BACTERIOLOGICAL REVIEWS, June, 1965Copyright ( 1965 American Society for Microbiology

Vol. 29, No. 2Printed in U.S.A.

Chromosome Transfer in Bacterial ConjugationEDWARD A. ADELBERG AND JAMES PITTARD'

Department of Microbiology, Yale University, New Haven, Connecticut

INTRODUCTION 161

MATING TYPES IN E. COLI 161

NATURE AND REPLICATION OF THE BACTERIAL CHROMOSOME.................... 162

NATURE AND REPLICATION OF F............................................... 162

INTEGRATION OF SEX FACTOR AND CHROMOSOME................................ 163

CHROMOSOME TRANSFER BY HFR STRAINS 165

FORMATION OF F-GENOTES..................................................... 166

TRANSFER OF F-GENOTES BY F' STRAINS....................................... 167

TRANSFER OF CHROMOSOME BY F' STRAINS 167

GENERAL MODEL FOR CHROMOSOME TRANSFER 168

DISCUSSION.................................................................... 169

SUMMARY...................................................................... 170

LITERATURE CITED............................................... 171

INTRODUCTIONThe transfer of chromosomal material from one

bacterial cell to another by direct cell-to-cell con-tact was discovered in 1946 by Lederberg andTatum (35). This process, called conjugation, hassince been shown to occur in many gram-negativebacteria. Most of our knowledge of the mecha-nisms of conjugation, however, comes from thework of numerous investigators on the organismfirst used by Lederberg and Tatum, Escherichiacoli strain K-12. Unless otherwise stated, the datadiscussed below have all been obtained with thisstrain and its derivatives.The first real clue to the mechanism of conju-

gation was provided by the discovery thatchromosome transfer requires the presence in thedonor cell of an autonomous, transmissible geneticelement (19, 34). This element was called F, forfertility. Since then, a number of other geneticelements have been discovered in bacteria which,like F, promote chromosome transfer. These ele-ments include the several different resistancetransfer factors (RTF) which carry a number ofgenes controlling resistance to antibacterialagents (2, 53), and certain colicinogeny factors,such as Col E and Col I (50), and Col V (31).The latter factors confer on the host cell theproperty of synthesizing colicines (toxins whichare highly specific for strains of E. coli other thanthe colicinogenic host).

Following the terminology proposed by Clarkand Adelberg (11), we will refer to all geneticelements which promote chromosome transfer assex factors, regardless of whether or not they

1 Present address: Department of Bacteriology,University of Melbourne, Victoria, Australia.

determine other functions, such as resistance todrugs or the formation of colicines. The presentpaper is an attempt to bring together the infor-mation now available concerning the replicationand transfer of sex factors and of the chromo-some; a general model relating all of the data willbe presented. For detailed discussions of many ofthese observations, the reader is referred to anumber of books and reviews (11, 17, 21, 30).

MATING TYPES IN E. C OLI

The discovery of F made possible the recogni-tion of two mating types in E. coli: F+, harboringF and behaving as genetic donors, or males; andF-, lacking F and behaving as genetic recipients,or females. When a population of F+ cells ismixed with an excess of F- cells, only about 1 in104 donor cells transfers chromosomal deoxyribo-nucleic acid (DNA) to recipients. This transferis due to the presence in F+ populations of raremutant types called Hfr, for high frequency ofrecombination. Upon isolation, such mutantsproduce populations in which 1 % or more of thecells are active donors (10, 18). After this dis-covery, Jacob and Wollman (27) described atechnique for isolating Hfr mutants from F-populations at will. Studies on Hfr X F- crossesled, by 1958, to the following picture of chromo-some transfer (29):

(i) The F+ chromosome is a closed (circular)structure; the F+ cell contains several identicalchromosomes, plus an undetermined number ofF particles. (Although the E. coli cell typicallycontains four chromosomes, these segregate atcell division in such a manner that all of thechromosomes in any given cell result from two

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successive replications of a single chromosome.E. coli thus behaves as a haploid organism.)

(ii) The conjugation of an FP cell with an F-cell leads to the transfer of one or more F parti-cles with an efficiency approaching 100% butonly rarely to the transfer of chromosomal DNA.

(iii) As a rare event, an F particle may becomeattached to the chromosome at one of manypossible sites; the cell in which this occurs iscalled an Hfr mutant. Such attachment has twoapparent consequences: F ceases to replicateautonomously; in the clone arising from the Hfrmutant, free F can no longer be detected; andthe chromosome breaks at the site of F attach-ment and is transferred to the recipient in anoriented manner characteristic of the particularHfr strain. The leading end of the broken chromo-some is called Origin; F, the genetic determinantof maleness, is transferred as the last chromo-somal marker. The entire process requires ap-proximately 100 min at 37 C.

(iv) In addition to attaching to the chromo-some and thus mobilizing it for transfer, F deter-mines at least two other properties required formaleness: the formation of receptor sites on thecell surface to which F- cells can attach, and theability to form the type of chemical union neces-sary for DNA transfer to take place.To explore the possible mechanisms by which

the attachment of F leads to chromosome trans-fer, it will first be necessary to consider what isknown about the nature and replication of eachof these elements.

NATURE AND REPLICATION OF THE BACTERIALCHROMOSOME

It is not intended to review here all of the in-formation available on the bacterial chromosome,but only that part which is relevant to the ques-tion of the transfer mechanism. For a broadercoverage, the reader is directed to a number ofrecent reviews and articles (6, 7, 20, 32).The first relevant fact is the circularity of the

chromosome. In 1956, Jacob and Wollman (28)discovered the closed nature (circularity) of thegenetic map of F+ E. coli; Hfr cells, however,were believed to have an open (linear) map.Furthermore, it was often pointed out that acircular map does not necessarily imply a circularphysical structure. In 1960, however, it wasshown b)y Taylor and Adelberg that Hfr cellswhich are not acting as donors also possess acircular linkage group (52), and in 1963 Cairnssettled the question once and for all by demon-strating that the DNA of E. coli can be extractedin the form of a circular molecule approximatelyone millimeter in length (6, 7). This structurecorresponds to the hypothetical chromosome

which had been reconstructed from recombina-tion data and from studies of the genetic effectsof P32 incorporation and decay (29).By the use of radioautographic techniques,

Cairns (6) also showed that the chromosomereplicates in the manner illustrated in Fig. 1. Theessential features of this process are the following.(i) Replication seems to proceed from only onepoint on the chromosome at any given moment.If there is more than one potential starting place,the activation of one apparently causes the sup-pression of the others. A single starting-pointfor replication has been established independ-ently by Nagata (36) and by Bonhoeffer andGierer (4). (ii) Although the two strands of DNAhave opposite polarity, replication in vivo ap-pears to proceed along both of them in the samedirection. (iii) The point at which replicationbegins [called the "replicator" by Jacob andBrenner (25)] can be inferred to provide at leasttwo functions essential to the replication process.It could allow the double helix to open, providingsingle strands to act as templates for the DNAreplicating enzyme, and it could also function asa swivel. The need for a swivel is a consequenceof the circularity of the chromosome; the un-duplicated region must rotate in order to un-wind, in a sense opposite to the rotation of oneof the two arms.The mechanism by which the replicator acts

as a swivel is not known, but it has been sug-gested by Cairns that the swivel might consist ofa single-strand break (6). In that case, the repli-cation process would create a new double helix inwhich both strands have free ends, since the endsof the newly forming single strands are necessarilyfree until completion of the replication processand ring-closure. The importance of this con-sideration for any model of chromosome transferwill become apparent in the following sections.

NATURE AND REPLICATION OF FThe sex factor (Fl) found in wild-type E. coli

strain K-12 has been shown to be a DNA elementapproximately 2%/o of the chromosome in length(15, 48). This corresponds to approximately 105nucleotide pairs. Recently, Rownd (personal com-munication) compared the profiles of the DNAfrom F- and F- cells by equilibrium density-gradient centrifugation, and found a shoulder inthe F+ profile corresponding to a DNA contain-ing 44%NO guanine-cytosine (GC). The chromo-somal DNA, on the other hand, contains 50%OGC. Of particular interest is Rownd's estimatethat the 44%7 GC material is equivalent to onlyabout 0.1 %7 of the chromosomal DNA, whereasthe measurements of the total sex factor sizeindicate it to be equivalent to 2%7 of the chromo-

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somal DNA. These differences can be reconciled,however, by assuming that the sex factor consistsof a small piece of 44%o GC DNA attached to amuch larger piece of 50%-- GC DNA. As will beshown later, such an assumption would accountfor many of the observations concerning F inte-gration and chromosome transfer by Hfr cells.Rownd's observations have been confirmed

and extended by Falkow and Citarella (16). Theseauthors measured the homology between FDNA and chromosomal DNA by isolating theDNA of F-genotes after their transfer to bacteriawith a widely different GC content, and measur-ing its adsorption to various types of bacterialDNA suspended in agar-gel columns. Theirresults show clearly that wild-type F is 1.9% of

FIG. 1. Replication of the bacterial chromosome,according to Cairns (1963). The squares representhypothetical replicators. Newly synthesized DNAstrands are shown as broken lines, and replication isshown as proceeding counter-clockwise from thereplicator at the top of the figure, which also servesas a swivel for the unwinding of the two old strands.

the bacterial chromosome in size, and consists oftwo regions: one region, making up 10% of F,contains DNA of 44%7c GC content; the otherregion, making up the bulk of F, contains DNAof 50% GC content. Half of the latter region ishybridizable with chromosomal DNA.Although the F genome has been neithe ve-

netically mapped nor physically isolated, thereare reasons to believe that it will ultimatelyprove to be circular. The reasons for so believingare as follows. (i) F shares with the chromosomeand with bacteriophage genetic material theability to replicate as an independent unit. It isthus, by definition, a "replicon" (25). Repliconswhich are now known to be circular structuresinclude the bacterial chromosome (6), as well asbacteriophage X (45). F further shares with X the

ability to integrate with the chromosome and toincorporate segments of chromosome upon re-verting to the autonomous state. All of thesesimilarities suggest a similar physical structure.(ii) A sex factor which has incorporated a chromo-somal segment (an F-genote) is subject to geneticmapping. (The nature and formation of F-genoteswill be discussed in Formation of F-Genotes.)Preliminary experiments using phage transduc-tion have given results which are compatiblewith a circular structure for the F-genote F14(Pittard, unpublished data). (iii) Finally, ananalysis of crossing-over between F14 and thechromosome has also provided suggestive evi-dence for the circularity of F14 (39).

Little is known about the replication of F, butsome experiments of Jacob, Brenner, and Cuzin(26) suggest that it is essentially similar to thereplication of the chromosome. Mutations werefound to occur on the sex factor which permit itto replicate at low temperature but not at hightemperature. Such experiments, together withobservations on the rate of F multiplication innewly infected cells (14), suggest that the repli-cation of F is independently controlled, pre-sumably by the action of a gene product on an Freplicator. In the absence of evidence to the con-trary, then, we shall assume that replication ofF, like that of the chromosome, proceeds in themanner shown in Fig. 1.The number of F replicons in an F+ cell has

never been directly determined. However, esti-mates have been made in the case of certainF-genotes, such as F-lac and F14, by measuringthe levels of enzymes controlled by genes whichhave been incorporated into the F-genote (26,41), or by measuring the relative probabilities ofa mutation occurring on the F-genote or on thechromosome (43). These estimates have variedfrom one F-genote per chromosome (26, 41) tothree per chromosome (43). To account for theregular distribution of F and chromosomal repli-cons at cell division, it has been postulated thatthese elements attach to the newly formingcross-wall at the time of their replication, insuch a manner that the replication products aresegregated (26, 46).

INTEGRATION OF SEX FACTOR AND CHROMOSOME

The autonomous replicons which are known tobe capable of attachment to the chromosome in-clude F and X. Campbell (9) presented a modelfor the attachment of X to the chromosome basedon pairing of the two circular structures followedby breakage and reunion (crossing-over). Al-though direct proof of the model is still lacking,it does account for the different marker order

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found when X is mapped by vegetative recombi-nation and by prophage recombination (8).

Campbell's model stems primarily from obser-vations on the structure of Xdg, the defectiveprophage in which the gal loci of the chromosomehave replaced a segment of the phage genome.Although less is known about the structure ofF-genotes, the similarities between them andsuch transducing phages as Xdg suggest a com-mon mechanism for their origin. We will assumethis to be so, and will further assume thatCampbell's model for Xdg formation is the cor-rect one. These assumptions constitute two ofthe postulates on which our general model forchromosome transfer will be based.

In Campbell's model, the crossing-over eventresponsible for X integration requires the pres-ence in the chromosome of a region of homology

0J,

FIG. 2. Integration of sex factor (heavy lines)and chromosome (light lines) by breakage and re-union.

with a site in the phage genome, i.e., a region ofidentical base-pair sequence. E. coli K-12 has oneattachment site for X, close to the gal locus. Inthe case of the sex factor, F1, there are 10 to 12known chromosomal sites at which integrationcan take place; these will be considered to beregions of homology with one or several regions ofthe F genome. According to our model, which isessentially the model proposed by Stern (51),pairing of F1 and chromosome occurs as a rareevent at one or another of these regions; oncepairing has occurred, a crossover within theregion integrates the two replicons. For example,one such pairing region is at the histidine operon.If we designate certain his mutational siteswithin the operon as A and B, integration of Fcan occur by a crossover between markers Aand B or to the left of both (Matney, personalcommunication). Similarly, a pairing region exists

at the lac operon, but the crossover which inte-grates the sex factor can take place either at oneend of the operon or between mutational sites inthe Z cistron (12).

Figure 2 illustrates the integration of F andchromosome according to these assumptions.The chromosome of the F+ cells is pictured ashaving several replicators; the chromosome of theHfr cell is shown with one additional replicator,that of the integrated sex factor. The existence ofseveral chromosomal replicators is inferred fromthe experiments of Nagata (36), in which syn-chrony of marker replication could be achieved

FIG. 3. Jacob-Brenner model of chromosometransfer. Replication has begun at the F replicatorat the top of the figure, and has proceeded in theclockwise direction. One of the daughter doublehelices is transferred in the direction shown by thearrow. The point at which DNA synthesis is takingplace would be the location of the DNA polymerase(P), which is pictured as being fixed to a point onthe bacterial membrane. The entire chromosomethen moves past that point, in the counter-clockwisedirection.

for a population of Hfr cells but not for a popula-tion of F- cells. This was interpreted to meanthat chromosomal replication can start at onlyone site in Hfr cells (at the F replicator) but atany one of several alternative sites in F- or F+cells. Positive evidence is lacking, however, andit is possible that only one chromosomal replicatorexists.

Regardless of whether the F+ chromosome hasone or several replicators, Nagata's experimentssuggest that chromosomal replicators cease tofunction in Hfr bacteria, replication beginningonly at the replicator of the integrated sex factor.Once replication has begun there, it proceedsaround the entire F-chromosome structure; in

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other words, F and chromosome now behave as asingle replicon. [It should be noted that in themodel of Jacob and Brenner (25) discussed below,F and chromosome are also proposed to consti-tute a single replicon in the Hfr cell. In theirmodel, however, replication is postulated to startat a chromosomal replicator rather than at theF replicator. This postulate is based on the ob-servation that acridine orange inhibits the repli-cation of autonomous F at a concentration towhich replication of the integrated replicon isresistant.]The chromosomal site at which F integration

takes place is known from studies of the transferof chromosome by Hfr strains during conjuga-tion. This process will now be examined in moredetail.

CHROMOSOME TRANSFER BY HFR STRAINSTwo questions can now be asked about the

transfer process: (i) what causes the circularchromosome of the Hfr cell to break at the timeof conjugation, and (ii) what causes the chromo-some to move from the male to the female cell?The first question can be answered by defining

chromosome "breakage" as the creation of a freeend. As discussed earlier, this is just what happenswhen the circular chromosome replicates. To ex-plain the fact that during conjugation the Hfrchromosome breaks at the inserted sex factor,Jacob and Brenner (25) proposed the followingmodel: chromosome replication in the nonconju-gating Hfr cell begins at a chromosomal repli-cator; the event of conjugation triggers the startof a new round or replication beginning at the Freplicator, such that the Origin (the leadingpoint in transfer) is duplicated first. One daugh-ter replica penetrates the female cell, moving atthe expense of the free energy liberated by thepolymerization of nucleoside triphosphates.Transfer is thus pictured as being directly gearedto replication (Fig. 3).The Jacob-Brenner model, however, does not

take into account Nagata's evidence that replica-tion of the Hfr chromosome begins at the Freplicator even in nonconjugating cells, and thatproceeds in the opposite direction; i.e., the Originis the last point to be duplicated, rather than thefirst. With this in mind, Bouck and Adelberg (5)proposed a somewhat different model. Accordingto their model, replication begins at F and pro-ceeds in the sense shown by Nagata. At themoment that a population of Hfr cells is mated,every cell is in a different stage of the replicationcycle. As each conjugating cell completes its repli-cation, one of the replicas fails to undergo ring-closure. Instead, the free end created becomes theOrigin, and transfer commences (Fig. 4). This

model says that DNA synthesis in a culture ofHfr cells is necessary for the initiation of chromo-some transfer, but may not be necessary for thetransfer process itself.The two models lead to a number of different

predictions. The Jacob-Brenner model, for exam-ple, says that if DNA synthesis is halted aftertransfer has been initiated, transfer should stop.The Bouck-Adelberg model, on the other hand,leaves open the possibility that transfer may con-tinue. To test these predictions, Bouck andAdelberg allowed Hfr cells to initiate transfer fordifferent lengths of time and then treated themwith phenethyl alcohol (PEA), which specificallyinhibits DNA synthesis (3). After allowing anadditional period of time for transfer to takeplace, the mating couples were sheared apart and

FIG. 4. Bouck-Adelberg model of chromosometransfer. Left: replication has begun at the F repli-cator at the top of the figure, and is proceeding in thecounter-clockwise direction. Right: replication hasbeen completed, but ring-closure of one daughterchromosome has been prevented by the initiation ofits transfer to an F- recipient.

plated to assay for recombinants. Their resultsclearly showed that some transfer does take placein the absence of net DNA synthesis; Jacob,Brenner, and Cuzin (26) demonstrated, however,that some DNA turnover takes place after PEAaddition, and applied their model to Bouck andAdelberg's results by attributing the observedtransfer to residual DNA synthesis. It is not yetclear which interpretation is correct.

Another prediction in which the two modelsdiffer has to do with the speed of chromosometransfer. The Jacob-Brenner model says that thespeed of transfer is directly related to the rate ofDNA synthesis, whereas the alternate hypothesismakes no such prediction. It has now been shownthat the time of entry of markers, which measuresthe speed of transfer, does not change when therate of DNA synthesis in the cell population is

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drastically lowered by thymine starvation (42) orby PEA treatment (Bouck and Adelberg, unpub-lished data). It is possible, however, that, for anygiven cell, DNA synthesis continues at its maxi-mal rate until it is stopped by the treatment inquestion. The decline in rate measured for thewhole population would then reflect the ac-cumulation of cells in which DNA synthesis hadstopped, and the observed residual transfer couldhave been effected by cells in which the rate ofDNA synthesis was maximal.

There are other differences in the predictionsmade by the two models, and these are currentlybeing tested in several laboratories. The twomodels agree, however, in one important predic-tion: they both say that, of the DNA which istransferred early, one strand is made duringconjugation. Thus, if the males are grown inheavy medium (C13N'5) and mated in lightmedium (C'2N'4), the early-transferred DNAshould contain one light and one heavy strand.The Jacob-Brenner model says that all of thetransferred DNA should be hybrid; the Bouck-Adelberg model says that the material trans-ferred earliest will be hybrid, whereas the materialtransferred later will be all heavy. The late ma-terial may not be found in detectable amounts,however, since chromosome breakage occursduring transfer to such an extent that the averagecell transfers only 16% of its DNA during anuninterrupted mating (11, 48).

Jacob, Brenner, and Cuzin (26) verified thegeneral prediction by carrying out the experi-ment described above, isolating the DNA fromthe zygotes after destruction of the male cells.The amount of heavy-heavy DNA which theyfound, in addition to the predicted hybrid DNA,could have come either from contaminating HfrDNA or from DNA transferred according to theBouck-Adelberg model. Very little of the latterwould be expected, since conjugation was inter-rupted at 25 min when transfer is only 30%complete.At this moment, then, it can be said with some

confidence that transfer of the Hfr chromosomeis initiated as a result of replication of the Origin,either as the end of a cycle of replication or asthe start of a new one. Whether the transfer itselfrequires DNA synthesis as a source of energy isstill open to question. A strong possibility existsthat both models are correct: conjugation maytrigger F-replication with consequent transfer,but this process may have to wait until the cur-rent reverse cycle of chromosomal replication hasbeen completed.A recent report by Roeser and Konetzka (45a)

is not incompatible with this view. Their datashow that PEA permits cells to complete their

current cycle of replication but prevents initia-tion of the next cycle. If the PEA is removedafter completion of the cycle, DNA replicationimmediately recommences; the addition of PEA20 min later permits completion of the secondcycle over a further 100-min period. This systemhas permitted a more refined test of the correla-tion between DNA synthesis and transfer. Donorcells which had begun a cycle of replication andhad then been treated with PEA were matedwith F- cells. No transfer was observed, eventhough most of the cells went on to complete thecurrent cycle of replication. Along with appropri-ate controls, this observation indicates thattransfer requires the initiation of a cycle of DNAsynthesis during mating, not just the completionof a cycle.

zY

z xY

B ~~~~xC AA

BBC C

FIG. 5. Formation of an F-genote by imperfectrelooping of the F-chromosome integrated replicon.F DNA is indicated by the heavy lines; chromosomalDNA by the light lines. A segment of F DNA re-mains in the shortened chromosome.

FORMATION OF F-GENOTESIn Integration of Sex Factor and Chromo-

some, Campbell's model was presented for theintegration of F and chromosome. The reversalof the process pictured in Fig. 2 would lead to achange from the Hfr state back to the F+ state.On rare occasions, however, the Hfr chromosomemight "reloop" imperfectly; breakage and re-union in such a structure would produce a sexfactor carrying a piece of chromosomal DNA, aswell as a chromosome in which a number of locihave been deleted and replaced by F DNA(Fig. 5).

This model, first proposed by Campbell to ex-plain the formation of Xdg, is compatible with allof the known facts about F-genotes. Strainscarrying F-genotes (F' strains) have been selectedin the following manner (24). A large populationof Hfr cells is mated for a limited period of time

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(e.g., 30 min) with a suitable F-, and is then de-stroyed by phage. If we consider the Hfr to carrymarkers A+ through Z+ in which A+ is trans-ferred first and Z+ last, no Hfr cell will transfermarker Z+ in the 30 min of mating. If, however,there are any "F' mutants" in the population inwhich marker Z+ has become integrated into thesex factor, the element F-Z+ will be transferredwithin the first few minutes of mating. If thefemale strain used is Z-, selection for Z+ re-combinants at 30 min will permit the isolation ofF-Z+/Z- heterozygotes. This is illustrated inFig. 6, in which markers X, Y, and Z are shownto have been transferred as part of an F-genote.

Campbell's model makes two other interestingpredictions. One is that the cell in which theF-genote is formed should now contain a chromo-some bearing a piece of sex factor DNA. Al-

FIG. 6. F-genote shown forming in Fig. 5 is nowshown in a recipient cell to which it has been trans-ferred. Pairing has occurred between homologousregions on the F-genote and on the chromosome. TheF-genote is shown attached to the bacterial membraneby its replicator.

though there is no way of selecting for the cell inwhich the original event took place, a clonearising from such a cell was accidentally dis-covered (1). When cells of this clone were treatedwith acridine orange to remove the F-genote (22),an F- strain was obtained which exhibits a re-markable property. When this strain is infectedwith wild-type F, it becomes a high-frequencydonor of chromosomal markers. The order ofmarker transfer shows that breakage alwaysoccurs at the site of original F attachment. Thisis just what is predicted if Campbell's model iscorrect and the chromosome contains a piece ofsex factor DNXA at the former attachment site.The new F put into the cell should pair with thehomologous F DNA in the chromosome, bringingabout breakage and transfer of the chromosomein the manner described in the following section.The incorporated fragment of F DNA has been

termed an sfa locus (for sex factor affinity). Twosuch sfa loci have been described (1, 44).The other prediction made by Campbell's

model is that early markers (e.g., A+) should beincorporated into F as often as late markers.Recently, a method for detecting such events wasdevised by Curtiss (12). He selected for the earlytransfer of both A+ and Z+ to a recipient, whichwas A-Z-; an F-genote was thus selected carry-ing both "early" and "late" Hfr markers, andthe diploid strain formed had the genotypeF-A+Z+/A-Z-. In this case the markers "A" and"Z" were two different loci concerned withproline biosynthesis.

TRANSFER OF F-GENOTES BY F' STRAINSWhen an F' strain such as that pictured in

Fig. 6 is mating with a suitably marked F- strain,the F-genote is transferred at a frequency ap-proaching 100%. Breakage of the F-genote occursat a specific site (inferred to be the F-replicator)and the F-genote markers are sequentially trans-ferred in the order X, Y, Z, F. This was firstshown by Hirota and Sneath (23) for a sex factorcarrying the markers ade, T6-r, and lac; more re-cently, Pittard and Adelberg (39) carried out akinetic analysis of the transfer of an exceptionallylong F-genote, F14. The DNA of F14 representsapproximately 10%c of the chromosome, and re-quires 9 min to be transferred. The earliestmarker to be transferred by F14 is met-i, and thelast marker is ile-1; the time required to transferthe met-ile segment is the same, whether it is partof the sex factor replicon or part of the chromo-some. The fact that both F-genote and Hfrchromosome are transferred at the same speedspeaks strongly for a common mechanism.

TRANSFER OF CHROMOSOME BY F' STRAINSWith one important exception, to be discussed

later, all F' strains transfer chromosome atmoderate frequencies. In each case, the chromo-some appears to break at the site characteristicof the Hfr in which the F-genote arose. For exam-ple, Hfr strain AB313 transfers xyl as an earlymarker with a frequency of about 40%. WhenF14, which arose in an AB313 cell, is put into anF- to make an F' strain, F14 itself is transferredat a frequency of 50%, whereas xyl is still trans-ferred early but at a frequency of only 5%0.

Pittard and Adelberg (38) have studied thekinetics of transfer of F14 markers and chromo-somal markers by F' cells. Zygotes were selectedwhich had received the chromosomal markerxyl+, and these were analyzed for the presence ofF14 markers met+, arg+, and ilv+. Large excessesof F- cells were used in these crosses, to ensure

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that each zygote arose from a mating involvinga single F' cell. The percentage of xyt+ recombin-ants containing F14 markers did not vary withthe time at which the matings were interrupted,indicating that the F14 markers preceded thechromosomal marker in transfer. However, thepercentage did vary with the F14 marker: met+,the earliest F14 marker to be transferred, was re-covered at 70%; arg+, the next marker to betransferred, was recovered at 30%; and ilv+, thelast marker to be transferred, was recovered at20%. These findings were interpreted to meanthat chromosome transfer causes the breakage ofF14 in transit.The true nature of this breakage was revealed

by the work of Scaife and Gross (47). Theirstudies on chromosome transfer by F' strainscarrying F-lac showed that the breakage is, inreality, a crossover between F-genote and

x

FIG. 7. F' cell is shown transferring its F-genoteto a conjugated F- cell. A crossover has taken placebetween the F-genote and the chromosome.

chromosome. Guided by this interpretation,Pittard and Adelberg (39) carried out exhaustiveanalyses of crossing-over events in both the F'donor and in the zygote. Their conclusions can besummarized as follows: F' strains transfer theirF-genotes with close to 100% efficiency. Chromo-some transfer occurs in such strains solely as aconsequence of crossing over with the F-genote.In the donor cell, the probability of a crossoveris constant per unit length of F-genote; in thezygote, the probability of a crossover is alsoconstant per unit length of the transferred ele-ment, with the exception that in the very smallregion adjacent to the leading end the crossoverfrequency is elevated about 40-fold. [The elevatedcrossover frequency in the proximal region ofthe F-genote in the zygote, but not in the donorcell, is consistent with the model of a circularF-genote in the donor and a linear (broken)F-genote in the zygote.] A given F' male cell cantransfer both chromosome (integrated with the

F-genote) and a second entire copy of theF-genote to the same recipient.The transfer of chromosome by F' strains thus

depends on crossing over in the region of ho-mology between the chromosome and F-genote(Fig. 7). If no region of homology exists, nochromosome transfer occurs. For example,strain AB1206, which harbors F14, has a chromo-somal deletion corresponding to the entireF-genote (41). Chromosome transfer by AB1206is about 10- that of other F14 strains, and isbarely detectable.

Examination of Fig. 7 leads to the predictionthat, if the crossing-over model is correct, a givenchromosomal marker should be transferred laterin an F' strain than in the parental Hfr strain bythe number of minutes required to transfer theF-genote. This prediction has been verified (40):

FIG. 8. Dynamic equilibrium between the inte-grated and nonintegrated states of the sex factor andchromosome is shown schematically. K1 and K2 arerate constants for the events of integration and sepa-ration. In the F' system, K1 and K2 are approxi-mately 0.1 and 0.9 per cell per generation, respec-tively. In the F+ :± Hfr system, K1 and K2 are bothapproximately 1 X 105 per cell per generation.

the xyl and mal markers are transferred at 18and 25 min, respectively, by Hfr strain AB313,but at 28 and 35 min, respectively, by the de-rived F' strain, AB1516. The F-genote in AB1516requires approximately 9 min to be transferred.

GENERAL MODEL FOR CHROMOSOME TRANSFER

In Chromosome Transfer by Hfr Strains wesummarized the evidence for believing that, inHfr cells, chromosome breakage and transfer areconsequences of replication beginning (or ending)at the replicator of the integrated sex factor.What has been inferred to be true for the F-chromosome replicon of the Hfr cell is presumedto be true also for the F-genote of the F' cell:replication of F leads to the transfer both of Fand of the chromosomal DNA with which it hasbecome integrated. In F' cells, as we have shownabove, the F-genote is the primary transferable

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element; the chromosome itself is transferred onlyby virtue of a crossover with it.The crossing over which takes place in F' cells

appears to be a very frequent and highly re-versible process (Fig. 8). Such crossing over doesnot, as a rule, produce clones of cells in whichchromosome and F-genote are stably integrated.Instead, each cell in an F' population grows intoa clone in which, at any given moment, some ofthe cells have an autonomous F-genote and somehave an integrated F-genote. As a rare event,however, one or the other of the two states canbecome stabilized. In AB1206, for example, theautonomous state has become stabilized as theresult of a chromosomal deletion which preventspairing and crossing over (see above), whereasCuzin and Jacob (13) reported a strain in whichthe integrated state has become stabilized afteran unknown mutational event.The results of the studies on the F' strains

throw new light on the integration of F andchromosome which converts F+ cells to Hfr. Itwill- be recalled (Nature and Replication of F)that F contains two types of DNA: one with aGC content of 44%, and the other with a GCcontent similar to that of the E. coli chromosome(50%). The latter material provides homologywith certain regions of the chromosome; a cross-over within one of these homology regions wouldlead to integration of F at a characteristic site.

F1, the sex factor found in the wild-type strainK-12 of E. coli, can thus be considered to be aunique F-genote in which the chromosomal DNAcorresponds to a number of small chromosomalregions instead of to one large one. The mannerin which such an F-genote might have arisen willbe discussed in the following section. The cross-over which produces an Hfr strain from an F+strain carrying F1 and the crossover which leadsto chromosome transfer by an F' strain may beconsidered to be completely analogous, and todiffer only in their apparent reversibility: in theF+ to Hfr transition the integration appears to behighly stable, whereas in the F' cell the integra-tion appears to be highly reversible.

Actually, as anyone who has worked with Hfrstrains will appreciate, the integration is reversi-ble in both cases. The rates of the transitions inboth directions are very different, however. InFig. 8, some approximate rate constants havebeen assigned to illustrate this difference. In theF' strain, the probability of a crossover leadingto integration during a single division cycle is onthe order of 1 X 10-1, and in the other directionis on the order of 9 X 10-1, based on the propor-tions of cells which transfer integrated and non-integrated F-genotes when F' populations are

mated. The two states are thus visualized asbeing in a state of rapid equilibrium.

In the F+ -+ Hfr transition, however, the ratein both directions is on the order of 10-5 per celldivision cycle. Thus, if one isolates an FP strain,the mutation rate to Hfr is about 10-5 per genera-tion and the proportion of Hfr cells in the cultureincreases very slowly. Conversely, if one isolatesan Hfr strain, the mutation rate to F+ may alsobe about 10-5 per generation (although this variesfrom one Hfr to another), and the proportion ofF+ cells in the culture increases very slowly. Inboth cases mutational equilibrium would bereached after about 105 generations, but this isnot likely to be observed. For one thing, dif-ferences in growth rate affect the proportion ofthe two types far more than does mutation pres-sure, and, for another, the constant reisolation ofstrains from single cells that is practiced by allbacterial geneticists usually prevents equilibriumfrom being approached. Neither factor, however,can interfere with the rapid attainment of equi-librium which occurs in F' cells.The most likely explanation for the different

rates at which F+ and Hfr cells, on the one hand,and the two types of F' cells, on the other hand,approach equilibrium would seem to be the dif-ferent amounts of chromosomal DNA which theF-genote contains in the two instances. The largerthe region of homology, the greater the frequencyat which pairing and crossing over will occur;hence the rapidity with which equilibrium isattained in F' cells.

DISCUSSION

Many features of the model described abovehave been either explicit or implicit in the recentpublications of Jacob, Brenner, and Cuzin (26)and of Scaife and Gross (47), as well ts in ourown. The purpose of this article has been tobring all of the current observations and sljpcula-tions together in one review.

It is apparent that practically all of the experi-mental work on sex factor function has been donewith F1, the sex factor found in E. coli strainK-12. The other known sex factors, such as RTF[Watanabe (53)] and col factors (31, 37) may bepresumed to promote chromosome transfer bythe same general mechanism, but direct evidencefor this belief is lacking. What is known aboutthese factors is compatible, however, with theview that they are analogous to F-genotes. Anumber of cot factors have been shown by Silverand Ozeki to consist of DNA and to be of thesame order of size as F (49), and one of the RTFagents has been shown to be capable of attach-ment to the chromosome (53).

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ADELBERG AND) PITTARDI)

According to the general model, then, a sexfactor must contain a unique segment of DNAwhich includes the genetic determinants for themating reaction and for the transfer of the sexfactor itself; in addition, it must contain one ormore segments of DNA which are homologouswith regions of the host chromosome. Either inthe unique segment or in the homology segmentsthere must be a replicator, capable of respondingto an initiator produced in the cell.The unique segment (e.g., the segment of F.

which contains 44% GC) is foreign to the hostcell, and its origin is completely obscure. It couldhave been derived from a bacterial virus, butother sources of foreign DNA are easily imagined.The homology segments, however, seem likely tohave been picked up as the result of chance inte-grations of sex factor and chromosome at novelsites, followed by an imperfect reversion to theautonomous state as pictured in Formation ofF-Genotes for the origin of F-genotes. In the caseof F1, such an event would have to have occurred10 or 12 times at as many different sites duringthe evolution of the F-K-12 system, to explainthe types of Hfr strains to which this system nowgives rise. It is not known how many homologysites there are in the case of the RTF and coli-cinogeny sex factors, although the presence in theformer of several unrelated resistance loci sug-gests that chromosomal fragment "pickup" hasoccurred more than once. The presence on onesex factor of several different chromosomal frag-ments could be the result of successive pick-upevents, recombination between sex factors, orboth. The biggest question remaining to beanswered is that of the mechanism by which sexfactors bring about their own transfer. A clue tothe problem may lie in the recently discovered at-tachment of bacterial DNA to mesosomes, whichare invaginations of the cell membrane (33, 46).It has been suggested (26) that the sex factor isalso attached to the membrane, and causes theformation of a receptor site for conjugation onthe cell surface immediately opposite the attach-ment site. Conjugation then triggers, in an un-known manner, the transfer of the sex factor aswell as of any other donor DNA which is inte-grated with it.The transfer process is still a mystery. Jacob

and Brenner (25) suggested that it is driven by Freplication. It should be pointed out, however,that the transfer of F is not unlike the injectionof phage DNA into a host cell, and it is possiblethat both phage injection and F transfer takeplace by the same mechanism. If so, replicationneed not be involved as the source of energy fortransfer, although the experiments described inChromosome Transfer by Hfr Strains show that

it is indeed necessary for the initiation of transfer,presumably because it provides a means of break-ing the "circular" chromosome.The general model which has been proposed

seems to be compatible with the observationsmade by Clark (la) on a double male strain ofE. coli K-12. This strain, prepared by crossingtwo different Hfr strains, has a sex factor inte-grated at each of two different sites on the bac-terial chromosome. When the double male ismated with an F- strain, its chromosome ap-pears to break at both sites; two chromosomalsegments are thus generated, and each male cellin the population transfers one or the other seg-ment (but not both simultaneously). These ob-servations can be explained by assuming thatF-replication and transfer start at one or theother of the sex factors, but that the chromosomeis broken by the initiation of replication at thesecond sex factor. A given male cell transferswhichever segment of the chromosome happensto begin replication first, as a consequence ofconjugation.The current hypothesis of chromosomal trans-

fer, in which the leading point is a site within theDNA of the integrated sex factor, seems at firstglance to contradict the often-repeated statementthat the sex factor is linked to the last marker tobe transferred. This statement is based on theobservation that only those recombinants whichreceive the entire donor chromosome are males,an observation which is fully compatible with thehypothesis of breakage within the sex factor.Indeed, evidence for a part of the sex factor beingpresent at the origin, or leading, end of thechromosome was presented several years ago(1, 54). These earlier findings lend further sup-port to the current hypothesis of chromosometransfer.

SUMMARYBacteria are host to a number of autonomous

genetic elements, or plasmids, with the followingproperties: (i) they are composed of DNA; (ii)they determine one or more phenotypic proi)er-ties of the host; (iii) they replicate in the hostcell autonomously; (iv) they promote conjugationwith other bacteria; and (v) conjugation leads totheir own transfer.

These elements, which we will call autotrans-ferable, include the Resistance Transfer Factors(RTF), colicinogeny factors (Col), and the Ffactor of E. coli strain K-12. Some autotrans-ferable elements appear to have incorporated seg-ments of bacterial chromosome which are capableof pairing with homologous regions of the hostgenetic material. Crossing over in paired regionsbrings about the integration of the host chromo-

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some with the autotransferable element, so thatwhen conjugation occurs, the entire integratedstructure is transferred. Autotransferable ele-ments which promote chromosome transfer inthis manner are called sex factors.

Studies on F+, F', and Hfr strains of E. coliK-12 indicate that replication of F is an essentialstep in the transfer process. Two models havebeen proposed: in one, replication is essentialonly for the initiation of transfer; in the other,the replication process provides the energy for thetransfer act itself. The experimental evidence ob-tained to date does not rule out either model; itis possible that both are correct. In both modelsit is visualized that autotransferable plasmidsattach to the cell membrane and govern the for-mation of conjugation receptors on the cell sur-face directly opposite the attachment sites.Conjugation triggers the transfer of the plasmid,along with any other DNA which has becomeintegrated with it. The bacterial chromosome istransferred in conjugation as a result of itsintegration with a sex factor.

LITERATURE CITED1. ADELBERG, E. A., AND S. N. BURNS. 1960.

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3. BERRAH, G., AND W. A. KONETZKA. 1962. Selec-tive and reversible inhibition of the syn-thesis of bacterial deoxyribonucleic acid byphenethyl alcohol. J. Bacteriol. 83:738-744.

4. BONHOEFFER, F., AND A. GIERER. 1963. On thegrowth mechanism of the bacterial chromo-some. J. Mol. Biol. 7:534.

5. BOUCK, N., AND E. A. ADELBERG. 1963. Therelationship between DNA synthesis andconjugation in Escherichia coli. Biochem.Biophys. Res. Commun. 11:24.

6. CAIRNS, J. 1963. The chromosome of Escher-ichia coli. Cold Spring Harbor Symp. Quant.Biol. 28:43.

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10. CAVALLI, L. L. 1950. La sessualita nei batteri.Boll. Ist. Sieroterap. Milan. 29:281.

10a. CLARK, A. J. 1963. Genetic analysis of a"double male" strain of Escherichia coli K-12. Genetics 48:105.

11. CLARK, A. J., AND E. A. ADELBERG. 1962.Bacterial conjugation. Ann. Rev. Micro-biol. 16:289.

12. CURTISS, R. 1964. An Escherichia coli K-12Hfr strain with the fertility factor attachedto or within the structural gene for ,-galac-tosidase. Bacteriol Proc., p. 30.

13. CUZIN, F., AND F. JACOB. 1963. Integrationr vereible de l'episome sexuel F' chez Esch-erichia coli K12. Compt. Rend. 257:795.

14. DE HAAN, P., AND A. H. STOUTHAMER. 1963.F-prime transfer and multiplication of sex-duced cells. Genet. Res. (Cambridge) 4:30.

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16. FALKOW, S., AND R. V. CITARELLA. 1965. Mo-lecular homology of F-merogenote DNA.J. Mol. Biol. (in press).

17. GROSS, J. 1964. Conjugation in bacteria. InI. C. Gunsalus and R. Y. Stanier [ed.], Thebacteria, vol. 5. Academic Press, Inc., NewYork.

18. HAYES, W. 1953. The mechanism of geneticrecombination of Escherichia coli. ColdSpring Harbor Symp. Quant. Biol. 18:75.

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20. HAYES, W. 1960. The bacterial chromosome.In W. Hayes and R. C. Clowes [ed.], Micro-bial genetics. Cambridge Univ. Press, Cam-bridge.

21. HAYES, W. 1964. The genetics of bacteria andtheir viruses. John Wiley & Sons, Inc., NewYork.

22. HIROTA, Y. 1960. The effect of acridine dyeson mating type factors in Escherichia coli.Proc. Natl. Acad. Sci. U.S. 46:57.

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25. JACOB, F., AND S. BRENNER. 1963. Sur la regu-lation de la synthese du DNA chez les bac-teries: l'hypothese du replicon. Compt.Rend. 248:3219.

26. JACOB, F., S. BRENNER, AND F. CUZIN. 1963.On the regulation of DNA replication inbacteria. Cold Spring Harbor Symp. Quant.Biol. 28:329.

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29. JACOB, F., AND E. L. WOLLMAN. 1958. Geneticand physical determinations of chromosomalsegments in Escherichia coli. Symp. Soc.Exptl. Biol. 12:75.

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30. JACOB, F., AND E. L. WOLLMAN. 1961. Sexualityand the genetics of bacteria. AcademicPress, Inc., New York.

31. KAHN, P., AND D. R. HELINSKI. 1964. Re-lationship between colicinogenic factorsEl and V and an F factor in Escherichia coli.J. Bacteriol. 88:1573-1579.

32. KELLENBERGER, E. 1960. The physical stateof the bacterial nucleus. In W. Hayes andR. C. Clowes [ed.], Microbial genetics.Cambridge University Press, New York.

33. LANDMAN, O., A. RYTER, AND R. KNOTT. 1964.On the chemical and physical basis of sta-bility of L forms. Bacteriol Proc., p. 59.

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35. LEDERBERG, J., AND E. L. TATUM. 1946. Novelgenotypes in mixed cultures of biochemicalmutants of bacteria. Cold Spring HarborSymp. Quant. Biol. 11:113.

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38. PITTARD, J., AND E. A. ADELBERG. 1963. Genetransfer by F' strains of Escherichia coliK-12. II. Interaction between F-merogenoteand chromosome during transfer. J. Bac-teriol. 85:1402-1408.

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40. PITTARD, J., J. S. LoUTIT, AND E. A. ADEL-BERG. 1963. Gene transfer by F' strains ofEscherichia coli K-12. I. Delay in initiationof chromosome transfer. J. Bacteriol.85:1394-1401.

41. PITTARD, J., AND T. RAMAKRISHNAN. 1964.Gene transfer by F' strains of Escherichiacoli. IV. Effect of a chromosomal deletion onchromosome transfer. J. Bacteriol. 88:367-373.

42. PRITCHARD, R. 1963. Discussion. Cold SpringHarbor Symp. Quant. Biol. 28:348.

43. REVEL, H. R., AND S. E. LURIA. 1963. On themechanism of unrepressed galactosidasesynthesis controlled by a transducing phage.Cold Spring Harbor Symp. Quant. Biol.28:403.

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