fig. 4. This buffersystem gave better resolution and inactivatedfewer enzymes than did Poulik's pH 8.6system.

All E. coli strains tested showed three bands

of alkaline phosphatase activity, their relativeintensities depending on the cultural condi-tions (40); these three phosphatases result fromminor posttranslational modifications of asingle gene product (39, 40). In most other spe-cies a single band of alkaline phosphatase ac-tivity was observed.

Intraspecific variability among strains inalkaline phosphatase mobility was generallyquite limited, as the results obtained withstrains of Erwinia amylovora, E. carotovora,and Serratia show. Furthermore, all E. colistrains tested were electrophoretically iden-tical (results not shown).

Intrageneric variability was marked. Proteusmorgani, for example, was very different fromother Proteus species in phosphatase mobility.Erwinia herbicola (?) was dramatically dif-ferent from other Erwinia species, whose alka-line phosphatases are unusually basic. In ourlimited survey, no two species had alkaline

Klebsiella

Citrobacter

T-

T1

-F

-F

-F

7

+-F

-F

T1

-F

-F

+H

T1 -H-+

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ALKALINE PHOSPHATASE: ENZYME EVOLUTION

Klebsiel la

Citrobacter

iSerratia

Proteusmorgasiii

T1FProteusvulgaris-mirabilis

I-I Erwinia

Enterobactermmm liquifaciens

anti Klebsiella aerogenes

FIG. 3. Cross-reactions detected by the Ouchterlony immunodiffusion technique with antiserum to Kleb-siella aerogenes alkaline phosphatase and the phosphatases of various enterobacteria.

phosphatases whose mobilities coincided attwo different pH values. This observation,coupled with limited intraspecific variability,indicates a possible use of phosphatase electro-phoresis in identification of bacterial isolates(G. T. Cocks, Ph.D. thesis, Univ. of Calif.,Berkeley, 1971). Others have already suggestedthat electrophoresis of specific enzymes beused in bacterial classification (4, 6).Acid phosphatase. Four of the bacterial

extracts exhibited no alkaline phosphatase ac-

tivity. However, these extracts did containacid phosphatases, identifiable both in crudeextracts and after starch gel electrophoresis bytheir sensitivity to inhibition by fluoride (45)and their low pH optimum, as shown in Table3. The electrophoretic mobilities of these en-zymes are given in Fig. 4. Among these specieswas Salmonella typhimurium which has al-ready been reported to lack alkaline phospha-tase (39). Extracts of these four strains failedto give precipitin lines with antibodies to E.coli alkaline phosphatase. We conclude thatthese strains do not have an inducible alkalinephosphatase under the usual conditions.

DISCUSSIONSequence-immunology correlation. Within

the past year a rationale has been provided forthe use of complement fixation and precipitintechniques in studies of biological classifica-tion and evolution. The rationale emerges fromthe demonstration that these techniques can

simply and economically measure the approxi-mate degree of sequence resemblance betweenrelated proteins. This has been shown mostclearly for a homologous series of lysozymes.The immunological distance (y) between anytwo lysozymes is related to their percentagedifference in amino acid sequence (x) by thefollowing equation y c 5x (32, 33). A correla-tion between sequence resemblance and im-munological resemblance is also evident fromprecipitin and complement fixation studieswith other proteins (33), including a homolo-gous series of cytochromes c (26), as Pragerand Wilson (33) have pointed out. There are

disadvantages to the immunological approachfor measuring sequence resemblance amongproteins. First, proteins that differ by more

than 30 to 40% in amino acid sequence gener-

Escherichia

-F

-F

T1

TF

-F

-T-

T1-

-F

T1

-1I

T1

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TABLE 2. Micro-complement fixation experimentswith enterobacterial alkaline phosphatases

Immunologicaldistance

Alkaline phosphatase source Anti-Anti- K eo

E. colia geneso-

Group 1Escherichia coli .........0...... 59Klebsiella aerogenes 1033 ....... 59 0Other Klebsiella strainsc ....... 48-65 0-32Citrobacter freundi ............ 59 58

Group 2Enterobacter liquefacienSd ...... 118 > 185Serratia marcescens ........... 121 186Proteus vulgaris ......... ...... 162 192Proteus morgani ............... 180 193Proteus mirabilis ............. 188Erwinia amylovora ............ 161 > 185Erwinia herbicola (?) ........... 150

a The antiserum used was a mixture composed offour antisera (prepared by immunizing rabbits 1, 2,001, and 002 for 16 to 19 weeks and mixed in inverseproportion to their homologous titers)."The antiserum used was a mixture of two anti-

sera (prepared by immunizing rabbits 2094 and 2095for 11 weeks and mixed in inverse proportion totheir homologous titers).

c Data taken from reference 43.d The phosphatase of this strain also reacted ex-

tremely weakly with antisera to K. pneumoniae andK. mobilis (Enterobacter aerogenes) phosphatases(43).

ally fail to exhibit cross-reactivity. Second, thecorrelation between sequence resemblance andimmunological resemblance is not perfect.Third, in order to obtain reproducible meas-urements of immunological resemblance, it isdesirable to use antisera prepared by immu-nizing at least four rabbits for a period of atleast 3 months with each reference antigen.Despite these disadvantages, the immunolog-ical approach is for many purposes a practicalalternative to the far more difficult and expen-sive approach of direct sequence comparison ofbacterial proteins.Evolutionary implications. Our immuno-

logical comparisons of enterobacterial alkalinephosphatases have evolutionary implications.The rate of evolutionary change in antigenicdeterminants of alkaline phosphatase seems tohave been roughly equal to that observed withother enterobacterial proteins. This hypothesisarises from the fact that the immunologicalrelationships reported here among the alkalinephosphatases of enterobacteria agree semi-quantitatively with those found for other en-

zymes. Five other enzymes have been surveyedby comparative immunological techniques inthis group of bacteria. In all cases the referenceantigen was the E. coli enzyme and in mostcases only a single antiserum was used. On thebasis of enzyme precipitation experimentswith antiserum to E. coli f,-galactosidase, ithas been stated that the E. coli enzyme showsstrong immunological resemblance to the f3-galactosidases of Shigella sonnei and Kleb-siella, but little or no resemblance to those ofbacteria outside the family Enterobacteriaceae,such as Aeromonas formicans (34), Lactoba-cillus bulgaricus, and Bacillus megaterium (9,35). In double-diffusion experiments with anantiserum to arginine decarboxylase, the en-zymes of E. coli and Shigella boydi were indis-tinguishable (E. V. Groman, Ph.D. thesis,Univ. of California, Berkeley, 1971). Each ofthese enzymes produced a precipitin line thatspurred weakly over the line given by the argi-nine decarboxylases of Salmonella, Arizona,and Citrobacter. The immunodiffusion reac-tions of the latter three bacteria were compa-rable in strength to those of Citrobacter phos-phatase with antisera to E. coli phosphatase.Similar experiments were carried out with an-tisera to two immunologically unrelated en-zymes (T and G) involved in protein synthesis(19). It was observed that extracts of Shigella,Salmonella, Klebsiella, and Serratia producedprecipitin lines with antisera to E. coli T andG, whereas extracts of Proteus gave a veryweak reaction. As was the case with alkalinephosphatase, extracts from bacteria not be-longing to the family Enterobacteriaceae failedto give precipitin lines with the anti-E. colisera. Another protein, the a subunit of trypto-phan synthetase, exhibits a similar range ofcross-reactivity, as determined by micro-com-plement fixation tests with antisera to the E.coli protein (29). These authors found averageimmunological distances of 2 for Shigella dy-senteriae, 64 and 65 for Salmonella typhimu-rium and Klebsiella and > 100 for Serratia.The Klebsiella and Serratia values are instriking agreement with the phosphatase re-sults.The above comparative studies on six di-

verse enzymes, with E. coli as a referencepoint, were all conducted with precipitin orcomplement fixation methods. These twomethods are now known to be equally effectivein detecting cross-reactions (33); that is, therange of the two methods is similar. In bothcases, the proteins that differ by more than40% in amino acid sequence generally fail toexhibit cross-reactivity (33). It thus appears,

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Escherichia coli C90F1

Shigella boydi

Salmonella typhimurium

Arizona arizonae

Citrobacter freundi

Citrobacter ap.

Klebsiella aerogenes 1033Enterobacter liquefaciens

Enterobacter cloacae

Serratia marcescens 274

Serratia sp. S

Serratia sp. pink

Serratia sp. white

Proteus vulgaris

Proteus mirabilis

Proteus morgani

Providencia alkalifaciens

Providencia sp.

Erwinia carotovora 495Erwinia carotovora EC-1

Erwinia amylovora 15580

Erwinia amylovora FB-1

Erwinia amylovora FB-9

Erwinia quercina

Erwinia herbicola (?)

Aeromonas sp.

Pseudomonas maltophilia

100

Relative Distonce Moved

FIG. 4. A diagram summarizing the results of horizontal starch gel electrophoresis experiments conductedat pH 7 with extracts of 27 bacterial strains. Dashed line indicates the position at which samples were ap-plied. Anode was on the right and cathode on the left. Closed ellipses represent alkalinephosphatase, open el-lipses represent acid phosphatase. Acid phosphatase .staining was faint under the usual alkaline conditions,was inhibited by the presence of 0.01 M NaF, and was greatly enhanced relative to alkaline phosphatase byuse of an 0.5 M sodium acetate buffer, pH 5.0, to dissolve the chromogenic reagents. The scale represents thedistance moved by each enzyme relative to the most anodal Escherichia coli enzyme (defined as 100).

that the E. coli proteins studied to date maydiffer from their counterparts in other bac-terial families by more than 40% in amino acidsequence. Although the data are fragmentary,none of the proteins tested seems to haveevolved markedly slower or faster than alka-line phosphatase.

Peptide mapping studies can sometimesgive semiquantitative information concerningdegrees of sequence resemblance among pro-teins. This technique has been applied to the a

subunit of tryptophan synthetase (10) withresults in qualitative agreement with theimmunological data cited above (29). A secondenzyme of tryptophan biosynthesis has beensubjected to a comparative peptide mappingstudy with results indicating that this enzymehas evolved at a rate equal to that observed forthe a subunit of tryptophan synthetase (28). Inboth cases, the Klebsiella and Salmonella en-zymes are equally distinct from the E. colienzyme. It thus appears possible that all sevenenterobacterial enzymes tested have been

TABLE 3. Tests for acid phosphatase

Effect of Effect ofSource of extract fluoride pH (acid/

(% inhli- alkaline)"bition)a

Escherichia coli C9OF 1 18 0.10Salmonella typhimurium 95 7.26Arizona arizonae 97 8.54Shigella boydi 90 1.94Enterobacter cloacae 56 79.6

aThe rate of hydrolysis of nitrophenyl phosphatecatalyzed by sonic extracts of bacteria was measuredat pH 8.5. The per cent activity remaining when theassay mixture was made 0.01 M in NaF was thendetermined.bThe ratio of the rate of hydrolysis of nitrophenyl

phosphate, in micromoles per minute, catalyzed bythe bacterial extracts at pH 5.8 and 8.5.

evolving at rates which are not greatly dissim-ilar.

This result might seem surprising becauseamino acid sequence arnalysis has shown that

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in vertebrates some proteins evolve muchfaster than others. Fibrinopeptides, for exam-ple, evolve hundreds of times faster than cer-tain histones (11). However, few of the verte-brate sequence studies have been performed onenzymes. These few studies indicate that ex-tracellular monomeric hydrolases, like ribonu-clease (11, 12) and lysozyme (12, 32), haveevolved more slowly than the fibrinopeptidesand a few times faster than intracellular meta-bolic enzymes such as triosephosphate dehy-drogenase and cytochrome c. Comparativeimmunological work has demonstrated thataldolase and glutamic, lactic, and malic dehy-drogenases evolve at a rate similar to triose-phosphate dehydrogenase (22, 36, 49). Al-though there is some immunologically detect-able variability in rates of enzyme evolution,in both bacteria (42) and vertebrates (49), andalthough it is desirable to obtain more defini-tive amino acid sequence information re-garding relative rates of evolution of differentenzymes, we suggest that a tendency for intra-cellular metabolic enzymes to evolve in ap-proximate unison is already evident in bothvertebrates and enterobacteria.DNA homologies. In vitro DNA hybridiza-

tion studies (7, 27) provide more direct meas-urements of overall genetic similarity amonggenera of enterobacteria. The pattern of inter-generic relationships measured by this tech-nique agrees with that derived from the pro-tein comparisons cited above. Stanier et al.(42) also pointed out a close correspondencebetween the results of DNA and protein com-parisons among species of Pseudomonas.

Taxonomic implications. It appears fromthe foregoing that in order to measure the ge-

netic relatedness of bacterial strains, one needsto compare only a few of their enzymes immu-nologically. Comparative studies of even a

single enzyme should be quite valuable. Ac-cordingly, we believe it is justifiable to recom-

mend the following revisions in the classifica-tion of enterobacteria on the basis of our alka-line phosphatase data and the other enzyme

and nucleic acid comparisons cited.Escherichia, Shigella, Salmonella, Arizona,

Citrobacter, and Klebsiella are a closely re-

lated group of genera. Their macromolecularsimilarities to one another are stronger thanthose to other enterobacteria. The numericaltaxonomic studies of Krieg and Lockhart (23)support this conclusion. We therefore propose

that all these genera be included in the tribeEscherichiae. Enterobacter liquefaciens andSerratia are excluded from this tribe and so

are all the species of Proteus, Erwinia, andProvidencia we tested. The idea that E. lique-faciens has been misclassified is supported byother evidence (5, 13).To define additional taxonomic groupings

within the Enterobacteriaceae it is essential toprepare antisera against purified proteins fromnumerous other species in the family.

Phylogenetic implications. Given the as-

sumption that the enzyme and nucleic acidsimilarities found among enterobacteria pro-

vide reliable estimates of their overall degreesof genetic similarity, it is worthwhile to draw a

tentative phylogenetic tree of this bacterialgroup. Other workers have shown that phyloge-

ESCHER/CHIA

SHIGELLA

SALMONELLA

CITROBACTER

KLEBS/ELLA

SERRATIA

PROTEUS

ERWIN/A

200 150 100 50IMMUNOLOGICAL DISTANCE

40 30 20 10% PROTEIN SEQUENCE DIFFERENCE

I

0

0

FIG. 5. Phylogeny of the enterobacteria, based on immunological resemblances among their alkaline phos-phatases and tryptophan synthetase a-subunits. The protein sequence difference scale is derived from theimmunological distance scale by using the Prager and Wilson equation (32).

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ALKALINE PHOSPHATASE: ENZYME EVOLUTION

netic trees based on macromolecular compari-sons often resemble those based on fossil evi-dence (11, 16, 37).The probable pattern of branching of line-

ages leading to the modern genera of entero-bacteria is shown in Fig. 5. As enterobacteriaof group I are genetically very similar it isproposed that they are derived from a common

ancestor that lived more recently than did thecommon ancestor of all enterobacteria. A stillmore recent common ancestry is proposed forShigella and Escherichia because of their ex-

tremely close macromolecular similarities (4,7, 10, 29). The tree is obviously incompletebecause of the fragmentary nature of the avail-able comparative macromolecular data andbecause most comparative work has been donewith only E. coli as a reference point.

ACKNOWLEDGMENTS

Cynthia Chan carried out some of the experiments andmade many contributions of encouragement, discussion, andmaterials. We thank Linda Ferguson for technical assist-ance.

G.T.C. was the recipient at various times of NationalScience Foundation and National Institutes of Health pre-

doctoral fellowships, and a National Institute of Healthtraineeship. The research was supported by National Sci-ence Foundation grant 13119.

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