THE ORIGIN OF BACTERIAL SPECIES · Origin of Species has drawn to a close, and the bacteriologist realizes thathehasbeensomewhat overlooked in the celebration of Darwin's im-portant

THE ORIGIN OF BACTERIAL SPECIES

GENETIC RECOMBINATION AND FACTORS LIMITING IT BETWEEN BACTERIAL POPULATIONS

ARNOLD W. RAVIN'

Department of Biology, University of Rochester, Rochester, New York

CONTENTS

I. Introduction ............................................................................IH. The Species Concept.....................................................................

III. Bacterial Mutation and Recombination....................................................A. Mutation.............................................................................B. Recombination.........................................................................

1. Conjugation .......................................................................2. Transduction .....................................................................3. Transformation......................................................................

4. Multiple methods of genetic transfer.................................................IV. Isolating Mechanisms......................................................................

A. General...............................................................................B. In Conjugation .....................................................................C. In Transduction .....................................................................D. In Transformation.....................................................................

V. Bacterial Speciation .....................................................................VI. References................................................................................

I. INTRODUCTION

The centenary year of the publication of TheOrigin of Species has drawn to a close, and thebacteriologist realizes that he has been somewhatoverlooked in the celebration of Darwin's im-portant contribution to scientific and intellectualprogress. Yet this neglect of the bacteriologist isundeserved, for as this review will try to show,recent discoveries in bacterial genetics haverevealed that the Darwinian view of evolutionencompasses not only the world of "higher"plants and animals, but the world of bacteria as

well. Heretofore the principal difficulty in inte-grating the bacteria within the Darwinianmodel of evolution was the apparently differentways of defining species for sexually reproducing,"higher" plants and animals, on the one hand,and for clonally reproducing, acellular organisms,on the other. This difficulty, however, is largelyovercome by recognizing that bacteria are in-deed capable of transferring and recombininggenetic material, which is accomplished throughbiparental, sexual reproduction in "higher"organisms, and by defining species universally

I Research of the author is supported by grantE-727 awarded by the National Institute of Al-lergy and Infectious Diseases.

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in terms of the capacity to exchange geneticmaterial.The plan of this article is to review current

concepts of speciation, to show how our knowl-edge of genetic recombination in bacteria affordsa definition of bacterial species in genetic terms,and then finally to consider the usefulness ofsuch a view of bacterial species for our under-standing of bacterial evolution.

II. THE SPECIES CONCEPTThe Darwinian hypothesis proposed that

species of plants and animals are the productsprincipally of two forces: the origin of heritablevariations in individual organisms, and theselection of those variations endowing theirbearers with advantage of one kind or anotherover natural competitors for survival and repro-

duction (18). The science of genetics, whicharose and has undergone rapid development sincethe days of Darwin, has revealed the nature ofthe genetic alterations, or mutations, whichserve as the raw materials of selection: their lowfrequency under natural conditions, and theirrandomness, that is, the lack of correspondencebetween the selective advantage of the mutantphenotype and the environment in which themutations are produced. We have learned also

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of the important role that sexual reproductionplays in inicreasing the rate of prodluction ofgenetic variety in a population, through bringingtogetherI, by recoml)ination, dlifferent mutatioinsthat aiise in (lifferent iildiviiluials.The high incidence of an adlaptive genetic

constitution within a species is the result, then,of spontaneous miiutatioin, recombination, andselection. However, once an a(laptive genotype(or, more properly speaking, a set of adaptivegenotypes) is acquired by a population, are thereany meehanisiims to prevent its rapid break(lownby the "blind" forces of mutation and recombi-nation? For it is obvious that mutation ancdrecombination can just as efficiently bring non-adaptive diversity to a population as they canadaptive diversity. Of course, selection couldcontinue to weedl out adverse or deleteriousmutations, but again thaInks to the investigationsof geneticists, we know that special geneticmechanisms may arise to reinforce the work ofselection. These miiechanisms, w hich are them-selves the produc(t of mutation aindl recombi-nation, serve to limit the ex(hange of geneticmateIrial by members of a given population.Recombination becomes effectively limited tothose individuals sharing a common adaptivegene pool. Thus, such mechanisms have beenterImed isolating neclhanisnms (21).There are a number of ways in w-hich two

populations of sexually reproducinig organismscan become genetically isolated from each other.Sperm-egg interactioins can become highlyspecific, so that sperm of one population areincapable of feIrtilizing the eggs of the otherpopulation. Copulatory organs can become so

specific, that the male genitalia of one pOpulatiOliare mechanically incomlpatible with the femalegenitalia of the other. Complex breeding be-havior, involving the precopulatory- antics ofmale-female pairs, may also become highlyspecific. In plants, flowers may be produced atdifferent times in (lifferent species, and so on.

This brief review of our knowledge of speciationin higher plants and animals has been set forthto see if we can find similar processes availableand utilized by bacteria. It is perhaps importantto distinguish between availabilitv and utili-zation, for as will be pointed out later, goodevidence exists that similar processes are availableto the bacteria, although the evidence is lesscertain that they are actually utilized in nature.

Wle might properly begin by inquiring wlhat a

species is in the wAorld of clonally reproduciiigbacteria. WA-e know holw this problem has beenlhandled for the hiiglher plants and ainimals, whiichreproduce sexually. Long before geneticistsbegan to speak of isolating mechaanisIims, taxoii-omists developed rules of classification ac-

cording to wlhich species were defined. In genieial,one may say that a population of individualsshariing a particular constellation of ecological,physiological, moirphological, and behavioral(haracteristics was called a species. The particuilarconstellation of characteristics adopted was oInthat was sufficient to demarcate a given lpol)-lation from others, and that was especially stable,that is, was reproducible in a given environ-mental situation and did not change appreciablyfrom one geneIration to the next. Today thlegeneticist would refer to a species as a p)opulationof potentially interbreeding individuals, that is,onle ill which genetic recombination can (cecuirbetween any pair of its members but is preveinted(between its mienml)rs and those of other speciespl)opulations. In genieral, it is probably tr'ue tlhatthe taxononmist's species corresponds veryclosely with the geneticist's species. But this isnot always the case. Some populations, regardledfor good and compelling reasons by the taxon-omist as different species, may be founid to becapable of interbreeding. The solution to thisproblem has usually been one of two types:

a. If the two populations are separated by a

sufficiently large geographical distance or barrierit is possible to assume that they descendled fromsome common anicestral species, that migrationseparated them, that mutation, recombinationi,and selectioi (lifferentiated themi for theirecological specialties, and that a mechanismisolating them genetically has not arisen becauseit has no selective advantage except in an areawhere, or at a time when the two populationsconfront each other. The two populations canithus be considered subspecies or races, as youwill. This is not to say that all geographicallyseparated populations descended from a commonancestral group are capable of interbreedinig.It is possible that some such groups acquired,possibly as a secondary consequeince of othermorphological and physiological differentiation,ain effective mechanism isolating them repi o-ductively. Such groups are usually termedallopatric species (see (56)).

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b. If the two populations are not separatedgeographically, one may assume again that theyare the descendants of a common ancestralpopulation, that mutation and recombination aredifferentiating them, and that unless the ad-vantage to be gained by an isolating mechanism(i.e., by preservation of the differentiation)outweighs whatever adaptive advantage theremay result from genetic flow between them, thetwo populations will remain nothing more thantwo samples drawn from a larger, heterogeneouspopulation. On the contrary, if an isolatingmechanism is of relative advantage, as in thecase of populations adapting to different eco-logical niches, and if it is actually beginning tooperate, the two populations may be regarded asincipient species.The problem of defining species among bacteria

would, of course, be fundamentally different ifno exchange of genetic material ever occurredbetween individual bacteria. Wze are just begin-ning to know something about these methods ofgenetic exchange, and so it is no surprise thatthe convention of naming species in bacteria issimilar to that of the classic taxonomist innaming species of higher plants and animals.One chooses, to distinguish a bacterial species,a particular constellation of ecological, morpho-logical, physiological, and biochemical charac-teristics that is unique and stable enough todemarcate it from other species, for whichother constellations of characteristics are found.It will be interesting to see how well the speciesof the bacterial taxonomist accord with whateverspecies the bacterial geneticist arrives at byutilizing the criterion of potential exchange ofgenetic material.

Before doing so, one must consider what weknow of bacterial mutation and recombination.

III. BACTERIAL MUTATION AND RECOMBINAT10N

A. Mutation

The bacteriologist rarely deals with theindividual bacterium. Rather he is nearly alwaysworking with a culture, or population, con-sisting of several millions or billions of indi-viduals, which, alas, he has often been wont inthe past to regard as a homogeneous collectionand hence as a unit. Thus, when bacterial popu-lations were found to undergo genetic changesunder varying environmental conditions, the

view developed that bacteria, unlike the "higher"organisms, were genetically flexible and couldadapt their genotype fairly readily to the exi-gencies of the environment (30).

It was not until the advent of the bacterialgeneticist that this view could be disproved.For the bacterial geneticist employed methodsto discriminate between what was going on atthe level of the individual bacterium and whatwas going on in the culture or population as awhole. Suffice it to say, his methods were ableto establish that bacteria are no different from"higher" organisms in their mutability. Geneticchanges are rare spontaneously, but when theydo occur there is no necessary correspondencebetween the change and the kind of environmentin which it appears. Thanks, however, to theserandom mutations, a culture is always sufficientlyheterogeneous that when the environmentchanges, mutants adapted to the new environ-ment are selected. As Stanier (79) has aptlypointed out, the haploid condition of bacteria(42) causes newly arising mutations to be ex-posed immediately to the action of naturalselection, thus enhancing the rapidity with whichselection can cull the genotypes within a popu-lation. The roles that mutation and selectionplay in the survival of a bacterial populationliving in a fluctuating environment are probablyvery important.The first evidence that bacterial mutations

occur at random was statistical in nature (40,54, 60). The argument was that, if mutationsoccur rarely (less than once per several gener-ations) and at random, then-in the absence ofenvironmental conditions fav-oring the repro-duction of the mutants-very large fluctuationswould be expected between the mean numbersof mutants appearing in parallel cultures inocu-lated with equal but small numbers of bacteriadrawn from a common population. On the otherhand, large fluctuations would not be expected ifdirect genetic adaptation accounted for theappearance of mutations. In the latter case, abacterium may have a small probability ofadapting genetically to a given environment,but, in any case, the number of adapted mutantsappearing in a culture should be dependentonly upon the total number of bacteria in thatculture. Suffice it to say that the prediction ofthe hypothesis of rare, random mutation wasfulfilled in all cases adequately studied [for

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example, bacterial resistanlce to virus (54, 59);bacterial rcesistance to drugs and antibiotics(61, 77, 83); l)acterial al)ility to fermnent sugars

(71)].AnIi elegaInt 1)roof of the ran(loniess of bacterial

mutations was provided by the Lederbergs (46).This lroof is ind(epend(eit of statistical considlera-tions and requiies only the simlplicity of replicaplating. By this methlod a large numliber of cellsfrom a bacterial population is plated onI the sur-face of a nonselectiv-e iie(Iiuimi (i.e., one in whiclhthe ImlutaInts (1o Iot have a selective a(lvantage)ani(l after a stitablep)erio(l of incubationi replicaplates are made by transferring impressions ofthe superficial growtlh to another plate by meansof a velvet (lisc (or other simnillar dleice). Thereplica plat' (cont.aillSn saletive medium thatpermits only mutaints of theldesire(l type to grow.By comparing the originial w-ithl the rel)lica plate,one can locate those (olonies On nloinselectivemedlium fromii wliclh the 1)acteria (apable ofgrowing on the selective me(lium were drawn.Only the hypothesis of random spontaneous mu-tations predicts that these colonies already coIn-tain mutant clones, wlhicll may be large or smallin size depending uponi how early or late, re-spectively, the spontaneous mutationis appearedin the growth of the colonies. By isolating thesecolonies aId( l)lating againi oIn InoInselectivemtedium, one shouldI fiindI by replica plating oInselective medium that the proportion of mutantcells is greatly increase(d. In a series of isolations,platings, and replica tests, oine should finally ob-tain a "pure" cultuie of imiutant cells. This findingwas obtained for a number of cases of bacterialmutation (46, 77, 83).

Either the statistical fluctuation test or the in-direct selection metlhod provides an estimate ofthe frequency of spontaneous mutations in agiven population. Generally, these frequenciesare low. Expressed as the probability of mutationper bacterium per generation, they vary usuallyfrom about 10-7 to 10-10. It is clear, therefore,that bacterial mnutations are rare events and occurin the absence of conditions which specificallyfavor the mutants. The bacteria represent no ex-

ception to the general rule observed among"higher" plants and animals that inherited varia-tions occur at randlom.

B. RecoamlbinationGiven the inherite(d variations provided by

mutation, the amiount of genetic VarietV possible

in a populatioin can be increased by combining,, inIthe various possible ways, mutated genietic miia-terial of (liffereint individuals. Such increatse ingenetic variety is generally provided by reconi-bination. Recombination has been define(d i(-cently by IPontecorvo (64) in a useful way. Heconsiders it '... any process wh}bich gives origillto cells or in(lividualls associating in new ways tw ooi' moIre hereditary (leterminants in which theirancestors (liffered: for instance, cells witlh (de-terminants Ab oI aB cleseending from other cellsw-ith AB oI ab." HeIr A and a, or B and b, referto the ailternative states of a given hereditarydeterminant affecting a particular property of thecell or individual. Thus, A may symbolize thedeterminant in a bacterium endowing it ivithstreptomycin resistance, while a symbolizes thealternative, or homologous, determinant makin-gthe bacterium sensitive to streptomycin; B, on

the other hand, may refer to the (ieternminaltmaking the bacteriumii capable of fermenting atspecific sugar, while b refers to its homologuerendering the bacterium unable to ferimleint thissugar.

In the case of bacteria, genetic recombinationloperates in the following way. From a particularkind of action occurring betweeni two populations,one that is genetically AbCd. . , and one thatis genetically aBcD.... , can be deriv-ed otherpopulationis which possess some or all ofthe othei possible combinations: ABCD.....abed...., ABCd...., abcD...., AbcLD....,aBCd...., Abed...., .BCD..... A.Bed.....abCD...., ABcD...., abCd.... AbCD....and aB(d..... The fact of genetic recombina-tion demonstrates that the hereditary materialof bacteria, like that of other organisms, conlsistsof separable elements, or genes, whiclh determiiinedifferent specific functions. These separable (le-terminaints are not ne(essarily ex(hange(l inle-pendently of each other during recombination.Two or more determinants may be exchanged in ablock, an(d the frequency of such block exchangesindicates the degree of linkage between any pairor set of genes. A number of lines of evidence (20,41) point strongly to the likelihood that the geinesof bacteria are organized in linear arrays. In somllecases, the bacterial genes can be mapped withconsiderable precision.The significance that recombination may halve

in bacterial evolution lies in the fact that thereare generally several genetic differences betweenany two recognize(l species. Each species-dis-

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tinguishing characteristic is usually capable ofmutation independently of the others. Two suchfairly similar species as Aerobacter aerogenes andEscherichia coli differ in their ability to produceindole from tryptophan, to attack citrate as asole energy source for growth, to produce acetyl-methylcarbinol during glycolysis, and to accumu-late organic acids in sugar fermentation. More-over, each one of these characteristics isgenetically complex. For example, whereas theinability of wild-type E. coli to utilize citrate isnot accompanied by any inability to utilize otherKrebs-cycle intermediates, such as a-ketoglu-tarate and succinate, mutation in A. aerogenesleading to inability to attack citrate is accom-panied by the inability to attack other com-pounds in the Krebs cycle. Further mutation mayrestore some of the latter ability (65). Thus,since a single mutation will rarely produce adifferent species, recombination could be an im-portant means of bringing together rapidly theconstellation of genetic factors that can constitutethe adaptive genotype of a new species.The particular processes by which genetic re-

combination is achieved in bacteria have beenthoroughly reviewed in recent times (27, 43, 67).For the purposes of the present discussion, thethree principal ways will be discussed briefly inturn.

1. Conjugation. In some strains of bacteria onefinds that certain cells are capable of attaching toeach other, and that during the period of attach-ment genetic material is transferred from one cellto the other across the "conjugation bridge."This process of conjugation has been shown toinvolve actual cell-to-cell contact (5, 19), and thetransfer of genetic material is unaffected eitherby the enzyme deoxyribonuclease or by antiseraspecific for the various kinds of viruses that in-fect the bacterium in question. In these respects,conjugation differs from the two other processesof genetic transfer which will be discussed below.Following the transfer of genetic material, bac-teria possessing genetic material combined fromboth parents appear in the culture. Althoughconjugation has now been reported in at leastfive genera of bacteria (Escherichia (41), Shigella(55), Salmonella (9), Serratia (10), and Pseudo-monas (31)), only the process occurring in Escheri-chia has been thoroughly studied. For that reason,only the picture of conjugation obtained bystudying it in Escherichia will be described here.

In order for one bacterium to conjugate with

another, it has been found that certain mating-type differences must exist between them. Onetype is referred to as F+, the other as F-. A mix-ture of F- with F- cells is infertile so far as theproduction of recombinant progeny is concerned.While a mixture or "cross" between F+ and F+has a low fertility (possibly due to the small per-centage of physiological F- variants known toexist in a genetically F+ population), the highestfrequency of recombination occurs when F+ cellsare "crossed" with F- cells. There is one interest-ing feature of the F+ characteristic: it is generallyhighly infectious, appearing in all of the F- cellswith which the F+ cells come into contact. Sucha high frequency of transfer is not observed forother hereditary characters.Another important fact which has been estab-

lished concerning the transfer of genetic materialoccurring during conjugation is its one-waynature. If an F+ bacterium is allowed to conjugatewith an F- bacterium, and if, after a suitableperiod of time is allowed for genetic transfer, theconjugating pair is physically separated by meansof a micromanipulator, recombinants are foundonly within the clones that are produced by theF- ex-conjugant, never within the clones pro-duced by the F+ ex-conjugant (4, 44). This findingis consistent with the view that genetic materialpasses only from F+ to F-, and never in the re-verse direction. It is also consistent with previousobservations by Hayes (28) that a number ofphysical and chemical agents can be used to killthe F+ conjugant without disturbing subsequentgenetic recombination; the same is not true forthe F- conjugant.

In an F+ X F- "cross," in which, say, the F+parent is Ab.... with respect to a given pair ofcharacters and the F- parent is aB..... , theproportion of recombinants of a given kind, sayAB...., within the total mixed population isusually rather low (less than 10-4). Wollman,Jacob, and Hayes (85) have provided severalpieces of evidence which support the followingexplanation for this low incidence of recombinantsof a specific kind. A given F+ population is het-erogeneous in containing a number of mutants,each capable of delivering at high frequency (in100 per cent of its contacts with F- cells) a partof the F+ genome. These mutants are called"Hfr." The part of the F+ genome that is trans-ferred is usually large enough to consist of severalgenetic markers, but the particular group of

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genetic markers carried depends on the particularHfr mutant involved (36).

It is possible, by a number of ways, to showthat the transfer of the particular group ofmarkers characteristic of a given Hfr strain ex-hibits polarity and occurs in an ordered sequence.Everything occurs as though the particular groupof markers were linked in a linear array, as thoughthe group transferred were a segment of the com-plete, linearly arranged Hfr genome, as thoughone end of the transferred segment regularly en-tered the F- recipient first, as though the segmentwere transferred at a uniform rate and eachmarker in the linear linkage group appeared in theF- cell at a fixed time. Any experimental pro-cedure (such as prematurely lysing the Hfr con-jugant) which disturbs this orderly transfer, re-sults in the appearance in the F- recipient of onlya fraction of the total Hfr segment that is capableof being transferred into it.

It is theoretically possible to construct a mapof the complete F+ genome by studying the link-age relationships of markers transferred by differ-ent Hfr mutants of the F+ strain. However, thereare several problems attending this task, andsuffice it to say the matter is not yet resolved.

Lederberg (42) points out that the availableevidence is also consistent with the view that theentire F+ genome is transferred by an F+ or Hfrconjugant, but that a segment of the transferredgenome is eliminated from the F- recipient priorto the formation of recombinant progeny. In thisview, not all the possible recombinations of F+and F- genetic material can be produced, al-though the F- ex-conjugant is a complete zygote(or holozygote) rather than a partial zygote (ormerozygote) as in AVollman, Jacob, and Hayes'view. Studies of the segregation and recombina-tion of F+ genetic material in the clones descend-ing from the F- ex-conjugant (4, 44) show that,indeed, not all recombinations occur nor are theyreciprocal (an AbC.... for every aBc. . , forexample). Results of such studies also show thateither an incomplete F+ genome is transferredor, if a complete F+ genome is transferred, aspecific part of it is lost shortly after conjugation.

Nevertheless, it must be emphasized that, al-though the conjugation of a given pair of F+ (orHfr) and F- cells results in recombinations thatare neither reciprocal nor reflect the total assort-ment possible from the particular F+ and F-genomes, conjugation in bacteria serves the same

function as sexual reproduction in higher organ-isms. It can achieve the diffusion of genetic ma-terial through a population, and it can increasethe rate of production of genetic diversity.

2. Transduction. This similarity of conjugationto sexual reproduction in higher organisms isequally true of the processes of bacterial recoin-bination now to be discussed, transduction andtransformation.

Transduction is defined as the transfer of aportion of the genome of the bacterium previouslyinfected by a temperate bacteriophage into itsnew host. This phenomenon was first observedamong the salmonellae using PLT22 phage (86).Here it was found that if the previously infectedbacterium (the donor) was genetically AbC. ...and the newly infected bacterium (the host or re-cipient) is aBc....., abc recombinants or ABc re-combinants or aBC recombinants may be pro-duced. It is to be noted that generally only asingle marker is trai sduced by a given salmonellaphage. Yet different phage particles in a particu-lar donor lysate carry different donor markers tothe host cells they infect, so that, on a populationbasis, most, if not all, markers of the donorgenome are transferrable to recipient bacteria.

Unlike conjugation, therefore, in which severalgenetic markers affecting quite distinct functionsof the bacterium are generally transferred fromF+ donor to F- recipient, transduction rarelyachieves the transfer of two or more functionallyunrelated markers from donor to host. Onlyclosely linked, and usually functionally relatedmarkers are sometimes found to be transducedtogether (20, 52).

Since the observation of transduction dependson the temperate nature of the bacteriophagevector, a brief mention must be made of the differ-ence between temperate and virulent bacterio-phages. For a full account, see (12). When a par-ticular bacteriophage infects a host bacterium ofa particular strain, the resulting host-bacteriumcomplex may enter upon either of two series ofevents. In one series of events, the so-called l)vticcycle, host bacterial metabolism is radically al-tered to produce numerous replica phage and theeventual dissolution of the bacterial cell. In theother series of events, the so-called lysogeniccycle, the host bacterium is not radically alteredin its metabolic properties although it possessesa genetic factor, called a prophage, which is reg-ularly inherited by all progeny of the infected

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host and which endows the cells bearing it withthe capacity of producing mature, infectiousphage of the type which infected the originalhost. This liberation of mature phage either oc-curs spontaneously (in one of about 105 lyso-genized descendants) or may be induced by avariety of chemical or physical agents, such asultraviolet light. For a given host strain, a par-ticular bacteriophage is said to be temperate ifthe probability is great that infection by it willresult in a lysogenic cycle; it is said to be virulentif the probability is great, on the contrary, thatinfection by it will result in a lytic cycle.

It should be pointed out that, although a bac-teriophage must of necessity be temperate if atransduction is to be observed, lysogenization ofthe host bacterium is not obligatory. Transduc-tion may or may not be accompanied by lyso-genization.

There are a number of lines of evidence demon-strating that, in a lysogenized bacterium, thesite where prophage replication is coordinatedwith bacterial reproduction happens to be aspecific locus in the host bacterium's genome.Whether the prophage is attached as a small side-chain to the longer linear genome of the host orw-hether it constitutes an integral part of thelinear structure of the host's genome is not yetresolved.As we have noted above, in salmonellae prac-

tically any genetic marker of the donor may betransduced from donor to host, but usually differ-ent markers are borne on separate phage particles.Another type of tranduction is observed in Esch-erichia coli strain K12, where only a very fewspecific markers can be transferred. In this strain,bacteriophage X transfers only the gal locus whichaffects the ability to ferment galactose. Interest-ingl enough, conjugation studies have shownthat the site of attachment of prophage X is veryclosely linked to the gal locus (58).These facts suggest that in salmonellae the

site of attachment of prophage PLT22 is variableand that the particular bacterial markers it willbe capable of transducing are those located in thebacterial genome immediately adjacent to itspoint of attachment. In E. coli K12, on the otherhand, the site of X prophage is presumably fixed,namely, to the gal locus. Whether a bacterio-phage is a generalized or specialized transducer(as with, respectively, PLT22 in salmonellae andX in E. coli K12), seems to depend, therefore,

upon whether it has fixed or variable sites ofattachment in the particular bacterial strain itinfects.

3. Transformation. In certain species, exposureof bacteria to the deoxyribonucleic acid (DNA)fraction extracted from mutant cells results inthe genetic incorporation of determinants nor-mally inherited by the mutants. Such genetictransfers are termed transformations. They differfrom similar changes effected in transductionand conjugation in that the agent responsiblefor transformation is sensitive to the enzymewhich depolymerizes DNA. Indeed, all of thechemical evidence points to the conclusion thatthese transformations in bacteria are mediateddirectly by DNA (32); in any event, they neitherrequire direct contact of the host and donor cells,nor bacteriophage as an intermediary agent oftransfer, nor are they prevented in the presenceof proteolytic enzymes, of protein-denaturingagents, of ribonuclease, or of specific polysac-charide-combining agents.

Neither the power of bacteriophages to infecta bacterium nor their power to transfer the genesof their previous hosts is sensitive to the enzymedeoxyribonuclease. At least, this is true for bac-teriophages in their extracellular form. This factis presumably due to the protein coat which pro-tects their internally located DNA. In any event,it seems reasonable that DNA bears the geneticinformation that is transferred in either transfor-mation, transduction, or conjugation. That DNAis the principal substance of phage that entersthe bacterial host is well demonstrated (29).Moreover, the transfer of genetic information ineither transduction or conjugation is highly sensi-tive to the decay of DNA-incorporated P3 andto ultraviolet light (23, 24), which result is to beexpected if DNA is the material that is actuallytransferred in these processes.

Transformations were first observed with thepneumococci (7, 26), but since that time similarprocesses have been found to occur among thegenera Haemophilus (2), Neisseria (3, 15), Agro-bacterium (38), Xanthomonas (17), and recentlyin Bacillus subtilis (78). In a transformation ex-periment, DNA is extracted from a donor straingenetically marked AbC.... ; an appropriaterecipient strain, one marked aBc, is then exposedto the more or less purified donor DNA. Shortlyafter exposure, the recipient cells are plated onsuitable selective media, on which the recombin-

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ant transformed recipients, of type ABc.... etc.,can be detected. As in transduction, the transfor-mation of a given recipienit b,acterium rarely re-sults in the replacement of more than oIne geneticdeterminant (A or b or C, in the case cited above).Nevertheless, closely linke(d determinants areknown to be transferired together (luring trans-formation (34, 35, 69).The process of transformation obviously has

great significance for the future study of thegenetics of somatic cells of higher organisms (66).Whatever informationi is elicited regarding themechanism of this process should prove usefulnot only in exten(ding transformation techniquesto more groups of bacteria, but also in investi-gating the hereditary differentiation of the so-matic cells of a higher plant or animal. To datewe know that, in ordler for transformations tooccur, the recipient cells must be in a physiologi-cal state of competence, whlich is geared to cellularreproduction ani-d apparently involves proteinsynthesis (22). Wh'enI competent, the bacteriacan make an effective contact with trainsformingDNA in a v-ery brief period of time (probablyless thani a minute). Haxving made this effectivecontact, the recipient bacterium is oIn the wayto becoming transformed, wlhich process can nolonger be reversed by deoxyribonuclease. How-ever, suitable physiological conditions and suffi-cient time must be provided for the expression ofthe phenotype (orresplondinig to the newly ac-quired genotype. Furtlhermiiore, it is kinow-n thatthe transformiIg DNA acquircd by the recipientbacterium is not immediately integrate(l intoits genome. This bacterium reprodluces an(d one ormore generations may elapse before the trans-ferred segment of (lonor I)NA is integrate(l intothe genome of whichever of the progeny cellshappen to receive it. AVith integration, this seg-ment of donoi DNA is relplicated in coordinationwith the total genome of which it is a part, itshomologue having been effectively rernoved(34, 68, 82).

4. Multiple miethods of genetic transfer. It isaltogether possible that a given bacterial speciesmay be capable of two or more methods of geneticrecombination, possibly under different condi-tions. For example, it is clear that both conjuga-tion and transduction can occur in certain strainsof Escherichia coli (58). Transformation is nowbeing studied in groups in which Ilysogeny isknown and, therefore, in which transduction is

likely (78). Undoubtedly, as further progress ismade, we shall learn of more andimore instancesof this kind. From an evolutionary point of view,of course, the significance is obvious. The greaterthe opportunities for genetic exchange betweenorganisms, the greater the spe(d with w -iicliadaptive genotypes can be produced in the pop-ulation to w-hiclh theyl belong.

IV. ISOLATING MIECHANISMSA. General

The significance of recombinatioIn that hasjust been pointed out has a corollary. Once an

adaptive genotype has been arrived at in a limlit-ing environment, sele(tion will imake predloini-nant those organisms possessing this genotvy)e.However, unless there is a I)ar to furthermutations and especially to recombinations withthe genotypes of poorl- adapted individuals, theadaptations will be transieInt on(es. Put anotherway, the efficiency of achievinig adaptationthrough selection is imp)airedl if there is no mech-anism for setting limits to the loss of whateveradaptive states are acquiredi at a given time. Inhigher organisms limits of this kindl are imposedthrough isolating mechanisms which allow tranIIs-fer of genetic material andl recombination tooccur onl1 Nx ithin certain circuniscribed popIula-tions. The living universe is nota vast comIllunityof genetically interacting organisms. Rather it is

divided into a large number of populations be-twN-een wh-]lichi "gene flow" is imipossible or mili-mized. The modern svstematist trained ingenetics would assign the ternm "biological spe-cies" to these populations.

Is there evidence th.at the baicterial universeis also differentiated into a number of populatioiiswAhose gene pools are separated from one another;that, in other wor(s, isolating miechanisms miatyoperate to limilit genetic recomb)ination in )ac-

teria?

B. In ConjugationThe F+ factor appears to affect the bacteriul

in such1 a way as to alter its surface antigenieproperties (51) and its motility (76). These effectsof the F+ factor are not surprising in view of thespecificity displayed by F+ cells in contactingP cells (lurinig conjugation. Several pieces ofevidence indicate that the F+ determinant isunlike other genetic factors that are transmittedduring conjugation (45). F+ shares, however, an

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important property of bacteriophage: after in-fection it behaves as though it were an additionto the bacterial genome. As an addition, it canfurthermore exist in one of two forms: in oneform, it is an unintegrated part of the bacterialgenome, and may be replicated either more orless rapidly than the "chromosomal" or linkedgenes (being infectious or lost through dilution,respectively); in its other form, it is integratedin the sense that its replication is coordinatedwith the replication of the host's genes, as thoughattached to them. In this latter form, found inHfr bacteria, the F+ factor is incapable of in-fecting other bacteria (while permitting con-jugation with them) and also prevents superin-fection of its host bacterium by a nonintegratedF+ factor. For genetic factors that are additionsto, rather than essential constituents of, thegenome, and which may exist either in an auton-omously replicating or in an integrated condi-tion, Jacob and Wollman (37) have proposed theterm "episomes."

It should be pointed out that the F+ factor,unlike bacteriophage, has not been obtained inextracellular form, although attempts have beenmade to do so (48), and F+ contagion requiresdirect contact between the F+ donor and F- re-cipient cells.What is particularly interesting about the F+

factor is the fact that there seem to be a numberof different kinds. An F+ factor making a bac-terium fertile with some F- cultures does notnecessarily make it capable of conjugating withothers (49). Furthermore, there is a good possi-bility that a single bacterium may possess morethan one F+ factor, and that one such factor maybe epistatic to another. Two strains may be inter-sterile, and yet differ in the possession of a factorcontrolling ability to conjugate with a thirdstrain (11). As a result, a large population of bac-teria capable of conjugation is separated intosmaller groups between which genetic transfercan occur to varying degrees, or not at all.How large a bacterial population may be inter-

related and connected by means of a system offertility (F) factors? There is now good evidencethat gene flow can spread beyond the boundariesof groups that are recognized as species by bac-terial taxonomists. Luria and Burrous (55) haveshown that a number of Shigella strains behaveas F strains with two E. coli F+ strains. As aresult of these conjugations, hybrids are found

which combine characters typical of E. coli withcharacters typical of Shigella species. Althoughconjugation is as efficient (up to 100 per cent) incrosses between Shigella F and E. coli F+ as incrosses between E. coli F and E. coli F+, thefrequency of recombination is lower in theShigella X E. coli crosses. Furthermore, somegenetic determinants of the E. coli parent failcompletely to be transmitted to the hybrid.These results suggest strongly that genetic ho-mology between the Shigella and Escherichiastrains is incomplete, as a consequence of whichintegration of E. coli genes into the recipientshigellae is not as efficient as in an "intra-spe-cific" cross. It may be hypothesized that in thecourse of evolution the processes of mutationand recombination occurring in separated pop-ulations of enteric bacteria that were originallygenotypically identical led to an increasing differ-ence in the structure of the DNA in the two pop-ulations. One might expect that this differencein structure, or lack of genetic homology, will bereflected in impaired pairing between DNA mole-cules of the two types. If such pairing is a neces-sary precursor to recombination between them,decreased efficiency in this step will result in alowered frequency of recombination. It may evenbe imagined that an F+ population may be cap-able of conjugating with an F population whilestill unable to have its genes integrated into thegenomes of the recipient bacteria, because of in-sufficient pairing homology between their re-spective DNAs. In this regard, Lederberg andLederberg (48) have reported strains that do notproduce recombinants with an E. coli K12 Fculture but that do possess an infectious F+ fac-tor. It was not ascertained whether those strainsphysically conjugated with the F tester, whilefailing to produce recombinations in it.

In summary, we can indicate at least two mech-anisms that may operate to limit genetic recom-bination in conjugation. The first of these isdifferences in fertility factors, which affect con-jugation compatibility. The second is differencesin structure of genetic material, which affectpairing compatibility and subsequent recombina-tions.

Luria and Burrous (55) also tried to obtainhybridization between E. coli F+ (or Hfr) cul-tures and several different Salmonella strainsbelonging to the antigenic group E2. These at-tempts failed to produce any hybrids. Recently,

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however, Baron, Spilman, and Carey (9) weresuccessful in obtaining hybrids from "crosses"between E. coli K12 (Hfr or F+) and 15 of about85 different Salmonella species or strains tested.The transfer investigated was that of the Lac+(lactose utilization) nilarker from the donor E.coli strain to the recipieInt Salmonella strain.2 TheSalmtionella strains that succeeded in serving asF- recipients belonged to a variety of serotypesin the Kaufmannl-Whlitc schema, so that therew as Ino apparent association betw-een known anti-genic components in Salmlonella species and theability to conjugate with and integrate the Lac+marker from E. coli K12. The frequency of Lac+hybri(Is was very low in the Salmonella popula-tions that proved to conjugate successfully (inthe order of 10-1 to 10-9). However, when a Lac+hybrid -as isolate(d an(l 'crossed" again with asuitabll marked E. coli K12 Hfr donor strain, thefrequency of hybrids was considerably higher (inthe order of 10-4). Utifortunately, it is not clearat the present timc whicther the low frequency ofhyNbridization in thc iitilial cross is clue to the inl-ability of the vast mnajority of recipient cells toconjugate with E. col Hfr or to pairing incom-patibility between the genomes of the donor andrecipient strains, a pairing incompatibility whichis at least partially alleviated following a success-ful iintegration of (doIoIr genetic material into therecipient cell.

lIn(ed, in the cas(s of falilure to hy-bridizeSalmtiontella strains with 1E. coli, rel)ortcdl both byLuria and Burrous (55) and by Blaron et al. (9),it was not determinedIwhich of the above-men-tionied mechanisnms, p)airinig incompatibility orconijugation incomn)atil)ility, resulted in thefailure to obtaini recombinations. It is altogetherpossible that other coliform organisms possessing

2 How\-ever, in the Salm2onella typhimuriuntlstrain TMI-9, the transfer was studied in greaterdetail (8). In this straiii it w-as found that geneticfactors for several (lifferent biochemical traits(incluiding lactose utilization and indole produc-tion) could be transferred, albeit, with differentefficiencies. The transfer of certain other markers(including those for X-sensitivity and for somaticand flagellar antigens) w-as not detected. Further-more, the hybrids produtced on using an E. coli F+donor did not appear to be infected by theF+ agent. Similar and additional results, includinghybridization of phage sensitivity and antigeniccharacteristics, have been reported by Miyake andI)emerec (57).

different F factors may succeed in hybridizingwith some Salmonella strains. In other words,only positive hybridization results can give anestimate of the extent of gene flow between pop-ulations belonging to different taxonomic species,and this estimate must of necessitv be a minimalone until there is convincing evidence that allthe fertility factors have been discovered (whichis certainly not true today). Ev-eII within w-hat isregardedl as a single species, Escherichia coli, onlyabout 80 out of 2000 strains tested behaved aseither F+ or F- to the particular fertility factorpresent in the E. coli K12 strain employed byLederberg and his associates (49).

It is altogether possible that conjugation in-compatibility due to differences in fertility factorsmay be the principal mechanism serving to re-strict genetic recombination between populationswhich are still fairly similar in their genomes andhence would be regarded as members of the samespecies by at taxonomist considering only pheno-typic platterns. Pairing incompatibility duie tomajor (lifferences in D.NA structure may be theprincipal mechanism restricting genetic reconi-bination between populations that have evolvedto a considerable extent from an originally similargenotype condition.

C. In Transduction

The ways in w-hich transduction can be limitedas a means of exchanging genetic informationbetween bacterial populations are not difficult tovisualize. Bacteriophages are fairly specific intheir ability to inifect bacterial hosts. Bacterio-phages capable of infecting most strains w-ithinthe same bacterial species are uncommon, buteven less frequent are bacteriophages capable ofinfecting different species within the same genus,and rarest of all are bacteriophages capable ofinfecting different genera.The ability of a bacteriophage to be adsorbed

by and to infect a bacterium is a genetic char-acter, and mav be altered b- a mutation in thephage genome or by a mutation in the bacterialgenome. Thus, resistance by a bacterium to aspecific bacteriophage can result either from abacterial mutation or from a phage mutation.Conversely, the pattern of infectivity by a givenbacteriophage in a number of bacterial strains(the so-called "host range" of the phage) is alter-able by mutation, and by recombination as well.

It is obvious that any mutation or recombina-

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tion resulting in the restriction of a bacterio-phage's ability to infect will thereby limit itsability to transduce bacterial genes. For example,Lennox (52) found that the wild-type phage P1,which does not form plaques on the K12 strainof E. coli, does not transduce genes into thisstrain; however, mutants of this phage, P1 k andP1 kc, which were selected for their plaque-form-ing ability on the K12 strain, can transducegenes into this strain. Furthermore, mutationsand recombinations in either phage or bacterialgenomes may affect the relative probability ofsetting up a lysogenic or lytic cycle for agiven bacterium-bacteriophage complex. Geneticchanges leading to a higher probability of thelytic cycle (or, in other words, making the phagemore virulent) will also lead to a limitation oftransduction. Although lysogenization is notobligatory for transduction to be carried out, thebacteriophage must obviously be temperate for atransduction to be effected. Indeed, there is goodevidence that transductions are produced by asmall defective percentage of a bacteriophagepopulation, by phages that can infect but cannotgive rise by themselves to mature infectious par-ticles (1, 14).

It is not yet settled whether all transductions,generalized as well as specialized, result from thecarrying by the transducing phage of that portionof its previous host's genome to which it had beenattached (12). The assumption that generalizedtransductions are due to phages that have vari-able sites of attachment to the host genome,whereas specialized transductions are due tophages that have a fixed site of attachment, isconsistent with the available evidence. If thisassumption is correct, moreover, it is possibleto imagine another mechanism for limitinggenetic transfer by transduction. Any geneticchange of temperate bacteriophage or of its hostthat alters the number of sites to which the phagecan be attached will affect the amount of geneticinformation it can transfer. Certainly bacterio-phage X in E. coli K12 is of less significance as ameans of increasing bacterial recombinationthan is bacteriophage PLT22 in the salmonellae.Although rare, bacteriophages are known that

cross taxonomic species and genus boundaries intheir infectivity. It has been shown that at leastsome of these bacteriophages with broad hostranges can also effect transductions betweenspecies or even genera. Lederberg and Edwards

(47) demonstrated that phage PLT22, derivedfrom a lysogenic strain of Salmonella typhimnu-rium, can bring about antigenic recombinationsbetween strains of Salmonella which have, ontaxonomic grounds, received species status. As aresult of such recombinations, several serotypeswere produced that had been previously dis-covered in nature and classified in the Kaufmann-White schema of classification. In addition, otherserotypes were produced that had so far been un-discovered in nature.Lennox (52) has shown that phage P1, origi-

nally isolated from the Lisbon-Carrere strain ofE. coli, and its mutant derivatives P1 k snd P1kc are capable not only of transducing characterswithin and between a number of strains of E.coli (B/r, C, W, K12), but also of transducingcharacters between E. coli and Shigella dysen-teriae strain Sh. Phage P1 grown on S. dysenteriaecan transduce such characters as galactose-utili-zation, pyrimidine-, tryptophan-, and arginine-independence into the appropriate mutants ofE. coli strains B, C, and W. Phage 1 kc grown onE. coli K12 can transduce such characters asarabinose-utilization and lactose-utilization intoShigella recipients. The Lac+ Shigella strain pro-duced by transduction still retained its othershigella characters, such as nonmotility and in-ability to produce indole. The Lac+ strain ofShigella is, therefore, a novel bacterial strain, andis especially interesting because the Lac- char-acter of shigellae found in nature seems to be quitestable, spontaneous Lac+ mutants not beingfound at least in the Sh strain.

Thus, hybridization can be effected between E.coli and S. dysenteriae by both conjugation andtransduction, which indicates at least partialgenetic homology between these organisms.

D. In Transformation

Apparently the physiological state of com-petence essential for transformability, whichseems to involve the synthesis of DNA-adsorbingsites on the surface of the host bacterium, is non-specific so far as the source of the DNA is con-cerned. DNA from a different species of bacteriumis picked up as avidly by competent bacteria asDNA obtained from the same strain as thatof the recipient bacteria (33, 72). Nevertheless,there may be other physiological barriers thatmay exclude potentially transforming DNAfrom entering a particular bacterium. It has

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b)een shown that the transformability of a pneu-mocoecus vraries inversely as its production ofcapsular polysaccharide (68), tlhus suggestingthat the nature of the (cell -vall ancd capsule canaffect a bacterium's aI)ilitv to recombine its ge-netic material with that of another organism bymeans of the latter's extracellular DNA. Thatsome populations of bacteria regularly liberateDNA into their environment has lbeen demon-strated. Catlin (15) lhas shown that certainneisseriae, UpoIn aginw, extrude DNA in theirslime layer. Furtherniiore, she has shown thatthis DNA is effective in transformation.

Probably the most important barrier to genetictransformations occurring between different pop-ulations of bacteria is their dlegree of genetichomology, as reflectecd in the structure of theirrespective DNAs. To (late a number of caseshave been reported( of transformations producedbetween populations regarded as distinct speciesby the bacterial taxoniomiiist. Schaeffer first re-ported interspecific tranisformation betweenHaentophilus influenzae and Haernophiluts para-influenzae (75). Leidy ct al. (50) extended thisfinding for a number of H. influenzae and H. para-influenzae strains. They also reported failure totransform Haemophiltis suis with DNA fromeitlher H. influenzae or H. parainfluenzae, al-tlhough it was transformied by autologous DNA.Bracco et al. (13) rel)orte(l transformation of twoviridans strains of Streptococcus, using DNAeither from these same strains, or from Streptococ-cus salivarius, or fromii pneumococci. Pakula andhis associates (62) h,a-e extended the investiga-tions on streptococci, and haxe reported that of45 viridans strains tested, only 13 were transform-able. Each of these 13 strains was transformed byDNA from any one oIr more of 5 different viridansstrains, 3 S. salivarius strains, 1 Streptococcus SBEstrain, 4 hemolytic strep)tococcus strains, and Ienterococcus strain. None of 16 S. salivariusstrains tested could he transformed, but sincethey were recently isolated from human salivaand presumably encapsulated, the failure couldbe due to poor penetrability of DNA into thesel)acteria. Of a number of hemolytic streptococci,only two strains belonging to group H were trans-formed; these two strains were transformed byDNA from viridans streptococci, S. salivarius, S.SBE, aind heterologouis hemolytic streptococci.Streptococcus SBE was also transformed by theseDNAs. Further work 1y Pakula's group demon-stnrated that intergenelrie transfo)rmations were

possible as well (63). The unencapsulated R36Astrain of pneumococcus was transformed by DNAfroim viridans streptococci, S. salivarius, S. SBE,ancl hemolvtic streptococci. Conversely, S. SBE,-iridans and lheimolvtic streptococci were trans-formed 1y pneumiococcal DNA. In addition,ssuccessful transforiiation was reported usingstaphylococcal DNA on one strain of S. SBEand one strain of hemolytic streptococcus. Thus,the relationships of the pneumococcus-streptococ-cus-staphvlococcus group of bacteria are suscep-tible of genetic analysis. Catlin (16) has also beenable to perform interspecific transformations ofATeisseria species. It is noteworthy that DNA lib-erated into the medium by autolyzing cells in anaging culture is as effective in induciing inter-specific transformations as DNA extracted fromthe cells by chemical means.

In general, where quantitati-e measurementswere made of the frequency of transformation,it has been found that for a given genetic char-acteristic the frequency is lower in inter- orheterospecific transformations than in intra-or homospecific transformations. Schaeffer (74)studied the question of whether the low frequencyof interspecific transformations was due to thelheterospecific origin of the genetic marker itselfor of the entire DNA transforming molecule, ofwhich the marker is a part. The particular markerhe used was one (Sm) conferring resistance tostreptomycin. To resolve the problem, he ex-tracted DNA from a lheterospecific transformant(XSmY) and tested it on the original recipientstrain (X), comparing it with the DNA extractedfrom the original streptomycin-resistant donorstrain (YSm). He found that suchIDNA does notbehave like the original donor DNA. On theoriginal recipient strain X it behaves like theDNA from that strain; that is, the DNA fromXSmY behaves like DNA from XSm (or XSmX)rather than like DNA from YSm (or YSmY), inits efficiency of transformation. Thus, in thecourse of transforming bacteria with DNA froma different species, the transformant itself isrelieved of whatever factors contributed to thelower efficiency of interspecific transformation.That the DNA of XSmY does retain some ofthe Y-specific structures, however, is suggestedby the fact that higher frequencies of transfor-mation are obtained in the reaction Y X DNAof XSmY than in the reaction Y X DNA ofXSmX.

These results tare consistent wsithi alhypoth-

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esis according to which pairing between theendogenous (or host bacterium's) DNA and theexogenous (transforming) DNA is a necessary

prelude to integration of a particular exogenous

marker (74). It is assumed that transformationdoes not necessarily involve integration of theentire molecule bearing the marker in question,which is supported by the results of linked trans-formations. It is further assumed that theexactness of pairing depends on the structuralhomology of the endogenous and exogenous DNAmolecules: the less complete the homology, theless exact is the pairing, which results in a loweredefficiency of integration of an exogenous markerinto the recipient genome. It will be noted thatthis hypothesis is essentially the same as theone proposed to account for lowered recombina-tion frequencies in interspecific conjugations(see above). In the case of heterospecific trans-formations, moreover, it appears that the inte-grated marker may be freed from those adjacentregions of the exogenous molecule which causeda lowered probability of integration.

Schaeffer (72, 73) then demonstrated, bymeans of DNA labeled with P32, that DNA fromone species of Haemophilus can be taken up bybacteria of another Haemophtlus species with thesame ease as DNA of the same species. Thesame number of DNA molecules, as measured byp32 uptake, is incorporated per competent cell ineither case. This follows from the fact that theDNA from a number of different species(although not all) have the same affinity for thesurface receptor of the host strain, and indeedcompete with each other for penetration into thecell when presented as a mixture to the recipientbacteria. Since DNA of, say, H. parainfluenzaehas no difficulty getting into H. influenzae, thenthe fact that the frequency of such interspecifictransformations is lower than the intraspecificones must be due to some process occurringafter penetration. This finding further supportsthe hypothesis of inexact pairing.Does such pairing incompatibility arise only

when bacterial populations have evolved genet-ically to a considerable extent, so that they are

recognized as different species? Recent evidenceindicates that pairing incompatibility may

arise within very closely related strains. Greenhas studied (25) the frequency of transformationby two different markers in two unencapsulatedstrains of pneumococcus, Rx and Rz, which were

independently isolated although closely related

in heritage (68). The two markers were ery2 andstr, which confer resistance to erythromycin andstreptomycin, respectively. It was observedthat, at any DNA concentration, the ery2 markertransforms both strains with equal frequency,but the str marker transforms the Rz strain witha fourfold less efficiency than it does the Rxstrain. Thus, there appears to be a specific effectdepressing the frequency of str transformationsin the Rz host strain. Green showed that, byvarying conditions under which the (loublymarked DNA was adsorbed to the Rz strain, hecould alter the frequency of transformation byboth markers by as much as a thousandfold, butthe ratio of ery2 to str transformations in the Rzstrain was independent of these changes. It didnot appear that selective nonadsorption of thestr marker by the Rz strain could explain thelowered frequency of str transformations. As amatter of fact, by examining the frequency oftransformants (recipient cells that adsorbed,integrated, and phenotypically expressed theacquisition of the marker) at various times afterthe recipient cells made contact with the trans-forming DNA, Green found that the initial rateof appearance of str transformants was the samein the Rx and Rz strains. At about 45 minutesafter contact, while str transformants continuedto be produced in the Rx strain, no further strtransformants were produced in the Rz strain.It appears as though some postinfection process,necessary for the completion of transformation,is terminated earlier in the Rz strain than in theRx strain. It is possible to imagine again thatpairing difficulties specific to the str region in theRz host lead to a premature termination of theintegration of the str marker in that strain.The question arises as to whether the factor

causing a depressed frequency of str transforma-tions is lost when the str marker is successfullyintegrated into the Rz host, as was found in thecase of the interspecific Haemophilus transforma-tions studied by Schaeffer. Green in-estigatedthis possibility by isolating 21 different strtransformants produced in the Rz strain by aDNA yielding str transformations at a depressedfrequency. Each str transformant was the resultof a separate transformation of a recipientbacterium. These transformants were thenindividually transformed by the ery2 marker.The DNA was then extracted from each of thesedoubly marked strains, and was tested on boththe Rx and Rz strains. All 21 DNAs prodluced

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('1J12 alwl(l str transformiations with equal frequencyin the R.x strain. How-exver, only one of theseD)NA p)reparationIs produced this result in theRz strain. Twenty of these)preplarations yleldle(lstir tran4I w4ormations at the previously observed,fourftold1 dlelpresse(l frequency. Thus, unlike thesituation in intersp)ecific Haetnophilus trans-formaitions, the str marker was found to be trans-fer'I'ed, in imiost eases, along with the "depressingfactor." The latter, while separable from the strniarker, was closely liniiked to it, anid behavedlike a transferable genetic factor.

Green (25) also showed that ultraviolet irra-ihation has anl inlteresting effect on the doublymilarked D-NA preparation from N-hiich the strnarker i tIraIsferred into the Rz strain at a

d(epresse(l frequency. Whenheanily irradited,such D)NA continueC'S to y-iekld str traInsformanitsat a d(ellressed( frequency, but the DNA subse-(qutently extracted froIml these str transformants is

often flounld to be relieved of the "'depressingfactor.' Seventeen sutch transformants wereexamineId, b first trainsforming them with thecry2 marker and( then extracting DNA from them.Tlhese l)NAs wvere tlhenI comIiparecd oIn the Rx aindRz strains. Fourteeni were found(l to be relieved ofthe ldep)ressing factor," transferring the strinarker at a nornmal frequency (relative to theCr!J Marker), while three continued to reveal thepresence (of the 'depressing factor.' Tlhus,uiltraviolet light appea;Irs either to destroysele('tivelv the 'depressing factor" or to increasethe prolbability of reconmbination away from it.

In any event, Green's work shoNs that DNAinco()ml)atil)ilitv factors can be found withini veryclosely related populations. It suggests that suchflaCtori's may be the ra\v materials fromi -whichstI'ronger btarriers to inte(rs)ecifi(c transformvationsmayI1t he (onstrut('(l.

V. B.ACT RIAL SPECIATION

The re1(ent investigations of recombination inb)acteria haave afforded an excellent opportunityto stu(ly the genetic relationships of differentbacterial populations. A particular result of theseinvestigations, which it has heeui the burclen ofthis article to demonstrate, is that for each modeof g(enetic recombination (conjugation, trans-duction. or transformation) specific isolatingmechanismis exist. Genetic recombination can,indeed, be severely limited between populationsof bacteria capable of the same mode of recom-billation.

The question arises, therefore, whether theseisolating mechanisms separate only populationisbelonging to wlhat the bacterial taxonomist hascalled species, andl are lacking in populationlsbelonging to the same taxonomic species. Inother words, the question is really the one posedat the outset of this review: whether the spee(ieSof the taxonomist correspond exactly to speciesdefined on the basis of geneti( recombination.Numerous instances have been cited in thepreceding sections that exact corresponclenice isnot observed.

B3efore proceeding to discuss why this finidingshould not surprise us, it is perhalps use(ful torecall wlhat van Niel (84) has so well stated: thetasks of the classifier and of the student ofgenetic relationships are not to be confused. Theconstruction of a practical key for the classifica-tion of the bacteria, wlhiicl has been the primarygoal of the bacterial taxonomist up to the presenttime, is a necessary and important task. Thesole criterion for the adoption of a particular keyshould be the empirical one of wlhetlher it xN-orks,whether one finds the dlesignation for theorganism one is study-ing. Other criteria mnust beuse(l, however, wheIn schemas are proposecl forthe plrobable evolutionary rilationships of thebacteria. Here we must have evidence for asseit-ing ". that group A is more closely related togroup B than it is to group C because the separa-tion in descent of group A from group B is More

recent than the separation of groups A anw 1Bfrom group C." It is precisely in questions ofthis sort that genetic evidence is imlportaInt. IIndiscussing bacterial evolution, moreover, itbehooves tis to utilize a definitioni of bacterialspecies that reflects the metechanisms involved inspeeiatilon.

In tl1e preceding sec(tions a mio(lel w as pre-sented'l for the conisequeinces to genetic materialof e(volutionary clhainge. As a bacterial populatiOInevolves, the predominant form of its geneticmaterial is changing. -Mutation and presumablyrecombination bring forth ne(w genotypes, an(lnatural selection causes the most adaptivce ofthese to prevail at any given time. Since DNAis a major component of the genetic material,one can expect that these processes will result illdlifferentiation or dlivergendce of the evolving DNAfrom that of the ancestral type. Lanni ((39) andlpersonal co7n munication) has recently performeiIan important scholarly job in bringing togethem'the available exvidence coincerining the w ays in

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which DNAs of a wide variety of organismsdiffer. The evidence he has compiled, which isadmittedly fragmentarv, points suggestively tothe conclusion that differentiation proceeds byway of alteration of the relative proportion ofadenine + thymine (A + T) to guanine +cytosine (G + C) nucleotides in the DNAmolecule. Groups of bacteria that are known onother grounds to be closely related generallypossess DNAs having similar (A + T)/(G + C)ratios. Thus, for example, this ratio for E. coli,salmonella and shigella DNAs is about 1, whileAerobacter aerogenes and Serratia marcescens haveDNAs with a ratio of 0.76. Proteus species, onthe other hand, have a DNA with an (A + T)/(G + C) ratio of 1.75.3At what point in the divergence of a bacterial

population from some ancestral type is it to beregarded as a distinct species? In answering thisquestion, one must first point out that if geneticdivergence alone occurred in nature, the taxono-mist would be unable to recognize discretepopulations of bacteria possessing characteristicsthat clearly distinguished one from another.Genetic divergence alone would make for abroad, extreme form of heterogeneity lacking inany discontinuity which would permit the humanobserver even to conceive of taxonomic cate-gories. Yet there is sufficient discontinuity in thebacterial universe to permit a fairly complexsystem of classification in which the variousorders and families are quite distinct (80, 84).Distinctness of boundaries falls off, on the otherhand, at lower taxonomic levels. Especially asone examines samples from similar ecologicalniches collected at different places, populations

3 An extraordinary finding of recent studiesusing density-gradient centrifugation has been thehigh degree of homogeneity in the population ofDNA molecules from a given species (70, 81). Ininvestigations of the relation between buoyantdensity in a cesium chloride solution and the basecomposition of DNA, several bacterial specieswere used in which the fraction of guanine-cyto-sine in their respective DNAs varied from 30 to70 per cent. It was found that the buoyant densitywas directly proportional to the guanine-cytosinecontent. But more remarkable still, in the wordsof Rolfe and Meselson (70), was the fact ". . . thatthe standard deviation of guanine-cytosine con-tent within the molecular population of any onebacterial species covers less than one-tenth of therange over which the mean guanine-cytosine con-tent, varies among the various species."

are found which show some degree of "overlap-ping," and "intermediacy" is common. It isprobably true, however, that in a particular localor micro-environment bacterial populations aremore sharply delineated from each other, andintermediate types are a less common occurrence.To what is this discontinuity of genetic types

due? Assuming that the modes of recombinationdescribed above operate in natural bacterialpopulations, it becomes obvious that the ac-quisition of isolating mechanisms would serve tolimit the amount of genetic divergence obtainablefrom recombination. The existence of a mechla-nism preventing "gene flow" between twobacterial populations could then serve as thecriterion for calling them distinct species. Yetwe have already noted that the taxonomist'sspecies do not always correspond to thosepopulations which are completely incapable ofexchanging genetic information, as evidenced byan experimental test of recombination. Thereason could easily stem from the very natureof evolution itself. Evolution is a dvnamicprocess, and speciation is sometimes unwittinglyobserved in transition. Two populations that arediverging may not yet have reached a stage ofcomplete isolation, and it is still largely aniarbitrary matter deciding whether the differencesbetween the two populations are sufficient towarrant their designation as separate species, atleast from a taxonomic point of view.

Furthermore, it needs to be understood thatisolating mechanisms differ and may possessintermediate degrees of efficiency as well asabsolute ones. Consider the circumstances underwhich bacterial evolution may occur. WVe mayvisualize a part (A') of some bacterial population(A) exploring a new ecological niche for which itis still poorly adapted. If no competitor speciesare present in this niche, or, if present, they arenot completely exhausting the possibilities forfurther expansion in the niche, evolution of anadapted form of A', or B, will arise in timethrough the gradual action of mutation, recom-bination and selection. Two possibilities mayexist, however, so far as the spatial separation ofB and A are concerned:

Possibility I: The nature of B's ecologicalniche does not assure complete separation of Bfrom its ancestral population A, or, for thatmatter, from an evolutionarily related population(i.e., another population also derived from A).

a. If in the course of evolving the adaptive

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genotype characteristic of 1B, the change in DNAstructure is great enough, pairing incompatibilitywill alssurc genetic isolation. This may be thewa-,!iy in whliclh certain strains of streptococci andplneumococci, which inlhbl)it very similar, if not(ltjoining niiiches (in the respiratorv tract of mannd(l other animals), have be)(ICome11 iincapable of

recoml)ininog w-itil ea.tch other.1). If the change in DNA struticture is not very

(cxten'siv. so that p)airing incompatibility is slight,other isolatin-g nmechanismssa-male selected forunltil slch incompatibility (levelops. This mayl)e the wa-yv in which (lifferent strains of E. coliwitlh presumably slightly (lifferent ecologicalp)referen''' , are genetically isolated from eachother. Differences in the systenm of fertilit-factors iffecting conlliigation could account forsuch isolation.

P'ossibilitv 11: The ntature of B's ecologicalnicheaissures a v-ery Iowx or negligible probabilityof contact by- fB xx-itlh its ancestral species A. Inthis (case, the ecok)gical isolition itself serv-es tol)ie('xnt (enetic recoml)ination between the twopopulati mn1-. No mechanism preventing recon-l)ination may ever arise unless the (livergencein the respective DNA structures is great enoughto assure complete incompatibility in the event(aiccidental oI exp)erimental) that the tx-opo})lations, or subelones of them, everconfronte(l each other. Tlis may be the xxav inxxilch twx o l)arasiti( l)ol)ulations, which lie in

lifferent parts of the lost's hody may be isolatedfrom each other.

Ieforc testing these idleas, hoxx e-ver, w-e firstneed to knoxw if the modles of genetic recombinia-tion that are knoxxn to occur in vitro actuallyoccur in nature. We know that certain bacteriaare )otentially capable of recombination, but itis not yet certain whether recombination isutilized in nature at all or xhlether the extent towxhich'it in utilizedI h.as any evolutionary force.Until xe can be sure of the extensive utilizationof the modes of recombination available to theba(terii. we (lo not knoxw if the isolating mecha-nUismis that h1a-v-e been discussed above have anyev-olutionary significance. A priori, however,there seemis little reasoni to (loubt that recombi-nation1 oCccurs to a signiificant extent amongbacteria in their naturlal environments. Austrian(6) lhas demonstrated that transformations ofp)neumococci (an occur in the bodies of a numberof animial species, and that such transformations

result in recombination of genetic factors affect-ing capsule synthesis and somatic antigens.Moreover, the frequencies of recombination thatare noxx observed in vitro are appreciable. Conju-gation in E. coli is nearly 100 )er cent efficient,ain(l recombination of genes betxx een a specificHfr (lonior and ani F recipient cani he as high as20 per (cnt. Transformation frequencies aicsometimes as hiiglh as 10 to 23 p)er cent.4

If, on the other hancd, genetic reeombinationdoes inot occur to any siginificaant extent in iiature,an(l rep)resents only an experiimental trick ofrecombining bacterial genes ini the bacteriologist'stest tube, xxve are then forcedl to consider themseaniing of the (apacity for recombination thatbacteria possess. It is conceivable that recombi-nation is an inherent property of the geneticapparatus of bacteria, by virtue of the chemicalnature and organizatioin of bacterial genes, andthat this property is expresse(l only amoing themore a(lvance(l forms of life v-hich utilized it inItlheir elacboration of meclhanisimis of sexual repro-(luction. In this viexvA, recoimbinlatioIi is a lateint,property of the genetic material of the im-oreprimitive forms of life, a pre-adal)tation, so tospeak, for the evolutionarilv more a(lvancedforms that folloxwed. Anotlher possibility,suggested by R. C. Lexvontin (personal coimmu-nidation) is that the capacity for recombination inbacteria is an evolutionary relic. In tlis viex-, thebacteria (descended(l( fIrOIm1 primitive formiiis of lifethat inclu(le(d geneti( recombination in theirprocess of reproduction, as the ''higher' p)lantsan(l animals (lo today in sexual reproduction, andsex xxas discar(lded in certain lines of evolution,including the ones leading to the bacteria.

Slhould furtlher study prove that geneticrecombination has little or Io ('volutioIlarvsignificance in natural bacterial populations, itxvill mean that mutation andl selection are the

The frequency of conjugation or transforma-tion (loes (lepend on environmental coinditionis. Inthis regard Lexwontin (53) has pointed out that inanimals wN-hose life cveles consist of alternatingasexu.al and sexual phases, the sexual phase isoften correlated wvith the appearailce of unfavor-able physiological conditions in the environment.The significance of saving recombination for eni-vironmental exigencies is obvious. It would beinteresting to see if recombination among bacteriain nature is also indtuced bv unfavorable environ-mental conditionis.

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primary forces permitting bacterial evolution.In order to account for the discontinuity ofgenetic types that we recognize among thebacteria, we would have to suppose that periodsof greatly relaxed selection occur from time totime in the bacterial world. Only under suchcircumstances could mutations accumulate tobring about new types diverging from the originaltypes in a sufficiently complex and multiple way.Then, when changing environmental conditionsmake selection more rigorous, bacteria withnonadaptive genotypes will be weeded out. Ifthe results of these evolutionary forces aredivergences in DNA structure, the capacity forgenetic recombination can still be used experi-mentally as a means of testing the degree ofdivergence. The extent to which recombinationis experimentally possible between two DNAstructures would be an index of the evolutionaryrelationship of the bacteria containing them.While perhaps not themselves forces directingevolution, mechanisms barring recombinationin vitro could nevertheless provide a criterion forspecies distinctions.Though the problems posed here are certainly

large and difficult, the means for studying themare not altogether out of reach. There are manyquestions that can be posed for experimentalattack. For example, it would be valuable toisolate cultures of the two related "species"from the same or adjoining ecological niches innature (say, a pneumococcal and a streptococcalstrain from the throat swab of some animal).Such freshly isolated strains can be multiplymarked genetically, and one can determine ifrecombinations occur in vitro. A positive outcomewould at least imply that two groups likely toconfront each other in nature are capable ofrecombining genetic information. To determineif this were indeed occurring, the two properlymarked strains could be re-introduced into thekind of ecological niche from which they weredrawn, and the production of recombinants couldbe checked. The same kind of experiment couldbe performed with strains obtained from differentbut similar ecological niches (say, the intestinaltracts of the dog and the horse).

Other questions need answering. Why is itthat of all the Salmonella serotypic combinationsthat are possible, only a fraction have beenreported (although new ones are reported fromtime to time)? Are only certain antigenic combi-

nations possible because they are the con-sequences of the only adaptive genotypes thatexist? Are the bearers of certain antigenic combi-nations strongly selected against, or is the pro-duction of such antigenic combinations unlikelybecause the only parental populations that couldrecombine to give rise to these combinationsare isolated in some way in nature?Why is it that Shigella Lac+ strains have not

been observed in nature although they arereadily produced in vitro by conjugation withE. coli, which is an enteric species that shigellaeare likely to confront in nature? Does selectionact strongly against such hybrids? Or are theremechanisms which genetically isolate thesespecies of which we are still ignorant?

It is clear that more extensive genetic studiesof an ecological nature must be carried out. Veryfew genetic experiments have been conductedutilizing the natural ecological niches inhabitedby the bacteria being investigated. With aresurgence of interest in bacterial ecology,reinforced by our new genetic knowledge andbiochemical techniques, a unification of ourknowledge of the bacteria is likely to be forth-coming.

VI. REFERENCES1. ADAMS, J. N. AND LURIA, S. E. 1958 Trans-

duction by bacteriophage P1: abnormalphage function of the transducing particles.Proc. Natl. Acad. Sci. U. S., 44, 590-594.

2. ALEXANDER, H. E. AND LEIDY, G. 1951 De-termination of inherited traits of H. influ-enzae by desoxyribonucleic acid fractionsfrom type-specific cells. J. Exptl. Med., 93,345-359.

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4. ANDERSON, T. F. AND MAZE', R. 1957 Ana-lyse de la descendance de zygotes formes parconjugaison chez Escherichia coli K12.Ann. inst. Pasteur, 93, 194-198.

5. ANDERSON, T. F., WOLLMAN, E. L., AND JACOB,F. 1957 Sur les processes de conjugaisonet de recombinaison chez Escherichia coli.III. Aspects morphologiques en microscopieelectronique. Ann. inst. Pasteur, 93, 450-455.

6. AUSTRIAN, R. 1952 Observations on the

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tI-ansformatioIn of pneumococcis in vivO.BUll. Johns Hopkins Hosp., 91, 189-196.

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25. (G'REEN, D. Al. 1959 A host-lsecific varia-tion afTecting frequency of transformationin pneimoeoccus. Exptl. Cell Research,18, 466-480.

26. (GIRIFFITI!, F. 1928 The significance of pneu-m11ococcal typIes. J. Hyg., 27, 113-159.

27. HARTAIAN, 1P. E. 1957 Transduction: a coim-parative evieiw. In The cheii/ical basis ofheredity, pp. 408-462. Johls Hopkins Press,Baltimore.

28. HA.YES, W. 195.3 The mechanisnm of geIneticrecoml)ination iII Escherich ia col i. Col(ISprinig Harb)or Symposia Qi/ant. Biol., 18,75--93.

29. HERSHEY, A. 1). AND CHASE, Ml. 1952 Inde-pen/lent fuinctions of viral proteini and nit-cleic acidl in groN-th of bacteIriophage. J.(Ten. Physiol., 36, 39-56.

30. HINSHELWOOD, C. N. 1946 ThI (hem/iical ki-netics of the bacterial cell. The Claren(loPress, Oxfordl.

31. HOLLOWAY, B. W. 1955 Genetic recomlina-t1iOI in P'se udomionas aer/ginosa. J. (Gteni.Microbiol., 13, 572-581.

32. HoTCHKIss, R. D. 1955 The biological iroleof the deoxypenitose nucleic aci(/s. In A-/iclei(acids, Vol. 2, p)p. 435-473. Academic Press,New York.

33. HOTCHKISS, 1t. 1). 1957 Criteria for ep/aniti-tative genetic transformation of b)aeteria.In Chemical basis of heredity, pp. :321-335.,Johns Hopkins Press, Baltimore.

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35. HOTCHKISS, It. I). AND EVANs, A. H. 1957(enetic an/I met al/lic mechanisms tinder-lying multiple levels of sulphonamide resist-anI1ce in plieuimoco/ci. In 1)rug resistan2ce iltii/icroorganisit/s, pp. 183-196. C(iba Fotinda-tioni Sympositim, London.

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36. JACOB, F. AND WOLLMAN, E.-L. 1956 Ite-combinasion genetique et mutants de fer-tilit6. Compt. rend., 242, 303-306.

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50. LEIDY, (., HAHN, E., AND ALEXANDER, H. E.1956 On the specificity of the desoxyribo-nucleic acid which induces streptomycinresistance in Hemophilus. J. Exptl. Med.,104, 305-320.

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58. MORSE, M. L., LEDERBERG, E. M., AND LEDER-BERG, J. 1956 Transduction in Escherichiacoli K12. Genetics, 41, 142-156.

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60. NEWCOMBE, H. B. 1949 Origin of bacterialvarianits. NatUre, 164, 150.

61. NEWCOMBE, H. B. AND HAWIRKO, R. 1949Spontaneous mutation to streptomyciii re-sistance and dependence in E. coli. J.Bacteriol., 57, 565-571.

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tose in Escherichia coli. J. Gen. Microbiol.,7, 69.

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Documents

THE ORIGIN OF BACTERIAL SPECIES · Origin of Species has drawn to a close, and the bacteriologist realizes thathehasbeensomewhat overlooked in the celebration of Darwin's im-portant