Aldolase Lactic Acid Bacteria: Case History Use ...iments was obtained by growing the various...

BACTERIOLOGICAL REVIEWS, Dec. 1973, p. 453-478Copyright 0 1973 American Society for Microbiology

Vol. 37. No. 4Printed in U.S.A.

Aldolase of Lactic Acid Bacteria: a Case Historyin the Use of an Enzyme as an Evolutionary

MarkerJACK LONDON AND KIMBERLY KLINE

Microbial Physiology Section, Laboratory of Microbiology and Immunology, National Institute of DentalResearch, Bethesda, Maryland 20014

INTRODUCTION ............................................................... 453MATERIALS AND METHODS ................................................. 454Organisms.454

Cultivation and Maintenance of the Organisms. 455

Purification of the FDP Aldolase.455Storage of Purified Aldolase and Cell-Free Extracts.456Disc GelElectrophoresis.456

Enzyme Assays.456Molecular Weight Determination.456Preparation of the Streptococcal Antialdolase Serum.457Immunoelectrophoresis.457Immunodiffusion.457Interpretation of Immunodiffusion Results.457Microcomplement Fixation.458

RESULTS.459Physical and Biochemical Properties of the FDP Aldolases of Lactic Acid Bac-

teria.459Demonstration of Immunological Relatedness Among FDP Aldolases of the

Lactic Acid Bacteria by Immunodiffusion.461Quantification of the Immunological Relatedness Among Aldolases of the Lacto-

bacillaceae by Microcomplement Fixation.467DISCUSSION.469Molecular Variation During the Course of Aldolase Evolution.469Preparation of a Phylogenetic Map for the Homofermentative Lactic Acid Bac-

teria Based on Structurally Related Proteins.470Does the Phylogenetic Map Accurately Reflect Interrelationships Among the

Lactobacillaceae?.471Implications of the PhylogeneticMap. 472

Protein Relatedness Studies Among Other Bacteria ....... . . . . . . . . . . . . . . . . . . . 473Rates of Evolution of Prokaryotic Proteins.475

CONCLUSIONS.475ACKNOWLEDGMENTS.476ADDENDUM.476LITERATURE CITED.476

INTRODUCTION

The first serious attempt to classify bacteriawas undertaken by Ferdinand Cohn in 1872(16). Although his system of classification isstill recognized as a scholarly work (77, 78),Cohn's lasting contributions to bacterial tax-onomy can be found in his penetrating philo-sophical discussions of this discipline (16, 17).The binomial system of nomenclature adoptedby Cohn for prokaryotes was similar to thatused for plants and animals; however, he wascareful to point out that fundamental differ-

ences existed between the two systems of classi-fication. Unlike the higher forms of life, bacteriaexhibit developmental stages of so rudimentarya kind that they cannot serve as guidelines forarranging them in a natural or phylogeneticsystem. Cohn emphasized this point and thefact that his classification scheme was basedexclusively on the morphological characteristicsof the representative bacterial types by referringto his genera as "form" genera. The accretion ofnewly described species of bacteria compelledMigula (51) to modify and enlarge Cohn'sclassification system. He used physiological as

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well as morphological traits to differentiate theever increasing number of recognizable bacte-rial species; in doing so, Migula established aprecedent which is still followed today. Orla-Jensen (57, 58) relied even more heavily on theuse of nutritional and biochemical traits todifferentiate the taxonomic groups in his classi-fication system.Taxonomic schemes grew apace with the

expanding biochemical and nutritional litera-ture and eventually evolved into determinativekeys which carefully avoided all allusions tonatural relationships. However, once it wasrealized that bacteria possess a limited numberof common biosynthetic and energy-producingpathways (37), the notion occurred to microbi-ologists (38, 77) that the grouping of microorga-nisms in accordance with their major physiolog-ical traits may represent a gross sort of naturalclassification. Two major scientific discoveries,a chemical interpretation of the structure of thegenome (80) and the deciphering of the geneticcode (55), provided a means by which theseintuitive systems of classification could be veri-fied. The role of the chromosome as regulator ofthe various development processes in plants andanimals had been appreciated, if not fullyunderstood, since the work of Mendel (50), andthe integration of this body of knowledge withthe newly discovered chemical basis of genefunction presented the first opportunity to com-pare simple or complex forms of life at the mostfundamental level yet attained. By determiningthe degree of physical homology between two ormore types of microbial deoxyribonucleic acid(DNA), the taxonomist could now preciselymeasure the extent of phylogenetic relatednessamong those bacteria from which the genome orgenome segments had been isolated.Within the last decade, however, the limita-

tions of this technique have become apparent,and thus far, only interspecific (23, 35, 52, 68)and, in one instance, intergeneric relationships(11) have been established among large familiesof bacteria thought to have natural affinities.Hybridizations between DNA and iibosomalribonucleic acid (rRNA) as suggested by Man-del (45) and Stanier (71) will probably circum-vent the restrictions imposed by the use ofDNAduplexes, since rRNA appears to be highlyconserved. The failure to demonstrate DNAhomology above the species or genus level canprobably be attributed to the degenerate andredundant nature of the genetic code (20, 21, 24,34, 69), whereby the structure of the genome, asmanifested by the nucleotide base sequence,can change significantly without altering thehereditary information contained therein. If thegenetic message is, in fact, conserved, then it

follows that the structure of the gene product isconserved to the same extent. Comparativestudies of isofunctional proteins of higher orga-nisms have revealed that in many instancesstructural homology still exists in evolutionarilydiverse organisms (2, 4, 24, 54, 84, 85). Elabo-rate phylogenetic classification systems basedon protein homologies have been created andthese, not surprisingly, are concordant with theclassification schemes based on paleontologicalstudies.

Using much the same rationale as the bio-chemical evolutionists, microbiologists be-gan applying the same *techniques to prob-lems of bacterial evolution. These began withcomparative physical studies of specific en-zymes in which such properties as proteincharge (8, 9, 25, 33, 83), kinetic characteristics(65, 74), or regulatory mechanisms (32, 40) werecompared. Although such studies yield usefulinformation which aids in the differentiation ofcertain microbial genera and species, in con-trast with DNA or protein homology studies,there is no way the data can be quantitated toprovide a means of accurately assessing intra- orintertaxon differences. Recently, it has beensuggested (42) that studies of physical proper-ties of proteins be combined with structuralhomology studies; such studies will provide afirm foundation upon which natural relation-ships of prokaryotes can be based. One of themajor benefits to be derived from a naturalsystem of classification is that it will enabletaxonomists to evaluate the accuracy of themore utilitarian determinative keys. Stanier(71) summarized this point in the followingfashion: "When sufficient evidence about aparticular phenotypic group has been accumu-lated at both the genetic and epigenetic levels, Ibelieve that we shall be in a position to decidewithout much ambiguity in how far the pheno-type resemblances among its members are theconsequence of evolutionary filiation, and inhow far they are expressions of convergence."

In this report, we discuss some of the ways inwhich contemporary biochemical and immuno-logical techniques are being used to create anatural system of classification for bacteria.The pitfalls and limitations of these proceduresare also discussed.

MATERIALS AND METHODS

OrganismsThe species of bacteria used in this study and

their sources are listed in Table 1. Wheneverpossible a typical representative of each specieswas selected for this work.

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TABLE 1. Strain designations and sources of microorganisms

Organism Source Organism Source

Genus StreptococcusS. agalactiae .............S. asalignus ..............S. bovis ..................S. cremoris ...............S. diacetilactis ...........S. durans ................S. dysgalactiae ...........S. equi ...................S. equinus ...............S. equisimilis ............S. faecalis ................

S. faecium ...............S. lactis....S. mitis ..................S. mutans ................S. pneumoniae ...........S. pyogenes ..............S. salivarius ..............S. sanguis ................Streptococcus sp. (groupH) .....................

Streptococcus sp. (groupL) .....................

S. thermophilus ..........

Genus PediococcusP. acidilactici ............

P. cerevisiae ..............P. homari ................P. parvulus ...............P. pentosaceus ...........Pediococcus sp............

ATCC 13813ATCC 8059ATCC 9809,15351ATCC 19257ATCC 11007ATCC 19432ATCC 9926ATCC 6580ATCC 9812ATCC 9542NIH, MR: R. Deibel, N83;

E. Sharpe, Cl; van Niel,H.3.1

E. Sharpe, K6A, CHI, N55ATCC 19435ATCC 15909,15910, 15912NIH, K1R, SL1, O1H1ATCC 6308ATCC 14289ATCC 13419,9222; NIH 112ATCC 10556

ATCC 8144

ATCC 9932ATCC 19258

ATCC 25740, 25741, 25742,25743

ATCC 8042ATCC 10400ATCC 19371ATCC 25744E. Garvie, 559,990

Genus LactobacillusL. acidophilus ............L. brevis .................L. bulgaricus .............L. casei ..................

L. coryniformis ...........L. curvatus ...............L. cellobiosus .............L. delbrueckii ............L. fermentum ............L. helveticus .............L.jensenii ................L. jurgurti ................L. lactis ..................L. mali ...................L. plantarum .............L. leichmannii............L. salivarius ..............L. xylosus ................L. zeae ...................

Other organismsArthrobacter globiformisBifidobacterium aster-

oides ..................B. bifidum ...............B. indicum .............Corynebacterium xerosis..Microbacterium thermo-sphactum .............

Sporolactobacillus inu-linus ...................

Staphylococcus aureus ....

ATCC 4356,19992ATCC 14869ATCC 11842Rogosa, OC91, OC45, Cl-11,Cl-15

Gasser, 64HKandler, M40van Niel, F.3.2, F.3.3, F.3.4Kandler, M30, M34Kandler, MlATCC 11739ATCC 9649ATCC 14931ATCC 15009ATCC 25258ATCC 5214ATCC 12315ATCC 27053ATCC 14917ATCC 4797ATCC 11741ATCC 15577ATCC 15820

ATCC 8010

ATCC 25910ATCC 15696ATCC 25912ATCC 7094

ATCC 11509

ATCC 15538ATCC 12600

Cultivation and Maintenance of theOrganisms

With the exception of members of the genusBifidobacterium, stock cultures of all lactic acidbacteria (lactobacilli, streptococci, and pedi-ococci) were carried in the reinforced litmusmilk medium of Rogosa (private communica-tion), which contains 10% (wt/vol) skim milkpowder, 0.5% (wt/vol) yeast extract, 0.5% glu-cose, 0.075% (wt/vol) litmus (Difco), and 7.1%(wt/vol) CaCO,. Cultures were incubated for 18h at 30 or 37 C and were subsequently stored at4 C. The bifidobacteria were cultivated in thiolmedium (Difco) at 37 C.

Material for small-scale enzymological exper-iments was obtained by growing the variousorganisms in 500-ml screw-capped bottles or in1-liter Erlenmeyer flasks containing 400 ml ofthe appropriate medium at 30 or 37 C. Thelactobacilli and homofermentative pediococciwere grown in MRS medium (Difco); most ofthe streptococci were grown in a complex me-dium which has been described elsewhere (41).

Streptococcus mutans was grown in the com-plex medium supplemented with 0.05% (wt/vol)Tween 80, whereas Streptococcus (Diplococcus)pneumoniae was cultivated in heart infusionbroth (Difco) supplemented with 0.5% (vol/vol)horse serum and 1% (wt/vol) glucose. Thecomplex streptococcal medium was also usedfor the cultivation of 400-liter batches of Strep-tococcus faecalis strain MR. Centrifugation ofthe cultures yielded cell pastes which providedthe starting material for large-scale purificationof the fructose diphosphate (FDP) aldolase.

All of the other microorganisms were grownand maintained according to procedures setforth in the American Type Culture Collectioncatalogue (10th edition).

Purification of the FDP AldolaseCell-free extracts of S. faecalis MR were

prepared by the sonic disruption of 25 g (wetweight) of cells suspended in 25 ml of 0.05 Mbis-Tris [2.2-bis(hydroxymethyl)-2, 2', 2'-

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nitrilotriethanol] buffer (Aldrich Chemicals),pH 6.5, containing 10 mM fl-mercaptoethanol(BTME). The crude extract was centrifuged for30 min at 30,000 x g, and the clarified superna-tant was decanted and centrifuged for 60 min at100,000 x g. An acid precipitate was obtainedby dialyzing the crude extract supernatantagainst 0.05 M tris(hydroxymethyl)aminometh-ane (Tris)-hydrochloride-acetate buffer, pH4.5, for 12 h at 4 C. The resultant precipitatewas removed by centrifugation and discarded.After the pH of the supernatant fluid wasadjusted to 6.5 by the addition of 2 N NaOH,0.05 volumes of M MnCl2 were added to theextract and the resultant precipitate was re-moved by centrifugation and discarded. Bymaintaining a temperature of 4 C, sufficient(NH4)2SO4 was slowly dissolved in the superna-tant fluid to give a concentration of 60% ofsaturation. After the removal of the precipi-tated protein, the (NH4)SO concentration ofthe supernatant fluid was increased to 80% ofsaturation. The protein precipitate was re-moved by centrifugation, dissolved in 10 to 15ml of BTME, and dialyzed for 12 h against 1liter of the same buffer.The dialyzed protein solution was applied to

a DE23 (Reeve-Angel) column (2.5 by 40 cm)which had been equilibrated with BTME, andthe enzyme was eluted with BTME and a linear0- to 0.6-M KCl gradient. FDP aldolase waseluted from the DE23 column by 0.38 M KCl.After the fractions containing the highest levelsof enzyme activity were pooled, the volume ofthe resulting solution was reduced to 5 ml byconcentration in a 10-M Amicon filtration appa-ratus, and the concentrate was applied to aSephadex G-200 upward-flow column (2.5 by 90cm). Enzyme activity was eluted with BTMEbuffer containing 0.1 M KCl. The peak activityfractions were pooled and reduced in volume forfurther treatment.The Buchler preparative polyacrylamide gel

(PAG) electrophoresis apparatus was used toobtain samples of FDP aldolase which wereelectrophoretically pure. Between 3 and 5 ml ofthe concentrated Sephadex column eluate (30mg of protein) were layered on a 10% PAGcolumn (9 cm long) prepared with the neutralbuffer system described by Chrambach andRodbard (13). A current of 22 mA was applied tothe column, and 5-ml fractions were collectedfrom the cathodal end of the column. Thisprocedure resulted in a loss of from 40 to 50% ofthe enzyme activity, whereas electrophoresis ofeluates in conventional anionic buffer (22)caused a 90% inactivation.

Storage of Purified Aldolase and Cell-FreeExtracts

Both in the pure state and in cell-free ex-tracts, the S. faecalis aldolase was extremelysensitive to freezing and thawing. Three cyclesof this treatment resulted in a 90% loss of theinitial activity. The addition of 20% (vol/vol)glycerol stabilized the enzyme in solution andpermitted repeated freezing and thawing withno detectable loss of activity. Thereafter, glyc-erol was routinely added to all aldolase prepara-tions prior to storage at -40 C.

Disc Gel ElectrophoresisThe electrophoretic mobility rates of the

various lactic acid bacteria aldolases were mea-sured with a Buchler disc gel polyanalyst byusing the anionic buffering system of Davis (22)or the neutral buffering system cited earlier.From 25 to 60 ,g of crude extract protein or 5 to20 Ag of pure streptococcal aldolase were layeredon PAG columns, and a current of 2.5 mA wasapplied to each column. Protein was detectedby staining with Coomassie blue (43). The posi-tion of FDP aldolase was located by bathing thePAG column in 5 ml of a solution containing:0.05 M Tris-hydrochloride buffer (pH 7.3), 0.01M potassium acetate; 0.3 mM fl-mercaptoetha-nol; 0.001 M nicotinamide adenine dinucleotide(NAD), 0.02 M FDP, 0.035 mg of triosephos-phate isomerase (Sigma), 0.5 mg of 3-phospho-glyceraldehyde dehydrogenase (Sigma), 0.01 Msodium arsenate, 0.004 mg of phenazine metho-sulfate, and 1 mg of nitroblue tetrazolium.Sodium dodecyl sulfate (SDS) polyacrylam-

ide gel electrophoresis was carried out by theprocedure of Weber and Osborne (81).

Enzyme AssaysFDP aldolase activity was determined spec-

trophometrically by using the method of Groveset al. (30). A unit of activity is defined as theamount of enzyme required to oxidize 1 ,gmol ofreduced NAD per min. The order of addition ofingredients in the assay mixture was generallynot critical. However, the usual practice ofstarting the reaction with the addition of sub-strate resulted in the complete inhibition of thealdolase activity of Lactobacillus plantarumcell-free extracts. Aldolase activity was onlydetectable when the cell-free extract was usedto initiate the reaction.

Molecular Weight DeterminationThe molecular weights of the various aldol-

ases were estimated by the molecular sieve

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technique of Andrews (3) by using an upward-flow Sephadex G-200 column which had beenstandardized with ribonuclease, chymotryp-sinogen A, ovalbumin, bovine serum albumin,muscle aldolase, and human 7S gamma-globulin. The estimation of the molecularweight was independent of the protein concen-tration within the range used in these experi-ments.

Preparation of the StreptococcalAntialdolase Serum

Four 6-month-old male New Zealand whiterabbits each received a series of four intrader-mal injections consisting of 0.2 ml of electro-phoretically pure S. faecalis MR FDP aldolase(420 psg), 0.2 ml of complete Freund's adju-vant (Difco), and 0.01 ml of 1% methylatedbovine serum albumin (MBSA) over a 4-weekperiod. These were followed by two intravenousinjections consisting of 0.25 ml of aldolase and0.01 ml of 1% MBSA given at 10-day intervals.The levels of aldolase-specific antibodies pro-duced by the rabbits during the course ofimmunization were monitored by semiquantita-tive precipitin tests. The rabbits were bled fromthe central ear artery 1 week after the finalintravenous injection, and the blood was storedat 4 C overnight. The clotted erythrocytes andfibrin were removed by centrifugation, and theantistreptococcal aldolase serum (anti-SA) wasdivided into 5-ml samples and stored at -40 Cuntil used.

ImmunoelectrophoresisOne of the methods used to establish the

purity of the S. faecalis FDP aldolase wasimmunoelectrophoresis. The technique hasbeen described previously (43), and the onlymodification in the present procedure involvedthe use of 0.04 M Tris-hydrochloride, pH 8.8,instead of 0.04 M barbitol buffer, pH 8.2. Theupper well of the agar plate received 12 gg ofpure aldolase, while the lower well was chargedwith 40 ;tg of crude extract protein. Afterpassing a current of 5 mA through the plate for75 min, the center trough received 40 Mliters of a1:5 dilution of anti-SA. The plate was incu-bated for 18 h at 4 C in a high-humiditychamber; the results are shown in Fig. 1. Witheither source of antigen, only a single precipitinline was formed, indicating that the purifiedaldolase was free of contaminating protein.

ImmunodiffusionImmunological homology between the various

lactic acid bacteria aldolases and the S. faecalis

n.,

FIG. 1. Protein stain of antigen-antibody complexon immunoelectrophoresis plate. The upper well con-tains electrophoretically pure S. faecalis MR aldolase;the lower well contains crude S. faecalis MR extract.

MR aldolase was first detected by using theStollar and Levine (75) modification of theOuchterlony immunodiffusion method. Minormodifications of the procedure are publishedelsewhere (44). The sample wells were chargedwith solutions containing from 0.1 to 0.8 units ofaldolase activity, whereas the center well re-ceived sufficient anti-SA to produce sharp linesof precipitation. The convention of Gasser andGasser (26), the essential points of which aredescribed below, was used to collate and sum-marize the immunodiffusion data.

Interpretation of Immunodiffusion ResultsThe theoretical aspects of immunodiffusion

as they apply to this study have been discussedrecently by Gasser and Gasser (26) and requireno detailed elaboration here. However, a briefdescription of the three basic precipitation pat-terns and their significance will greatly simplifythe presentation of the data. If the interactionsof the reference (homologous) protein, an un-known protein, and the antiserum preparedagainst the homologous protein produce fusedor confluent lines of precipitation (Fig. 2A), theunknown protein possesses the same antigenicdeterminants as the homologous protein andthe two form.a group of identical specificity. It isalso possible for two or more heterologous pro-teins to interact and produce fused precipitinlines. For example, if heterologous proteins 1and 2 bearing antigenic determinants, A, B, C,D, U, V, and W and A, B, C, D, X, Y, and Z,respectively, are made to react with an an-tiserum prepared against a protein with thedeterminants A, B, C, D, E, F, and G, theantibodies will only recognize the first fourantigenic sites of the heterologous proteins andproduce confluent precipitates. Such proteinsare assigned to groups of apparent identicalspecificity. A precipitate with a single spur isproduced when one of the two antigens beingcompared has more determinants in commonwith the homologous antigen than its neighbor(Fig. 2B). This pattern is known as a reaction ofpartial identity and usually occurs when two or

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A.

C.FIG. 2. Three basic types of immune precipitates.

A, Confluent lines or reaction of identity. B, Singlespur or reaction of partial identity. C, Double spurs or

reaction of nonidentity.

more proteins have evolved in a sequential or

unidirectional fashion. The double-spurred pre-cipitate or reaction of nonidentity results from

the interaction of two heterologous proteinswhich share a number of antigenic determi-nants with the homologous protein but very fewor none with each other (Fig. 2C). This patternindicates that the two heterologous proteinshave evolved in a random or divergent fashionwith respect to the reference protein.

Microcomplement FixationThe microcomplement fixation assay (mCf)

of Wasserman and Levine (79) provides anaccurate means of measuring the immunologi-cal differences between two partially homolo-gous proteins; however, the information contentof the results is quite different from that ob-tained by immunodiffusion. In contrast to im-munodiffusion, mCf cannot differentiate be-tween sequential and random changes in theantigenic composition of two or more heterolo-gous proteins because this technique measuresthe interaction of the antigen and antibodyindirectly. The amount of complement fixed issimply a function of the number of antigenicdeterminants recognized by the antiserum. Forexample, if an antiserum prepared against areference protein bearing the determinants A,B, C, D, E, and F is used to compare twoheterologous proteins possessing the determi-nants A, B, C and D, E, F, respectively, theextent of immunological reactivity as measuredby mCf would be very similar or identical.However, the divergent nature of these twoproteins would not be detected by this test. Forthis reason, mCf is used in conjunction withimmunodiffusion experiments to corroborateand quantitate the gross immunological varia-tions detected by the latter.Guinea pig complement (GPC) was standard-

ized by the procedure of Hook and Muschel(29), and 2.5 50% complement fixation unitswere routinely used in the experiments. ThemCf system was standardized with the purealdolase as antigen and anti-SA; by using theformer in a range of 0.1 to 0.8 ,g and the latterat a dilution of 1: 75,000, 80% of the added GPCwas fixed at equivalence (0.3 Atg of antigen).Thereafter, the source of antigen was a cell-freeextract which had been first centrifuged for 30min at 30,000 x g followed by centrifugation for90 min at 200,000 x g to eliminate anti-comple-mentary activity (44). Cell-free extracts con-taining the homologous antigen were diluted togive a range of 0.8 to 8 ,gg of protein; extracts ofpediococci were diluted to give a range of 2 to 20mg of protein; and extracts of lactobacilli wereused in the range of 4 to 40 Atg of protein.Extracts of each organism surveyed were testedat least twice at three antiserum concentra-tions.


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The results of the mCf experiments were firstanalyzed to learn whether the percent of com-plement fixed was a linear function when com-pared with the log of the antiserum dilution. Ifthe value of the slope of the line obtained withthe heterologous (HET) antigen is the same asthat calculated for the homologous (HOM)antigen, a direct comparison of the data can bemade (67); an example of several such determi-nations is depicted in Fig. 3. The indices ofdissimilarity (ID) were calculated for the heter-ologous aldolases by using the following mathe-matical expression derived by Wilson et al. (85).

Index of dissimilarity

AB dilution HOMx % complement fixed HOM

AB dilution HETx % complement fixed HET

Since the ID value is an expression of the degreeof antigenic homology between the homologousand heterologous aldolase, the results representa comparative measure of structural homology.

RESULTS

Physical and Biochemical Properties of theFDP Aldolases of Lactic Acid BacteriaCharacterization of the pure S. faecalis

aldolase. The purified aldolase exhibited max-imum activity at a pH of 7.5. At this pH, theapparent Km for the substrate, FDP, is 0.8mM. Addition of 25 mM NH4Cl produces atwofold increase in the reaction rate in the pres-ence of 20 mM KCl. A kinetic analysis of thedata reveals that the ammonium salt only in-creases the maximum velocity of the reaction,the Ki, for FDP is not altered. As is the casewith other class II aldolases (65), ethylenedia-minetetraacetic acid (EDTA) inhibits catalyticactivity; however, the inhibition is unconven-tional in that the enzyme and EDTA do not in-teract immediately. To produce a 50% inhibi-tion of aldolase activity, the enzyme had to bepreincubated with 0.1 mM EDTA for 15 minprior to the addition of substrate.A number of nucleotide phosphates and inter-

mediate products of glucose catabolism weretested as potential regulators of the FDP aldol-ase. The following nucleotide phosphates-adenosine 5'-triphosphate (ATP), deoxyATP, adenosine diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, cy-tidine 5'-triphosphate, and inosine 5'-triphos-phate-had no demonstrable effect on aldolaseactivity in the range of 2.5 x 10-' M to 1 x108 M. Similarly, at concentrations of 5 x

90

80

60

Z 40uwtt:

I\sfoejoll

1N

ANTISERUMh DILUTION (0xl3)

FIG. 3. Relation of complement fixation to the logof the antibody concentration.

10' M to 1.5 x 10-2 M glucose-6-phosphate,fructose-6-phosphate, 6-phosphogluconate, ri-bose-5-phosphate, xylose-5-phosphate, pyru-vate, and phosphoenol pyruvate neither en-hanced nor inhibited the enzyme activity.Acetyl coenzyme A and acetyl phosphate werealso tested at levels ranging from 5 x 10- I M to5 x 10-3 M; neither had any effect on enzymeactivity.Compared with other aldolases (65), the

streptococcal enzyme with a molecular weightof 56,000 as estimated by Sephadex G-200chromatography is relatively small (Fig. 4).This value is concordant with that obtained byusing crude cell-free extracts. While the enzymemigrates as a single protein band in SDSpolyacrylamide gels (Fig. 5), the rate of migra-tion indicates that the aldolase has dissociatedinto two subunits which have a molecularweight of 28,000 each (Fig. 6).

Size determinations of other lactic acidbacteria aldolases. A comparative survey ofother lactic acid bacteria revealed the existenceof five molecular weight classes ofFDP aldolase(Table 2). These vary in molecular weight from56,000 to 176,000, and a minimum of two weightgroups has been found for each genus in thefamily. For example, the majority of speciestested in the genus Streptococcus possess analdolase which has a molecular weight of ap-proximately 56,000; however, the aldolase ofStreptococcus salivarius is greater by a factor of2.5 (molecular weight = 135,000). With threemolecular weight groups of aldolase (56,000,76,000, and 118,000) found among its members,the genus Lactobacillus is distinguished by thediversity of enzyme size. The pediococcal aldol-ases, on the other hand, exhibit the greatest sizedifference; the aldolase of Pediococcus parvulushas an estimated molecular weight of 58,000,

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8r

Muscle FDP Aldolose

Bovine Serum AlbuminS. foecolis FDP Aldolose

Ovlbumin

Chymotrypsinogen A

Ribonucleose

240 280 320 360 400ELUTION VOLUME (ml)

FIG. 4. Estimation of the molecular weight of thepurified S. faecalis MR aldolase by Sephadex G-200filtration.

A BFIG. 5. Protein stain of SDS-polyc

showing the position of the S. faecalsubunits. A, Aldolase alone. B, Aldcbumin standard.

6to0

-4

-J-J

02uj

BACTERIOL. REV.

B Bovine Serum Albumin

OvalbuminGlyceroldehyde -3-P

DehydrogenaseS foecolis FDP Aldolose

Ribonucleosehymotrypsin

02 04 06MOBILITY

08 10

FIG. 6. Determination of the molecular weight ofthe S. faecalis MR aldolase subunits by SDS-polyac-rylamide gel electrophoresis.

whereas the enzyme from other species has an

approximate molecular weight of 176,000.Aldolase activity could not be detected in

cell-free extracts of Pediococcus homari, Bifido-bacterium bifidum, Bifidobacterium asteroides,Bifidobacterium indicum, and the heterofer-mentative lactobacilli (Lactobacillus fermen-tum, Lactobacillus cellobiosus, and Lactobacil-lus brevis).

Representatives of each aldolase size group

OVALBUMIN were subjected to chemical treatments, whichincluded incubation in 0.1 M acetate bufferadjusted to a pH of 4.5, heating at 56 C for 10min, and absorption and elution from dieth-ylaminoethyl-cellulose. None of these treat-

ALDOLASE ments altered the molecular weight of therespective aldolases. Furthermore, the mixingof two cell-free extracts containing a small andlarge aldolase, respectively, had no effect on

either enzyme; Sephadex G-200 chromatogra-phy easily separated both activities.FDP affinity constants of the five classes of

aldolase. By using cell-free extracts of S. fae-calis MR (aldolase molecular weight = 56,000),Lactobacillus xylosus ATCC 15577 (aldolasemolecular weight = 70,000), Lactobacillus casei64H (aldolase molecular weight = 118,000), S.salivarius 112 (aldolase molecular weight =

130,000), and Pediococcus acidilactici (aldolasemolecular weight = 176,000), Km values forFDP of 0.8 mM, 0.45 mM, 0.45 mM, 0.8 mMand 0.38 mM were observed with the respectiveenzymes. These preliminary data suggest thatthe affinity of the enzyme for its substrate is not

acrylamide gels a function of enzyme size. However, with thelis MR aldolase exception of the S. faecalis aldolase experi-)lase plus oval- ments, confirmation of these results must await

further studies with the pure aldolases.

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TABLE 2. Molecular weightsa of the FDP aldolases found among lactic acid bacteria

Streptococcus Mol wt Lactobacillus Mol wt Pediococcus Mol wt

S. faecalis (MR) 56,000 I. L. casei (64H) 118,000 I. P. cerevisiae (8042) 176,000S. faecium (K6A) 57,000 L. casei (OC91) 116,000 Pediococcus sp. (559) 176,000S. bovis (9809) 59,000 L. zeae (15820) 118,000 Pediococcus sp. (990) 176,000S. equinus (9812) 58,000 L. delbrueckii (9649) 118,000 P. pentosaceus (25744) 178,000S. dysgalactiae (9926) 58,000 L. leichmannii (4797) 116,000 P. acidilactici (25740) 176,000S. sanguis (10556) 57,000 L. acidophilus (4356) 122,000-S. mutans (6715) 64,000 L. jensenii (25258) 122,000 II. P. parvulus (19371) 58,000S. pneumoniae (6308) 56,000 L. helveticus (15009) 122,000Streptococcus sp. 56,000 Others

(group H) 8144 Microbacterium ther- 56,000mosphactum (11509)

S. salivarius (13419) 138,000 II. L. plantarum (14917) 78,000S. salivarius (112) 130,000 L. salivarius (11741) 80,000 Sporolactobacillus in- 56,000S. salivarius (9222) 135,000 L. xylosus (15577) 70,000 ulinis (15538)

I________ III. L. curvatus (Ml) 56,000

a Determined by Sephadex G-200 chromatography.

Comparison of electrophoretic mobilityrates of aldolases. Cell-free extracts of variouslactic acid bacteria were applied to anionic PAGcolumns and after electrophoresis, the positionsof the FDP aldolases were located by activitystains. The electrophoretic mobility constants(Rf) were calculated from the distances traveledby the respective aldolases. In some instances,only a single dye band was observed; however,in most cases a major dye band and one or moreminor bands appeared. Although the R. valuesof the minor components are presented in thesummary of the data (Table 3), the size andintensity of the major dye bands leave littledoubt that they are, in fact, the FDP aldolases.This supposition is supported by the fact thatthe Rf of the major dye band produced bycell-free extracts of S. faecalis is identical withthe R, value of the pure aldolase. The nature ofthe minor components is still unknown, andalthough they may be nonspecific dye-reducingproteins, it is also possible that they are iso-zymes or dissociated aldolase subunits.The data presented in Table 3 indicate that

the aldolases can be assigned to one of fourelectrophoretic mobility groups; these divisionscan be seen more clearly in the diagrammaticpresentation of the results (Fig. 7). Apart fromthe streptococci, the intrageneric groupingsbased on the aldolase R. values coincide withthe molecular weight divisions; that is, thesmaller aldolases of the lactobacilli and pedi-ococci migrate more rapidly than the largeenzymes. Surprisingly, the larger S. salivariusenzyme migrates at roughly the same rate as thesmaller streptococcal aldolases.

Temperature sensitivity of the aldolases. Itis apparent from the preceding results that,within each molecular weight group, many ofthe enzymes have readily detectable chargedifferences. Since charge differences are usuallyindicative of amino acid substitutions withinthe respective proteins, an attempt was made tocorrelate the mobility coefficients and molecu-lar weights with a third property of the en-zymes, thermostability. The simple procedureis appended to Table 4, where the experimentalresults are summarized. No clear relationshipbetween response to heat and protein charge ormolecular weight was found. The wide varia-tions in heat sensitivity which were observedwithin each molecular weight class of aldolaseprecludes the formulation of any general hy-pothesis relating enzyme size to heat response.Likewise, the protein charge differences cannotbe used to predict whether a particular enzymeis heat sensitive or resistant; the amino acidmutations which confer heat stability upon anenzyme apparently do not necessarily alter theprotein charge.

Demonstration of ImmunologicalRelatedness Among FDP Aldolases of theLactic Acid Bacteria by ImmunodiffusionSeparation of the streptococcal aldolases

into five antigenically distinct groups. Theextent of relative immunological homology be-tween the various FDP aldolases was approx-imated by an extensive comparative studyusing cell-free extracts of strains representing 21species of Streptococcus as sources of antigen.

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TABLE 3. Electrophoretic mobility rates of the FDP aldolases found among members of the lactic acid bacteria

Species tested Strain no. J Rf major band(s) R, minor band(s)

Streptococcus sp. (group H)S. sanguisS. pyogenesS. bovisS. diacetilactisS. bovisS. lactisS. mitisS. dysgalactiaeS. equinusS. salivariusS. cremorisS. faecalisS. faecalisS. mutansS. mutansS. pneumoniaeS. salivariusS. duransS. mitisS. agalactiaeS. thermophilus

Lactobacillus jenseniiL. acidophilusL. jugurtiL. casei var. caseiL. casei var. caseiL. casei var. rhamnosusL. lactisL. delbrueckiiL. bulgaricusL. helveticusL. leichmanniiL. plantarumL. xylosusL. salivariusSporolactobacillus inulinusPediococcus pentosaceusP. cerevisiaePediococcus sp.Pediococcus sp.P. parvulusMicrobacterium thermo-sphactum

ATCC 8144ATCC 10556ATCC 14289ATCC 15351ATCC 11007ATCC 9809ATCC 19435ATCC 15909ATCC 9826ATCC 9912ATCC 13419ATCC 19257MRCl

KlRO1H1ATCC 6308112ATCC 19432ATCC 15910ATCC 13813ATCC 19258

ATCC 25258ATCC 19992ATCC 52164HF.3.2OC-91ATCC 12315ATCC 9649ATCC 11842ATCC 15009ATCC 4797ATCC 14917ATCC 15577ATCC 11741ATCC 15538ATCC 25744ATCC 8042559990ATCC 19371ATCC 11509

0.630.640.650.650.680.690.690.690.720.730.730.730.730.730.740.740.750.750.760.770.78, 0.750.79

0.320.380.40, 0.310.400.440.440.440.470.470.47, 0.420.500.68, 0.420.690.710.570.450.500.530.550.620.67

0.69, 0.780.670.780.810.750.750.770.740.810.810.860.800.81, 0.85

0.850.430.85

0.820.530.86

0.480.480.540.32, 0.180.29, 0.150.52

0.260.460.74, 0.80, 0.860.75,0.80, 0.86

Since the antistreptococcal aldolase serum(anti-SA) had been prepared against the S.faecalis MR aldolase, this enzyme automaticallybecame the point of reference for all compari-sons. Only two of the three possible precipita-tion patterns were observed in this set of experi-ments; examples of confluent (fused) and sin-gle-spurred precipitin lines are shown in Fig. 8aand b and 9a, b, and c. The absence of double-spurred precipitates suggests that the strep-tococcal aldolases have not yet undergone anymajor antigenic divergence. On the basis of thepaired cross-reactions, the various aldolases

were assigned to one of five antigenic groups(Table 5); these are arranged in a descendingorder of antigenic homology. Group I is acollection of enzymes of identical antigenicspecificity, whereas groups II, III, IV, and Vcontain aldolases of apparent identical specific-ity. Group IV is unique in that it containsaldolases of different molecular weights (56,000and 135,000) which cannot be differentiatedserologically. The results also show that thealdolase of S. pneumoniae can readily be classi-fied with those of the other streptococci.Antigenic grouping of the aldolases found

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0

0.2 _I-

a07-)co

0

Ir0x

0

wtl:

J

0.4

0.6 k

0.8

1 .o

IStreptococcus

U

Pediococcus

I Group I

Groupl

L octobocillus

FIG. 7. Summary of the electrophoretic mobilityrates (R,) of lactic acid bacteria aldolases.

among the homofermentative lactobacilli.The anti-SA reacted with crude extracts of allthermo- and streptobacteria tested, indicatingthat some degree of antigenic homology existsbetween the aldolases of the lactobacilli and S.faecalis. Although the majority of cross-reac-tions produced either confluent or single-spurred precipitates (Fig. lOa, b, c), double-spurred patterns were observed in two in-stances. The immunodiffusion patterns segre-gated the homofermentative lactobacilli intonine antigenically distinct groups. The im-munological hierarchy of eight of the groups isshown in Fig. 11. The -species Lactobacillusacidophilus was divided into two groups in theorder L. acidophilus 19992 > L. acidophilus4356. These correspond to Gasser's L. acidoph-ilus groups Ib and III, respectively (26). In-cluded in this arrangement are three groups ofapparent identical specificity. The aldolases ofLactobacillus casei var. casei and L. casei var.rhamnosus comprise the first group (Fig. 10).Confluent precipitin lines were also producedbetween extracts of Lactobacillus leichmannii,Lactobacillus lactis, Lactobacillus bulgaricus,and Lactobacillus delbrueckii (Fig. 10); Lac-tobacillus jugurti and Lactobacillus helveticus(not shown) exhibited a similar pattern ofidentity.

In contrast to the sequential precipitin reac-tions observed with the streptococcal aldolases,several instances of antigenic divergence weredetected with aldolases of the lactobacilli. Dou-ble spurs were produced when comparing ex-tracts of L. acidophilus 4356 and L. plantarum(Fig. 12c); the same type of reaction occurredbetween extracts of L. xylosus and Lactobacil-lus salivarius (not shown). The result of thecomparison was completely unexpected since

both enzymes belong to the same molecularweight class.The constitutive nature of the FDP aldolase

precluded any direct demonstration of an-tiserum specificity using cell-free extracts ofhomofermentative lactic acid bacteria. An in-

TABLE 4. Effect of heat on the stability of FDPaldolases from lactic acid bacteriaa

Remaining activity(%) after 15-min

Organism incubation at:

48C 56C

Streptococcus faecalis MR 0 0S. agalactiae ATCC 13813 5 0S. durans ATCC 19432 65 0S. lactis ATCC 19435 90 0S. thermophilus ATCC 19258 84 44S. bovis ATCC9808 96 52S. mutans 01H1 86 85S. salivarius 112 90 92

Pediococcus pentosaceus 50 0ATCC 25744

Pediococcus sp. 559 95 2P. acidilactici ATCC 25740 96 7

Lactobacillus lactis ATCC 100 012315

L. xylosus ATCC 15572 81 13L. casei 64H 100 98

a Procedure: 0.5 ml of crude extract containing 10 to20 units of aldolase activity were mixed with 0.5 ml of0.05 M BTME buffer, and a zero time sample of 0.1ml was removed and mixed with an equal volume ofthe BTME buffer. The remaining solution was thenincubated in a water bath which was maintained at 48or 56 C, and 0.1-ml samples were removed every 3 minfor 15 min. The samples were diluted with 0.5 ml ofchilled BTME buffer and rapidly cooled to 4 C.Approximately 10 Mliters of the samples was subse-quently assayed for aldolase activity.

S. feecalis S. faecalisMR (1) MR (1)

S. faecium S. faecalis L. casei v. casei S. bovisCHI (6) N83(2) M40 (5) 9809 (2)

S. fsecakis & faecium S. faecium S. fealsC1 (5) KSA (3) NSS(41 N83 (3)

S. haecalisMR (4)

FIG. 8. Nature of the cross-reaction between vari-ous aldolases of lactic acid bacteria.

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19268 (1)S. seoguis S selivrius1066(6) 13419 (2)

S mitis16909 (1)

S. bots S. s ngua

909(6) 10656(2)

S. thtmophisa mists

19268)4) 16910(3)

S. pyogeno19289 (1)

S. bovis S. Uanua909(6) 10696 (2)

S. dysplacwti S.us#Wa9926(5) 112(33

S. pyogenes19289 (4)

FIG. 9. Immunological comparison of eight streptococcal aldolases. Experiments such as these were used todetermine the antigenic hierarchy of the streptococci.

TABLE 5. Hierarchical grouping of aldolases ofrepresentatives of the genus Streptococcus based onprecipitin cross-reaction with S. faecalis antialdolase

Specificity Organismsgroup

I Streptococcus faecalisS. faeciumS. durans

II S. lactisS. cremorisS. diacetilactisS. thermophilusS. mitis (I)

III S. bovisS. equinusS. pneumoniaeS. sanguisS. mutans

IV S. salivariusS. agalactiaeS. mitis (II)Streptococcus sp. (group H)

V S. pyogenesS. dysgalactiaeStreptococcus sp. (group L)

S. faecalis

L casei

A

A

A

A

A

A

A

A

A

A

A

A

L. ,ylo

A

A

A

A

)SiS

L. salivarius

L. Ieichmanniilgroup

A

A

A

A

A

A

L. acidophilus

x

AI

L. plantarum

X L. ,ensecenii

L. he/veticus 2

group

1. Group of identical specificity consisting of L. Ieichmannii,L. delbrueckii, L. lactis and L. bulgaricus.

2. Group of identical specificity consisting of L. helveticus andL. jugurti.

FIG. 11. Hierarchy of immunological relatedness ofStreptobacterium and Thermobacterium FDP aldol-ases based on precipitin cross-reactions with S. fae-calis antialdolase. The arrowheads indicate the domi-nant antigen of the paired cross-match; the X indi-cates double-spur formation.

L Ccui v. caseuM40 )1)

L. caei v. aslctosta L. caei v. rlemnmossaOC-46() CL-15 (2)

L. cai v. rhemcsos L. <pe v. iOC-91 (4) 64H (3)

L aidophilhs439681)

L. lichmennii L. bulricsa4797 9) 11842 )2)

L.12c1 L. d

L- (3)12316 (4) CL-29 (3)

L. jugunti921 (1)

L. lact L. delbruackii12315 )5) CL-29 (2)

L. wCsOpilUS L solimtss4359 )4) 11741 (3)

FIG. 10. Reactions of apparent identical specificity and partial identity between aldolases of the lactobacilli.

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L ddbubckiiCL-23(1)

a niAWII I dbwdhctkIt utsw ls I

L Awds123151(9

L ci v. me;M401

121

I awnkls dyspbcrie Ac*ccaasA L debW ckli1 (a 92( 30(4) CL-2( 3)

L dpbuskCL-29 (4)

PWtococca sp.

so (1)L debr ii L cidbplWlsCL-28 2J (2)

L jutti L p/anmtwm521(9 14 17()

Pediococcza sp.M (4)

FIG. 12. Demonstration of antigenic divergence among the three genera of lactic acid bacteria by immuno-diffusion.

direct test was conducted with extracts of threespecies of heterofermentative lactobacilli. Ex-tracts of these aldolase-deficient bacteria pro-vided an excellent control system and, as ex-

pected, produced no lines of precipitation withanti-SA (Fig. 13a).Demonstration of partial antigenic homol-

ogy with pediococcal aldolases. Cell-free ex-tracts of the various strains of pediococci werecompared in the same fashion as the lactoba-cilli. Exhibiting either confluent or single-spur-red precipitates, the immunological compari-sons detected three groups of apparent identi-cal specificity. The first group is comprised offour strains of P. acidilactici, the second groupcontains Pediococcus sp. 559, Pediococcus sp.990, Pediococcus cerevisiae, and Pediococcuspentosaceus, and the third group is representedby the aldolase of P. parvulus.Circumscription of the family Lactobacil-

laceae. The relative ease with which the al-dolase-antialdolase system detected interge-neric relationships among the three morpho-logically distinct groups of lactic acid bacteriamade it essential to exclude the possibility ofgeneral or nonspecific reactions with aldolasesof gram-positive bacteria not belonging to theLactobacillaceae. To this end, cell-free extractsof Staphylococcus aureus, Corynebacteriumxerosis, Arthrobacter globiformis, Sporolacto-bacilus inulinus, and Microbacterium ther-mosphactum were tested against the antisera.Only the aldolase of M. thermosphactum, afacultative psychrophile associated with meatspoilage (49), produced a precipitate; the othersproduced no visible cross-reaction (Fig. 13b).The inclusion of this gram-positive rod in the

survey was not accidental. Recently, a biochem-ical characterization of the genus Microbacte-rium by Collins-Thompson et al. (19) revealedthat M. thermosphactum, unlike the other

L cellobiosus11739 (1)

L casei v. casei L casei v. case940(5) 40(2)

_

L brWe1U4 (4)

A globiformis8010 (1)

L cellobiosw11730 (6)

C xerosis7094(2)

L Fwnw L Ifw.nts I wus1431 (3) 14931(5) 12300(3)

S. inulints15538(4)

FIG. 13. Results of cross-matches between aldol-ase-deficient lactobacilli or extracts of nonrelatedgram-positive organisms. The sample well containsapproximately 50 ug of crude extract protein; thecenter wells contain 40 Mliters of anti-SA. The plateswere incubated for 72 h.

members of this loosely structured group, is a

strictly homofermentative bacterium whichlacks a tricarboxylic acid cycle. At a value of 36,the moles percent guanine plus cytosine ratio ofits DNA is vastly different from the valuesobtained with the DNA of other species in thegenus and is similar to that of most strepto-cocci. Despite the similarities, the authors didnot suggest that M. thermosphactum be as-signed to the lactic acid bacteria. This reluc-tance stemmed from certain differences be-tween M. thermosphactum and the lactobacilli;at variance were cell wall and lipid compositionand the presence of catalase in the former.However, it is clear from the reaction betweenthe M. thermosphactum aldolase and the anti-SA that the organism is related to the lacticacid bacteria.No immunological evidence was found to

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support the natural relationship proposed forSporolactobacillus and Lactobacillus by Kita-hara and Suzuki (36).Evidence for antigenic divergence between

aldolases of the lactic acid bacteria. Theimmunodiffusion experiments suggest that,within each genus, the aldolases of the constitu-ent species have evolved in a relatively sequen-tial fashion; the two exceptions found amongthe lactobacilli are described above. To deter-mine whether any significant antigenic diver-gence had taken place at the intergeneric level,aldolases from each of the three genera werecompared with one another. The creation of animmunologically based hierarchy made it possi-ble to select aldolases from members of eachgenus whose reactivity with the antiserum wasroughly equal. Several examples of such inter-generic comparisons are shown in Fig. 12a, b,and c. When extracts of L. delbrueckii or L.lactis were paired with extracts of streptococcalantigen group II (Streptococcus cremoris orStreptococcus diacetilactis) or streptococcalantigen group m (Streptococcus bovis) andmade to react with anti-SA, double spurs wereproduced. Similar results were obtained whencomparing L. acidophilus extracts with extractsof S. salivarius (streptococcal antigen group IV)or Streptococcus dysgalactiae (streptococcalantigen group V). The precipitation patternsobtained with enzyme extracts of L. delbrueckiiand S. salivarius or S. dysgalactiae show singlespurs directed towards the wells containing theenzyme with fewer antigenic determinants (Fig.12a), whereas doubled-spurred bands wereformed between extracts of Pediococcus sp. 990and L. lactis (Fig. 12b), Pediococcus sp. 559 andL. delbrueckii (Fig. 12b, c), L. xylosus and P.acidilactici, and M. thermosphactum and Pedi-ococcus sp. 559. Figures 14 and 15 summarizethese double diffusion experiments, and fromthese data it is possible to discern at least fourdistinct lines of aldolase divergence. The data

S faecalis

K 7j | S. diacetilactis

A

AIA

A

A

A

x

A

x

A

A

A

M. thermosphactum

x

A

A

A

A

Pediococcus sp. 559

x

A

A

A

L. /eichmannii

S. bovis

A A s. dysalactiae

A A X L. acidophilus

FIG. 14. Summary of intergeneric cross-matchesbetween the various aldolases of lactic acid bacteria.

S. faecalis

A S. lactis

A X

A

A

A

xx

x

P. acidilactici

xX

x

M. thermosphactum

X L. xylosus

X X L. salivarius

FIG. 15. Summary of intergeneric cross-matchesbetween aldolases from four morphologically distinctforms of lactic acid bacteria.

also show that many of the streptococcal aldol-ases are not as closely related to the S. faecalisenzyme as are aldolases found among severalspecies of pediococci and lactobacilli.

Effect of anti-SA on aldolase activity. Theaddition of relatively small amounts (3 to 5Tliters) of anti-S. faecalis aldolase serum toreaction mixtures containing 0.2 to 0.4 units ofaldolase present as either the pure enzyme orcell-free extracts produces a 50% inhibition ofcatalytic activity; the degree of inhibition isproportional to the antiserum concentrationover the range of 1 to 10 Mliters. Since thealdolase activity in extracts of Streptococcusfaecium, S. bovis, S. pneumoniae, Pediococcussp. 559, L. casei, L. delbrueckii, and L.plantarum is also sensitive to anti-SA, theinhibition by antiserum appears to be a generaleffect. Two types of controls serve to demon-strate the specificity of this system. First,substitution of equivalent amounts of unimmu-nized rabbit serum for anti-SA in the reactionmixtures did not result in an inhibition ofactivity. Second, the inhibition is specific foraldolases of lactic acid bacteria. The enzymeactivity in extracts of S. inulinus is not inhib-ited by 40 ;liters of anti-SA.Although it is possible to establish an order of

antigenic relatedness based on the extent ofantiserum-induced inhibition of enzymatic ac-tivity (48), no attempt was made to quantitatethe immunological differences between aldol-ases of the lactic acid bacteria by this methodfor the following reason. The two proceduresemployed in these experiments, immunodiffu-sion and microcomplement fixation, measurethe total antibody-antigen interaction, whereasimmune inhibition only measures the effect ofsuch interactions on the catalytic site of theenzyme. This effect could conceivably be pro-duced by antibody binding to only a few keyantigenic sites on the enzyme molecule which

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mask or modify the active site. Moreover, anamino acid substitution in one or more of theantigenic sites, which usually alters the configu-ration of the active site, could obliterate theinhibitory effect of the antiserum. In either ofthese circumstances, the total complement ofantigenic determinants is probably not beingmeasured.

Quantification of the ImmunologicalRelatedness Among Aldolases of theLactobacillaceae by Microcomplement

FixationDetermination of indices of dissimilarity

by microcomplement fixation. The data frommCf assays were used to calculate the ID for thealdolases of the various lactic acid bacteria.These results both quantitate and corroboratethose obtained by immunodiffusion experi-ments (Table 6). Based on their respective IDvalues, the aldolases were organized into clus-ters which generally coincided with the im-munodiffusion groupings.The streptococcal aldolases were segregated

into five major clusters which, with only threeexceptions, are comprised of the same enzymesfound in the corresponding identity groups ofthe immunodiffusion experiments (compareTable 5 with Table 6, column 1). A clearseparation of the individual clusters wasachieved without further mathematical treat-ment of the data. In every instance the serologi-cal distance between the nearest neighborswithin a specific cluster is always less thanone-half the distance between the two neighbor-ing clusters. The results also show that theconstituent enzymes of each of the original fiveidentity groups are not, in fact, immunologi-cally homologous to one another; each cluster iscomposed of a spectrum of ID values ratherthan a single value. Even the aldolases of thefirst cluster, which corresponds to identicalspecificity group I, have undergone a measura-ble degree of antigenic divergence. The extent ofantigenic divergence is more striking in theother clusters or groups of apparent specificity.Only the aldolases of Streptococcus mitis

15909, S. mitis 15912, and Streptococcusequisimilis could not be accommodated withinone of the five major clusters. The S. equisimilisaldolase is obviously less antigenically relatedto the reference protein than are the aldolases ofapparent specificity group V. For the present,this enzyme has been used as a nucleus for asixth cluster. The failure to detect spur forma-tion when comparing the S. equisimilis aldolasewith the group V aldolases can probably beattributed to the relative insensitivity of theimmunodiffusion method. The S. mitis aldol-

ases pose a special problem. Although S. mitisstrains 15909 and 15912 can be assigned to asubgroup intermediate between apparent iden-tity groups II and III (or clusters 2 and 3), thealdolase of a third strain, 15910, is clearlyantigenically distinct from the two former en-zymes. These data can be interpreted to meanthat the species as presently constituted is anamalgam of heterogeneous organisms which donot belong under a single species heading. Thesituation is probably analogous to that de-scribed by Gasser et al. (27), in which thespecies Lactobacillus jensenii and L.leichmannii had been confused with one an-other because they were physiological lookalikes.An attempt was made to relate the antigenic

groupings of the aldolases to the type of Lance-field antigen associated with the various speciesof Streptococcus. A rough correlation betweenthe two antigenic systems can be seen from thedata presented in Table 6, column 1.A comparison of Fig. 11 with Table 6, column

2, reveals that the antigenic hierarchy of theLactobacillus aldolases based on mCf assays isconcordant with that of the immunodiffusionexperiments. The results of the mCf studiesestablish unequivocally that the aldolases of thegroup D streptococci and L. casei share thegreatest degree of immunological homology. Inaddition to the three L. casei subspecies, thiscluster also contains the aldolases of Lactobac-terium zeae, Lactobacillus coryniformis subsp.coryniformis, and L. coryniformis subsp.torquens. Since the immunodiffusion cross-matches of this group always produced lines ofidentity and the ID values cluster between 3.4and 6.5 with an internal variation of 2.8 IDunits, this group of aldolases appears to have arelatively high degree of antigenic homology. L.zeae has been characterized recently by Ghernaand Mills (private communication) and has aphenotype identical with that of L. casei subsp.rhamnosus. The two subspecies of L.coryniformis differ from the conventional L.casei phenotype according to Abo-Elnaga andKandler (1). Unlike L. casei, L. coryniformissubsp. torquens and coryniformis produce D andD,L-lactic acid, respectively, rather than L-lac-tic acid. At this time, the relative importance ofthese phenotypic differences cannot be as-sessed. The aldolases of L. xylosus and L.salivarius are shown as distinct entities in Table6, column 2, in spite of their physical similari-ties; the precipitation pattern of nonidentity(double-spurred precipitate) between these twoenzymes dictates such a separation. The orderand aggregation of the remaining Lactobacillusaldolases are in accord with the hierarchical

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to 0c

-H -H H 1-H -H -H

OCn

C_, t-

c r tr o

U0 L LLa.

3 U.) .

=IICODE r B-~~~~~c

oL'11 I; c; m oocq I0O.O O O O O O O O O C~l0CC-

+ + -H -H 4i -H -H 4 ++++++

q lp L": c" > > ooq C;0 oq oi O 0 CD CD m 00 w C UD 00

X,2s _ NN

O o sr ~~~~~os LO Cs 4ea t- u Cqrt~~~~~~~~C C C) O sr0n=e;e>0c4ra a

0O _toI

S~~~~~~~~~~~~~~~~~~~~~~~~~CIt C,]Lo2Z~w

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O--C) 0 v LO 00 o v M m (M 0 C O_0 0-N0 C w

Q _ _ N~~~~c Csi 0 6 (7 CO _ clD Cm Cm Cm Cv nC-D IO (DtR 00 0 00 00 CO

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.~c CA)=~~~~~~~~~~~~0~~~~~~

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o- CO co CO

C6C 6 i u u i'uiu C6C/3C') CO CO6C 6 C )u i )U iU i U

a03 L; l

L.ttr3XE 00c> E cEu.

0cc cm oecctcoectte ctctC

468

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scheme proposed in an earlier section andrequires no further explanation here. Two orga-nisms, Lactobacillus curvatus and Lactobacil-lus mali, were added to the list of bacteriasurveyed by mCf assay. Both aldolases appearto be intermediate between the L. leichmanniiand L. acidophilus groups; these positions weresubsequently confirmed by immunodiffusiontests.As was the case with the Lactobacillus aldo-

lases, the arrangement of the pediococcal aldo-lases in accordance with either their ID values orreactions of partial identity was identical. Thethree clusters shown in Table 6, column 3, arecoincident with the three groups of apparentidentical specificity.With ID values of approximately 10, the

degree of homology between the group D strep-tococcal aldolases and the aldolases of the groupN streptococci, P. acidilactici and M. thermos-phactum, appears to be equal. Since the aldo-lases of the three organisms belong to differentmolecular weight classes and produce lines ofnonidentity when compared with one anotheron immunodiffusion plates, it seems safe toconclude that they represent three distinct linesof aldolase evolution.

DISCUSSION

Molecular Variation During the Course ofAldolase Evolution

It has become general practice to estimate theextent of structural similarity of isofunctionalproteins by comparing one or more of theirvarious physical characteristics; the two proper-ties most frequently used are the electrophoreticmobility coefficient and molecular weight.Closely related or homologous proteins are usu-ally invariant, or nearly so, in either parameter;conversely, variability is associated with a de-crease in structural homology. Do the FDPaldolases of lactic acid bacteria constitute anexception to this well-established rule? Thestriking diversity in size and protein charge ofthe aldolases found among the three genera oflactic acid bacteria would suggest that verylittle structural homology exists between thephysically distinct forms of the enzyme. This isobviously not the case, however; in addition toestablishing the existence of structural homol-ogy between the various groups of aldolases, theresults of the immunological studies suggestthat the degree of structural homology is virtu-ally unrelated to the above-mentioned twomajor physical properties of these proteins. Acomparison of the S. faecalis and L. caseialdolases best typifies this anomaly. The L.

casei aldolase is twice as large and one-half asmobile in PAG columns as the reference S.faecalis enzyme but, despite these differences,the former shares a degree of immunologicalhomology with the latter which is surpassedonly by aldolases of other Lancefield group Dstreptococci. Fortunately, a relatively simpleexplanation of this apparent enigma can beextracted from the available data. Dividing theaverage molecular weight of each of the fivealdolase size classes (see Table 2) by 28,000, themolecular weight of the S. faecalis aldolasesubunit, the integers 2, 3, 4, 5, and 6 areobtained. If the assumption is made that thevarious representatives of five molecular weightclasses of aldolase simply represent the associa-tion of two or more identical subunits, theresults of the physical and immunological stud-ies are no longer incongruent. The variations inmolecular weight merely reflect the differentmodes of assembly of the structurally conservedsubunit, and since the polyacrylamide gels alsoact as molecular sieves (13) the electrophoreticmobility rates are influenced, at least in part,by the size of the enzyme. Although this point ofview is generally consistent with the data, thepossibility that the net protein charge has beenaltered by amino acid substitutions in noncon-served areas of the peptide chain cannot beexcluded. Implicit in this hypothesis is theassumption that subtle mutations in the pri-mary amino acid sequence of the monomer areresponsible for the gross alterations in thequaternary structure of the enzyme. There isalready some evidence from preliminary studieswith the aldolases of L. casei and P. cerevisiae(unpublished data) to indicate that these en-zymes are, in fact, composed of subunits ar-ranged in a tetrameric and hexameric configu-ration, respectively.Although variations of this magnitude in the

quaternary structure of partially homologousproteins are quite rare, such occurrences are notcompletely without precedent. Sadoff and co-workers (66) found that the FDP aldolase fromspores of Bacillus cereus is roughly one-half aslarge as the major molecular weight species ofaldolase present in the vegetative cell, yetantisera prepared against the purified sporeenzyme produced confluent lines of precipita-tion when the former was compared with thelarger aldolase of the vegetative cell on im-munodiffusion plates. This result indicated thatthe two forms of the enzyme are immunologi-cally homologous. A more detailed descriptionof the molecular evolution of the lactic acidbacteria FDP aldolases will be presented in aseparate report.

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Preparation of a Phylogenetic Map for theHomofermentative Lactic Acid BacteriaBased on Structurally Related Proteins

Basis for relating immunological homologyto structural homology. The rationale forusing immunological techniques to measureamino acid sequence homology of isofunctionalproteins has been amply discussed by a numberof workers, including Wilson and Kaplan (84),Prager and Wilson (60), Stanier et al. (72),Gasser and Gasser (26), and London et al. (43,44), and a detailed reiteration of these points isnot necessary here. However, several issuesrelated to the precision of the methodology, itslimitations, and the restrictions these factorsplace upon the interpretation of the resultsdeserve elaboration at this time. Studies withproteins of known amino acid sequence haveestablished that quantitative immunologicaltechniques can readily detect substitutions ofone (14, 61), two (6, 7), or three (5) amino acidsin the primary structure of partially homologousproteins. The superb sensitivity of the micro-complement fixation assay prompted Pragerand Wilson (60) to suggest that the immunolog-ical distance (log ofthe index of dissimilarity x100) between two related proteins can be cor-related with a specific number of amino acidsubstitutions in the primary structure of theproteins. By using avian egg white lysozymes asa model system, these workers established thatfive immunological distance units are equivalentto an alteration of one amino acid. The authorswent on to suggest that this relationship may begenerally applicable to all species of proteins iftheir stringent experimental procedures are fol-lowed.The general attitude towards the suitability

of using immunological procedures to estimateprotein homology is somewhat less enthusiasticthan that cited above and is probably bestexpressed by some concluding remarks in areport by Margoliash et al. (47): "With eucar-yotic cytochrome C it was found that thecorrelation of primary structure and immuno-logical cross-reaction is, in general, good but notperfect. This probably reflects the fact that notall structural changes in cytochrome C, onlythose that are effectively within the antigenicdeterminants, effect the immunological reac-tion..... Despite these limitations, immunologi-cal cross-reactions among sets of ancestrallyrelated proteins from different species mayprovide useful taxonomic indications."

Until the recent report by Prager and Wilson(60), little was known of the extent to which thestructures of related proteins could be alteredand still retain immunological cross-reactivity.

Based on their own work and a survey of theliterature, these authors found that a primarysequence difference of 25 to 40% resulted in acomplete loss of immunological activity in allspecies of proteins examined thus far. A furtherrestraint is placed on the usefulness of theimmunological approach by the knowledge thatthese techniques only measure the similaritiesin the surface or external portions of the pro-teins, and it is precisely these areas which tendto evolve most rapidly (46, 60). Hence, theimmunological procedures appear to be limitedto relating sets of proteins with relatively highsequence homologies (60% or greater).The number of antigenic determinants found

on a protein can also influence the reliability ofimmunological homology data. For example, ifa group of structurally related proteins possessonly a few antigenic determinants per molecule,any alterations in those sites could greatlyexaggerate the differences between the actualsequence homology and that determined byimmunological tests. In these instances, thedegree of sequence homology might be relativelygreat, although the immunological techniqueswould indicate that little or no homology existsbetween the various ancestrally related pro-teins.Despite its limitations and possible pitfalls, it

will become apparent from the ensuing discus-sion that immunological procedures provide arapid, convenient, and reliable means of com-paring the structures of isofunctional proteins.Phylogenetic map of the lactic acid bacte-

ria. The phylogenetic map of the homofermen-tative lactic acid bacteria (Fig. 16) was assem-bled in four stages by using both physical andimmunological properties of the FDP aldolases.(i) The immunodiffusion patterns of noniden-tity delineate, the branches of the four majordiverging lines of aldolase evolution and providethe backbone of the scheme (solid lines, Fig.16). (ii) Within each of the lines, the positions ofthe individual species are first approximated bythe immunodiffusion reactions of partial identi-ty; (iii) a more precise placement is madepossible by quantifying the distances along eachline by using the microcomplement fixationdata (ID values). (iv) Finally, minor divergingbranches (dotted lines) corresponding to thedifferent aldolase molecular weight classes areincluded. The significance of these subgroups isas yet unknown. Since the size of the enzymeappears to be a function of subunit arrange-ment, the differences in molecular weight mayrepresent little more than the substitution ofseveral amino acids in the primary structure ofthe enzyme. However, these apparently trivialdifferences may produce functional changes in a



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particular size class of aldolase which confer aselective advantage upon the organisms pos-sessing them.The results of using these criteria for charting

evolutionary relationships are readily apparentin Fig. 16. For example, the reactions of noni-dentity (double-spurred precipitates) betweenthe aldolases of L. xylosus and L. salivariusnecessitate their placement on two distinct anddiverging lines of evolution despite the fact thatthese aldolases belong to the same molecularweight group. Differences in the molecularweights of their aldolases have resulted in theplacement of S. salivarius, P. parvulus, L.plantarum, and L. curvatus in minor evolution-ary outcroppings.Although a large number of organisms have

been examined thus far, this map cannot beconsidered complete. With the exception ofButyribacterium rettgeri (unpublished data),the obligately anaerobic gram-positive non-sporeforming rods have not yet been surveyed.At this juncture it should be pointed out thatthe map will probably have to be enlarged sincethe aldolase of B. rettgeri reacts strongly withthe antistreptococcal aldolase serum. The rela-tionships between this large group of gram-posi-tive rods and the homofermentative lactic acidbacteria shall be treated in a separate report.

Does the Phylogenetic Map AccuratelyReflect Interrelationships Among the

Lactobacillaceae?Phylogenetic maps of the lactic acid bacteria

as elaborate as this one have not yet beenassembled; however, Gasser and Gasser (27)have used D- and L-lactic dehydrogenases

(LDH) to chart the natural affinities whichexist among a large number of species of homo-and heterofermentative lactobacilli, and a partof their map overlaps with the aldolase map. Areproduction of one of their maps is shown inFig. 17, and this is to be compared with themain Lactobacillus branch of the aldolase map(Fig. 16). If the two branches of homofermenta-tive lactobacilli are rotated 900 towards eachother to form a single linear sequence, the orderof the constituent species in the sequence isessentially identical with that found in the FDPaldolase map. Moreover, both immunologicalsystems recognize the same groups of identical

L. jugurti

L. acidophil. III

.

/L. acidophilus 11

L. Ieichmannii

L. acidophilus lb

// L. jensenii

L. acidophilus la

-L L. fermenti I

Heterof. lactob. gr I(H.L. I)

FIG. 17. Antigen relationships of LactobacillusLDH based on microcomplement fixation studies(after Gasser and Gasser, reference 26).

L helveticus

.ocidophi/us X

acidophilus Ib

P cerevisioe L. /eichmonni /P pentosoceus \ L. curvotus

Pediococcus sp 559 \ L. locis L deibrueckiiP Ocidl toctici L. bulgoricus

P porvu/us ------GroupD- '_-L.xylosusS sotivorlus s Group -L. solivorius

Sbois -

S. equinus, S soequis- 5, b.ov/sStreptococcus sp (HA M thermosphoctum

S pyogenes S. pneumonioe - L. plontorumS. dysgotoctice .S ogotoctoeeSdysgolactio

S mitis (ff1Streptococcus sp (L)

FIG. 16. Proposed phylogenetic map of the homofermentative lactic acid bacteria.

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specificity, namely, the L. Ieichmannii groupconsisting of L. leichmannii, L. bulgaricus, L.lactis, and L. delbrueckii, and the L. jugurti-L.helveticus group. The failure to detect the twodiverging lines of lactobacilli with the anti-streptococcal aldolase serum can probably beattributed to the relatively great immunologicaldistance between the aldolases of the L. acido-philus groups and the reference protein. Theantigenic determinants which these enzymesnow share are probably few in number andhighly conserved. The discrepancy in the twosets of data emphasizes the importance of theinternal reference protein. By using a group-specific aldolase or an aldolase of a closelyrelated Lactobacillus, the divergence at the L.acidophilus groups would probably be detecta-ble. On the basis of weak cross-reactions withanti-L. acidophilus L-LDH serum, the authorsconcluded that the LDH of L. casei and L.salivarius are related, but immunologically dis-tant from the L. leichmannii group. This obser-vation is also in accord with the aldolase map.The inability of the anti-L-LDH serum to cross-react with the LDH of the group D streptococcican be explained simplistically by suggestingthat the antiserum titer was comparativelylower than that of the anti-aldolase serum.However, the possibility cannot be excludedthat the FDP aldolase is a more structurallyconserved protein than the L-LDH and thereforeevolves at a lower rate.Although it is apparent that portions of both

schemes require much refinement, it is equallyimportant to acknowledge the fact that thoseareas of the maps which overlap exhibit a highdegree of coordination. The excellent agreementin the ordering of nine species of Lactobacilluspermits both maps to be interlocked and thehomofermentative streptococci and lactobacillican readily be related to a number of aldolase-less heterofermentative species of Lactobacillusand Leuconostoc. A precise integration of thetwo maps is not possible at this time, since theimmunological distances of the aldolases areprobably not equivalent to those of the lactatedehydrogenases. Despite this limitation, thealdolase map can be united with the expandedLDH map of Gasser and Gasser (26) (Fig. 18) toreveal the evolutionary sequence of the constit-uent species. From the points of overlap, thehomofermentative members of this familyspread out in one general direction, whereas theheterofermenters lie in the opposite direction;the diverging groups form two large discreetclusters. Studies with other conserved proteinswill be needed to extend and confirm bothphylogenetic maps.

Implications of the Phylogenetic Map

Evolutionary interrelationships. For thepresent, no significance should be attached tothe direction of the respective evolutionarybranches. The spacing and distance betweeneach of the lines merely represent the mostconvenient way of displaying the data. Simi-larly, the data are presented in a two-dimen-sional plane, and the arrangements withingroups of apparent identical specificity areprobably three dimensional. Without internalmarkers, it is not possible to arrange theseantigenically related groups with a high degreeof confidence. For this reason, the positionswithin groups of apparent identical specificityare drawn in a linear fashion. The map doesshow that the four genera are related throughsome common ancestor, and it allows the rela-tive evolutionary distances between groups andindividual species to be easily determined.However, the most provocative aspects of themap are not readily apparent, and these arediscussed below.Morphology versus protein homology. It

can be seen from Fig. 16 and Table 6 that S.faecalis and L. casei form a bridge between therods and the cocci; but, in addition to this fact,it is also apparent that the latter is the speciesmost closely related to the group D streptococci.Because one species is rod shaped and the otherspherical, a dilemma involving a basic preceptin microbial taxonomy immediately arises. Howimportant a taxonomic character is the shape ofmicroorganisms? Clearly, in schemes not re-flecting natural relationships a property of thissort can assume any degree of importance, andit may provide a convenient method of distin-guishing two groups of organisms whose physio-logical characteristics are very similar. If, how-ever, the protein homology studies of the malicenzyme (43, 44) and aldolase accurately reflectthe natural affinities between the species L.casei and S. faecalis, then one must concludethat shape is a relatively trivial character whichshould not be used to divide closely relatedgroups.The morphological differences which exist

between the tetrad, P. acidilactici, and theStreptococcus, S. faecalis, also appear to be ofminor importance because the immunologicaldistance between P. acidilactici and S. faecalisis the same as that between the latter and S.lactis, its nearest streptococcal neighbor (seeTable 6). The same relationship can be shownfor the three species, S. faecalis, M. thermo-sphactum, and S. lactis. Unlike Arthrobacterspecies (39), the lactic acid bacteria breed true,

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I L. leichmannii group J

L. jugurti } L acidophilus 111 - L. acidoph. Ia - L. acidoph. lb L. jensenii |

Heterof. Lactob. gr. lila

I L. fermenti group

Heterof. Lactob. gr. I

Heterof. Lactob. gr. If Leuconostoc gr 1L. buchneri

I ILeuconostoc gr 2

L. brevis

FIG. 18. Interrelationships between heterofermentative Lactobacillus and Leuconostoc species based onimmunological similarities of their LDH (after Gasser and Gasser, reference 26).

rods producing rods and spheres producingspheres. However, it now appears that theestablishment of a stable spherical or rod-shaped genotype among this group of organismsmay involve relative minor genetic mutations.The argument has been framed succinctly byvan Niel (77), who wrote: "To believe that mereresemblance in morphology represents a soundbasis for revealing phylogenetic trends-as isimplicit in the arguments of priority of morpho-logical over physiological characteristics-restson a misconception of the fundmental value ofsuch characters when successfully employed forthe classification of higher plants and animals,where they are intricately linked with the entiredevelopmental history of the organisms."Comparative evolutionary age of the re-

spective genera. If it is assumed that (i)isofunctional sets of prokaryotic proteins aresimilar to isofunctional sets of eukaryotic pro-teins and evolve at relatively constant rates,and (ii) that the rate of amino acid substitutionwithin the antigenic determinants accuratelyreflects the rate of amino acid replacement ofthe entire protein, it is possible to use theimmunological distance as a rough measure ofrelative evolutionary time. The microbial lineexhibiting the greatest immunological diver-gence as manifested in the span of ID valuesshould be considered the most ancient group inthe family. Among the lactic acid bacteria, thegenus Lactobacillus is the most immunologi-cally diverse group found and is, therefore, theoldest group in this family. The streptococciappear to be a more recent group, whereas the

pediococci exhibit the least divergence of thethree morphologically distinct groups. This lineof reasoning leads to a rather obvious conclu-sion, namely, that the rod-shaped forms are theprogenitors of the spherical forms. Benecke (10)postulated that the coccoidal forms are thesimplest morphological entities in nature, andthat these gave rise to the more complex tetradand rod-shaped bacteria; the evidence pre-sented here argues against this point of view.However, the conclusions must be qualified.Although the survey of species belonging to theLactobacillaceae was comprehensive, it is possi-ble that there are streptococci in nature whichpossess aldolases with a greater degree of evolu-tionary divergence than those studied here, andthese organisms would expand the evolutionarytime scale of this genus.

Protein Relatedness Studies Among OtherBacteria

Enterobacteriaceae. Five species of isofunc-tional enzymes have been used to demonstratethe natural relationships which exist among thevarious genera of enteric bacteria. Cordes andLevine as cited by Wilson and Kaplan (84)prepared antisera against the purified induciblealkaline phosphatase of Escherichia coli andestablished the following order of relatedness ofthe four genera studied: Escherichia > Aero-bacter > Serratia > Proteus. A comparativeimmunological study of the tryptophan synthe-tase a subunit (53) of a number of entericbacteria with antiserum prepared against the E.coli a subunit revealed that the five genera

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under investigation were related in the order:Escherichia > Shigella > Salmonella > Aero-bacter > Serratia. Recently, Cocks and Wilson(15) confirmed and extended the work of Cordesand Levine by comparing the alkaline phospha-tases of a larger number of Enterobacteriaceaewith antisera prepared against the pure alkalinephosphatases of E. coli and Klebsiellaaerogenes. A much enlarged phylogenetic mapshowed the following intergeneric relationships:Escherichia > Shigella > Salmonella > Cit-robacter > Klebsiella > Serratia > Proteus >Erwinea. Where the three sets of data overlap,the ordering of the genera is identical. Thepatterns of immunological cross-reactivity ob-served when comparing the enteric fl-galactosi-dases (18, 64), L-asparaginases (59), and the Tand G factors involved in protein synthesis (28)are in general accord with the results citedabove. Taken together, the five studies circum-scribe and define a phylogenetic group com-posed of eight bacterial genera. The data arealso consistent with DNA homology studies(11).The alkaline phosphatases from species

within the genus Klebsiella have been com-pared immunologically by Steffen et al. (73).These studies indicate that K. aerogenes andAerobacter (Enterobacter) aerogenes are oneand the same organism. However, the genusEnterobacter appears to be relatively heteroge-neous, containing species such as Enterobacterliquefaciens, which are only distantly related tothe reference strain E. aerogenes.Pseudomonadaceae. Stanier and co-workers

prepared antisera against purified muconolac-tonizing enzyme (MLE) and muconolactoneisomerase (MI) from Pseudomonas putida bi-otype A in an attempt to clarify the taxonomicstructure of this genus. Although the isologousenzymes found in Pseudomonas multivoransand Pseudomonas acidovorans did not cross-react with either of the antisera, the MLE ofPseudomonas aeruginosa, Pseudomonas stut-zeri, and Pseudomonas fluorescens biotypes A,B, C, D, and E reacted definitively with theanti-P. putida MLE. In addition, extracts of anumber of taxonomically indeterminate strainsof Pseudomonas also reacted with the anti-MLE serum. Only three of the species possessedMI which reacted with the anti-P. putida MIserum. On the basis of this study, the authorsexcluded the organisms designated P. putidabiotype B from the species.Micrococcaceae. A comparative study by

Whiteside et al. (82) of a membrane-boundadenosine triphosphatase (ATPase) foundamong Gram-positive aerobic cocci revealed

that the micrococci and certain Sarcina arerelated. As in the case of the gram-positivehomolactic cocci, the morphological differencesbetween Micrococcus and Sarcina do not consti-tute a natural division or boundary of thisgroup. The ATPase of Sarcina flava and Sar-cina lutea reacted strongly with the Micrococ-cus lysodeikticus anti-ATPase serum, as didmany of the Micrococcus species. The micro-cocci were arranged in the following order ofrelatedness: M. lysodeikticus > Micrococcustetragenus > Micrococcus roseus > Micrococ-cus conglomeratus > Micrococcus varians. Thefailure of the ATPase from Micrococcus rho-dochrous, Sarcina ventriculi, Sarcina maxima,and Sporosarcina ureae to react with the anti-ATPase serum came as no surprise to theauthors. Of the organisms studied, only Mi-crococcus caseolyticus remained taxonomicallyindeterminate; M. rhodochrous phenetically re-sembles the actinomycetes and had alreadybeen reassigned to that family (29). Similarly,the anaerobic sarcinae had been clearly differ-entiated from their aerobic counterparts byCanale-Parola (12) and have since been rele-gated to the family Peptococcaceae (63). Fi-nally, the authors pointed out that the S. ureaeproperly belongs among the gram-positivesporeforming bacteria.Suprafamilial relationships. Thus far, the

discussion has centered on the establishment ofnatural relationships among organisms withinspecific taxonomic families (as they are definedin Bergey's Manual of Determinative Bacteriol-ogy, 7th ed.). However, several recent studiesindicate that it may be possible to demonstrateinterfamilial relationships. McFadden and De-nend (48) prepared antiserum against pureHydrogenomonas eutropha ribulose diphos-phate (RUDP) carboxylase and showed that itwould inhibit RUDP carboxylase activity inextracts of Rhodospirillum rubrum, Chlorellapyrenoidosa, Chromatium D, and Thiobacillusdenitrificans to varying degrees. Unfortunately,immune precipitates were not produced whenthe various extracts were made to react withanti-RUDP carboxylase serum in immunodiffu-sion experiments. The haptene-like behavior ofthe antigen suggests that the RUDP carboxyl-ases of the respective photo- and chemolitho-trophs share very few antigenic determinantswjth the homologous protein.

Tronick et al. (76) reported that the gluta-mine synthetase of nine diverse species of gram-negative organisms shared antigenic determi-nants with the isofunctional enzyme of E. coli.The anti-E. coli glutamine synthetase serumreacted with the partially homologous enzymes

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in the order Enterobacteriaceae > Pseudomo-nadaceae > Azotobacteriaceae (private com-munication, S. Tronick and J. Ciardi). Gluta-mine synthetases of several gram-positive orga-nisms did not react with the antiglutaminesynthetase serum, indicating that the diver-gence which ultimately produced gram-positiveand gram-negative organisms is beyond thelimits of detection of this immunological systemand is, therefore, probably relatively ancient.These two preliminary reports suggest thatamong prokaryotes there exist enzymes withhighly conserved primary structures. Studies ofsuch enzymes may permit bacteriologists toeventually prepare phylogenetic maps which en-compass many of the known bacterial families.

Rates of Evolution of Prokaryotic ProteinsNoting that the indices of dissimilarity for

several sets of isofunctional enzymes found inenteric bacteria were very similar, Cocks andWilson (15) theorized that intracellular meta-bolic enzymes might evolve approximately inunison. Shortly thereafter, Rocha et al. (62)demonstrated that this was by no means ageneral rule. Within the same protein, trypto-phan synthetase, the a and f2 subunits frommembers of five genera of enterobacteria wereshown to evolve at markedly different rates byimmunological procedures.

Stanier and co-workers (72) found a similarsituation among enzymes from the pseudomo-nads. The inducible muconate-lactonizing en-zyme appears to have sustained fewer antigenicalterations than its companion enzyme, muco-nolactone isomerase. Although the titers of theantisera prepared against each of the P. putidaenzymes were essentially identical, the an-timuconolactone isomerase serum reacted withfar fewer extracts of Pseudomonas species thandid the antimuconate-lactonizing enzyme se-rum.

It also appears that catabolic enzymes of thelactic acid bacteria may not evolve in unison.The species L. casei can be easily differentiatedinto its two constituent subspecies, L. casei var.casei and L. casei var. rhamnosus, by a series ofphysiological tests (44). Similarly, antisera pre-pared against the inducible malic enzymes ofeither S. faecalis or L. casei var. casei recognizetwo antigenically distinct forms of enzymeamong members of the species L. casei (Table7). One of the antigenic forms of the malicenzyme is found only in L. casei var. casei, andthe other form is found only in strains of L. caseivar. rhamnosus. DNA homology data are incomplete agreement with this division of thespecies (52, and J. Johnson, private communi-

TABLE 7. Comparison of the indices of dissimilarity ofmalic enzvme and FDP aidolase

ID for malic enzyme Aldolafe

Anti- Anti- Anti- Anti-Strain tested S. fae- L. casei L. casei S. fae-

MRll 64H M40 calisMa malic malic FDP

enzyme enzyme enzyme aldolase

Streptococcus fae- 1.0 20 19 1.0calis MR

Lactobacillus casei 52 1.0 1.0 4.8var. casei 64H

L. casei var. casei 51 1.0 1.0 4.7M40

L. caseivar. alac- 44 1.0 1.0 5.8tosus OC45

L. caseivar.rham- 78 5.7 7.2 5.4nosus OC91

L. caseivar.rham- 72 11 9.4 6.3nosus CL-11

cation). In sharp contrast, the FDP aldolase. IDvalues are relatively uniform and cannot beused to differentiate the two subspecies (Table6). From the lower indices of dissimilarity andabsence of a clear divergence between the L.casei subspecies, it is clear that the constitutiveFDP aldolases are evolving at a significantlyslower rate than either the inducible malicenzyme or gross nucleotide sequence of thegenome.

CONCLUSIONSThe absence of paleontological records and

intricate development histories can no longerdeter the microbiologist from establishing natu-ral relations between physiologically and mor-phologically distinct groups of prokaryotes. Thebacterium's macromolecules, i.e., DNA, RNA,and proteins, have become the "fossil records"with which contemporary taxonomists arecharting natural affinities in the microbialworld. In contrast to similar studies with eu-karyotic proteins, there is no way at present toascertain whether a species of bacterium is ofrecent or ancient origin. To determine the age ofa species of organism or protein, paleontologicalrecords are needed as reference points in time.For the present, microbial taxonomists mustcontent themselves with the preparation ofphylogenetic maps with only comparative timescales. Relative time scales can be based onimmunological or amino acid sequence differ-ences. However, within this limited framework,

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the amount of work to be done is formidable.Our present level of technology is such that

DNA homology studies can provide the basis fora realistic and modern definition of a bacterialspecies (see Stanier, reference 71). Once acontemporary species concept has been for-mulated, relatedness studies similar to this onecan be integrated in such a way as to define thehigher taxonomic units. Thus, infraspecific andinfrageneric relationships can be established bycomparing the nucleotide base sequence ofDNA molecules or rapidly evolving proteins.More conserved proteins will be useful in dem-onstrating relationships at the tribal or familylevel. Suprafamilial groupings may be achievedby comparing highly conserved proteins or de-termining RNA homology. A classification sys-tem based upon these criteria will also have theadvantage of permitting microbiologists to de-tect the creation of new microbial species aswell as following the evolution of well-estab-lished lines of bacteria.

ACKNOWLEDGMENTSWe gratefully acknowledge the helpful comments

and criticisms of C. B. van Niel, R. Y. Stanier, M.Rogosa, and F. Gasser, in the preparation of thisarticle.

ADDENDUM

Subsequent to the completion of this manuscript, apaper by S. Tafler, P. Setlow, and L. Levine describ-ing an immunological study of bacterial DNA polym-erases was published (J. Bacteriol. 113:18, 1973).Antisera prepared against homogeneous E. coli DNApolymerase I were used to determine the degree ofimmunological relatedness between similar enzymesfound in other enteric bacteria. The following order ofrelatedness can be ascertained from the reportedindices of dissimilarity: Escherichia = Shigella >Salmonella > Klebsiella > Aerobacter > Serratia =Erwinia > Proteus. This ordering differs from theothers cited in this manuscript only in the placementof Erwinia and Proteus.

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ERRATUINMAldolasev of Lactic Acid Bacteria: a Case History in the Use

of an Enzyme as an Evolutionary MarkerJACK LONDON AND KIMBERLY KLINE

Microbial Physiology Section, Laboratory of Microbiology and Immunology, National Institute of DentalResearch. Bethesda, Maryland 20014

Volume 37, no. 4, p. 459. The equation used to calculate the index of dissimilarity was erroneouslyattributed to Wilson et al. (85). The expression was, in fact, derived from the definition of the indexof dissimilarity presented in references 67 and 85. A derivation of the correct equation and thejustification for calculating the ID by this method is given by A. B. Champion, E. M. Prager,D. Wachter, and A. C. Wilson (1974; Micro-complement fixation, p. 397-416; In C. A. Wrightled.], Biochemical and immunological taxonomy of animals; Academic Press Inc., London). Theequation is as follows:

Log ID = + log Xhm XH

where ID = the index of dissimilarity; m = slope (defined below); YH(Yh) = percent complementfixed at the peak obtained with the homologous (heterologous) antigen; XH(Xh) = antiserumconcentration used in the homologous (heterologous) reaction. The slope m is defined by theequation, Y = m log X + b, and for the aldolase system is 170.

Since it is routine procedure in our laboratory to raise the concentration of antibody in theheterologous system so that the percent of complement fixed (Yh) is equal to, slightly less orslightly greater than the percent of complement fixed by the homologous system (YH), the value

YH-Yhfor the term becomes insignificant and the discrepancies between the two equations are

mvery small or nonexistent. In all but three instances, recalculating our data with the equation ofChampion et al. yielded ID values which fell within the confidence limits of the values presentedin Table 6 (p. 468). However, the ID values for the aldolases of Lactobacillus jensenii, Lactobacillushelveticus, and Lactobacillus jugurti were 10 to 15% lower than those cited in Table 6 because itwas not possible to raise the antibody concentrations in these heterologous systems to a levelwhich would have made YH = Yh.

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