JOURNAL OF BACTERIOLOGY, July 1975, p. 1-6Copyright 0 1975 American Society for Microbiology

Vol. 123, No. 1Printed in U.S.A.

Conservation of Salmonella typhimurium DeoxyribonucleicAcid by Chromosomal Insertion in a Partially Diploid

Escherichia coli HybridE. M. JOHNSON,* BRUCE P. PLACEK, NORMA J. SNELLINGS, AND L. S. BARON

Department ofBacterial Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20012

Received for publication 25 February 1975

A partially diploid Escherichia coli hybrid recovered from mating with aSalmonella typhimurium donor was converted to an Hfr strain, designatedWR2080, as a means to examine the manner in which the added Salmonellagenetic material was conserved in it. The Salmonella argH+, metB+, and rha+alleles contained as supernumerary genes in WR2080 were transferred together toE. coli recipients in interrupted mating experiments approximately 25 min afterinitial parental contact; transfer of the allelic E. coli genes by a haploid Hfr of thesame transfer orientation occurred between 23.5 min (argH+) and 25 min (rha+)after initial contact. Entry of the E. coli ilv+ marker of WR2080 in theseexperiments occurred at 29.5 min, 1.5 min later than the entry time of thismarker from the haploid E. coli Hfr. When unselected inheritance of the recessiveE. coli argH- and rha- alleles of WR2080 was examined among ilv+ selected E.coli recipients in which unselected inheritance of the Salmonella donor genes wasshown to be low (8%), inheritance of argH- was only 7%, whereas 51% inheritedthe neighboring rha- gene. In a comparative cross employing a haploid E. coliHfr, in which rha inheritance was similar at 56%, argH inheritance was 41%. Itwas concluded that the Salmonella genes contained in WR2080 were conservedon a genetic segment about 1.5 min in length chromosomally inserted near theallelic E. coli genes, thus creating a duplication on that region within the hybridchromosome.

In a previous study (7) we examined thenature of partially diploid Escherichia coli hy-brids formed in matings with a Salmonellatyphimurium Hfr strain. We identified threetypes of partially diploid hybrids, differing withrespect to the manner in which the Salmonellachromosomal fragments were conserved inthem. The first type consisted of F' strains inwhich the Salmonella genetic material wasmaintained extrachromosomally by associationwith a donor-derived F factor, as part of afunctional F-merogenote. Such hybrids werelysed by the male-specific phage R-17, and wereable to transfer their inherited Salmonella genesat high frequency in conjugation experiments.Diploid hybrids of the second type were notsensitive to R-17, nor were they able to transferthe Salmonella genes. Nevertheless, they re-sponded as males when tested with the female-specific phage 0II, and the Salmonella geneswere conserved in them, extrachromosomally,as parts of covalently closed circular (CCC)molecules of deoxyribonucleic acid (DNA);these circular elements were considered to bedefective F-merogenotes unable to direct syn-thesis of the F-pili upon which male-phage

sensitivity and conjugal transmissibility de-pend. In diploid hybrids of the third type, whichcomprised the majority of those examined, theSalmonella DNA was conserved in some man-ner which involved neither association with Fnor assumption of the CCC configuration. Itwas with a diploid hybrid of this latter type tihatwe carried out the experiments detailed in thepresent communication. Our findings indicatedthat the added Salmonella DNA in this hybridis not maintained extrachromosomally, but isconserved instead as a genetic segment insertedwithin the hybrid chromosome.

MATERIALS AND METHODSOrganisms. With the exception of the hybrid Hfr

WR2080, the bacterial strains employed are describedin Table 1. Transfer orientations of the Hfr strainslisted in this table, as well as of the hybrid HfrWR2080, are shown in Fig. 1. The characteristics ofWR2080 are detailed in the Results section. To avoidpossible confusion, we have used only the E. coli genedesignations throughout the presentation, and thusrefer to a homologous Salmonella gene by the samedesignation given its E. coli allele. For the sake ofpropriety, however, we point out here that theSalmonella genes which we refer to as argB and argH

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2 JOHNSON ET AL.

TABLE 1. Pertinent characteristics of thebacterial strains

Strain Description Relevant genotype

WR2004 E. coli Hfr metBWR2005 E. coli Hfr purEWR2017 E. coli Hfr metB, argH, thiA, his, strAWR3026 E. coli F- ilv, metB, argH, thiA, his,

xyl, malA, strAWR3029 E. coli F- ilv, metB, argH, thiA, his,

xyl, rha, malA, strAWR3040 E. coliF- ilv, trp, xyl, rha, strAWR3041 E. coli F ilv, trp, xyl, strAWR3051 E. coli F- proA, thr, leu, argB, thiA,

his, strAWR4016 S. typhi- serA

muriumHfr

FIG. 1. Chromosome map of E. coli K-12 showingthe positions of relevant genetic loci and the transferorientations of the Hfr strains employed. Time unitsare those of Taylor and Trotter (13).

are, in the Salmonella nomenclature (12), referred toas argC and argF, respectively.

Noninterrupted mating and unselected markeranalysis. As described previously (8), bacterial mat-ings were carried out in Penassay broth for 2 h at 37 Cafter which the mating suspensions were dilutedappropriately and plated on minimal agar selectivemedium. Hybrids were purified by streaking on mini-mal medium of the same composition as that used fortheir selection. Unselected inheritance of auxotrophicmarkers was scored by determining the ability of thehybrids to grow on minimal medium lacking theamino acid or vitamin in question. The ability toferment a carbohydrate was scored on MacConkeyagar indicator medium containing 1% of that carbohy-drate. This medium was used also to identify partiallydiploid hybrids by their segregation of fermenting andnonfermenting clones as described previously (1).

Interrupted mating procedure. The procedure

employed for interrupted matings was similar, essen-tially, to that described by de Haan and Gross (5).Prewarmed donor (5 x 108 cells/ml) and recipient (2x 109 cells/ml) suspensions in Penassay broth weremixed with gentle agitation at 37 C for 5 min. Thismating mixture was then diluted 1:200 into pre-warmed liquid minimal medium and kept at 37 Cwithout agitation for the duration of the experiment.At 3-min intervals, 0.5-ml samples of the minimalmating suspension were added to tubes containing 0.5ml of Penassay broth, subjected to vigorous agitationfor 60 s by a Vortex Jr. mixer, and plated (0.1-ml/plate) on appropriate selective media containing 625gg of streptomycin sulfate per ml. The numbers ofexconjugants found in successive plated samples plot-ted agairtsi the times of plating revealed the time ofentry for the marker selected.

Bacteriophage testing. The procedures for testingsensitivity to the male-specific bacteriophage R-17 (3,11), and for determining the male or female responseof a hybrid to the female-specific bacteriophage 0II(4), were described in our previous communication(7).Examination for the presence of CCC DNA.

Examination of hybrids for the possible presence ofCCC DNA was accomplished by the dye-buoyantdensity centrifugation procedure of Bazaral and He-linski (2).

RESULTSConstruction of an Hfr derivative of a

partially diploid S. typhimurium x E. colihybrid. In previous crosses (7) between S.typhimurium Hfr WR4016 and the E. coli K-12recipient strain WR3026, in which hybrids re-ceiving an early transferred donor marker (xyl+)were selected, approximately 40% of the E. coliexconjugants examined exhibited the segrega-tion behavior of partially diploid hybrids. About25% of those partially diploid hybrids werefound to conserve the added Salmonella DNAas part of an F-merogenote. In some cases theseF-merogenotes were functional, i.e., transmissi-ble by conjugation, and in others they were not.Most of the partially diploid hybrids, however,were seen to conserve the added Salmonellagenes in some manner which did not involveassociation with F. Nevertheless, because of theinstability of the sex factor of S. typhimuriumHfr WR4016, many of the diploid E. coli hybridsin which conservation of the added Salmonellagenes did not involve F did contain a superin-fecting F factor.

For the purpose bf the present study, it wasnecessary to obtain a partially diploid E. colihybrid in which the added Salmonella geneswere not conserved by association with F and inwhich no superinfecting F factor was present. Itwas desired also that the Salmonella geneticsegment contained in this hybrid should bear

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S. TYPHIMURIUM DNA IN A DIPLOID E. COLI HYBRID

those markers which would allow the bestdetermination of its length, with reference tothe available markers of the recipient. To ac-complish these ends, we first mated S.typhimurium Hfr WR4016 with E. coli WR3029,a Rha- derivative of WR3026, selecting for thosehybrids receiving the argH+ allele of the donor.We then tested these Arg+ hybrids for sensitiv-ity to the male-specific phage R-17, a procedurewhich eliminated from consideration (becauseof the indication of F-infection) the majority ofthe hybrids examined. Among the remainder,whose resistance to R-17 suggested that theyhad escaped F-infection, we narrowed furtherthe scope of our consideration to those whichalso had acquired the donor rha+ allele (for useas an indicator of diploidy), but not the adja-cent (see Fig. 1) donor ilv+ allele (to help definethe limits of the Salmonella gene segment andto allow the subsequent introduction of a posi-tive E. coli allele at this locus). We thensearched among these hybrids for any whichshowed evidence of diploidy, as indicated bytheir segregation (on MacConkey rhamnoseagar), of daughter cells from which the Salmo-nella rha+ allele had been lost.We selected for further study a partially

diploid E. coli hybrid containing the Salmonellaalleles rha+, argH+, metB+, and thiA+. Thishybrid responded as a female when tested withthe female-specific phage 01. It was examinedby dye-buoyant density centrifugation for thepossible presence in it of CCC DNA, and nonewas detected. Loss of the Salmonella rha+marker from this hybrid occurred spontane-ously at low frequency, generating one segregantper 500 to 1,000 cells. Examination of Rha-segregants of this hybrid revealed that they hadlost also the Salmonella genes argH+ andmetB+. None of the segregants, however, wereobserved to lose the thiA+ gene.

Anticipating the conversion of this partiallydiploid hybrid to an Hfr, we desired two addi-tional allelic alterations, namely, its conversionfrom streptomycin resistance to streptomycinsensitivity, and the substitution of a positive E.coli allele at its ilv locus. The hybrid was mated,therefore, with the streptomycin sensitive E.coli Hfr WR2005, without streptomycin counterselection, and exconjugants were selected forreceipt of the donor malA+ allele. An exconju-gant was chosen which had received, in additionto the selected malA + character, the strA-sand ilv+ alleles of the donor. The responses ofthis exconjugant to R-17 and OII remainedthose of a female, and again, no CCC DNA wasdetected in it. It segregated Rha- daughter cells

at a low frequency which, as before, had lostalso the Salmonella argH+ and metB+ alleles.Again, all segregants remained Thi+. Conver-sion of this exconjugant to an Hfr was thenaccomplished by the F-linked terminal marker(in this case lac+) selection procedure (6) usingthe E. coli Hfr WR2017. The new Hfr generatedby this procedure was designated WR2080. Asdid each of its predecessors, WR2080 segregateddaughter cells exhibiting en bloc loss of theSalmonella rha+, metB+, and argH+ alleles. Asbefore, all segregants remained Thi+.Interrupted mating experiments with the

partially diploid E. coli Hfr WR2080. In non-interrupted matings between the diploid E.coli Hfr WR2080 and E. coli recipient WR3029,the Salmonella rha+ and argH+ markers of thedonor were transferred at approximately thesame frequency (2 x 10-3), and the transferfrequency of its E. coli ilv+ marker was some-what lower at 5 x 10-4. In interrupted matingexperiments performed with this pair, the fol-lowing entry times were determined for theSalmonella markers of WR2080: rha+, 24.7 min;metB+, 24.8 min; argH+, 25.3 min. The thiAallele of WR3029 proved unsuitable for use inthese experiments, and no entry time could bedetermined for this donor marker. Entry time ofthe E. coli ilv+ allele of WR2080 was 29.5 min.In comparative experiments using the samerecipient in interrupted matings with the nor-mal haploid E. coli Hfr WR2004, which has thesame transfer orientation as WR2080, we re-corded the following marker entry times: argH+,23.5 min; rha+, 25.0 min; ilv+, 28.0 min. Thecomparison of the marker entry times of thisdonor with those of WR2080 is shown in Table 2.To reassure ourselves that the later entry

times of the argH+ and ilv+ markers of WR2080were not due to a difference in the injectionspeed of its chromosome from that of WR2004,we employed both Hfr strains individually ininterrupted matings with the E. coli recipientWR3051 to examine the entry of a markerlocated proximal to the argH locus in thetransfer order. The results of these experimentsalso are shown in Table 2. Entry of the thr+-leu+(joint selection) alleles of Hfr WR2080 occurredat the expected time of 12 min, the same time asobtained for WR2004, thus indicating that therewas no difference between these Hfr strains withrespect to the speed of chromosome injection.Nevertheless, whereas entry of the argB+marker of WR2004 occurred at the expectedtime of 23.5 min (the argB and argH loci areinseparable by interrupted mating), entry of theargB+ allele of WR2080 did not occur until 25.1

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TABLE 2. Comparative marker entry times (min) for the partially diploid E. coli hybrid HFR WR2080 and thesimilarly oriented haploid E. coliHFR WR2004a

Entry time (min)Interrupted mating pairs

thr+-leu+ argB+ argH+ metB+ rha + ilv+

WR2080 x WR3029 25.3 24.8 24.7 29.5WR2004 x WR3029 23.5 25.0 28.0WR2080 x WR3051 12.0 25.1WR2004 x WR3051 12.0 23.5

a The argB+, argH+, metB+, and rha+ markers of WR2080 are derived from S. typhimurium.

min. The entry of the Salmonella alleles ofWR2080 near the 25 min mark, coupled with the1.5 min delay in the entry of its E. coli ilv+ allele(29.5 min versus 28.0 min for WR2004) isconsistent with the view that a segment bearingthe Salmonella genes is inserted in the WR2080chromosome near the region occupied by theallelic E. coli genes.

Inheritance of unselected markers derivedfrom Hfr WR2080. We examined 200 of theearliest-appearing argH+-selected WR3029 ex-conjugants formed in the interrupted matingswith the diploid Hfr WR2080 for unselectedinheritance of Salmonella metB+ and rha+alleles; all were found to have inherited bothof these alleles in addition to the selectedargH+ gene. We examined also 200 of theearliest appearing rha+-selected WR3029 excon-jugants formed in those interrupted matingsfor unselected inheritance of the SalmonellametB+ and argH+ alleles. Again, all had in-herited both of these Salmonella alleles inaddition to the selected rha+ gene. Finally, weexamined 100 ilv+-selected exconjugants from aWR2080 x WR3029 cross for unselected inheri-tance of the Salmonella rha+, metB+, andargH+ alleles. There were 42 exconjugants inwhich unselected inheritance of Salmonella al-leles occurred and each of these 42 acquired allthree Salmonella markers.The inseparability of the Salmonella alleles

in these experiments contrasts with the behav-ior of their E. coli counterparts when transferredby a haploid E. coli Hfr. An example of thenormal inheritance picture is seen in the crossbetween the haploid E. coli Hfr WR2017 and theE. coli recipient WR3040 shown in Table 3.Examination of 100 WR3040 exconjugants se-lected for receipt of the donor ilv+ marker showsthat although the rha+, metB-, and argH-donor alleles were most frequently inherited enbloc, separation of one or more of these markersdid occur in 17 of the 56 hybrids in which theirunselected inheritance was observed.

Unselected inheritance of the recessive E. coli

TAmBL 3. Unselected marker inheritance among 100ilv+-selected E. coli WR3040 exconjugants from a

cross with haploid E. coliHFR WR201 7a

SummationHybrid class No. of marker No.

inheritance

ilv + 44 rha+ 56ilv+ rha+ metB- argH- 39 metB- 45ilv+ rha+ 9 argH- 41ilv+ rha+ metB- 6ilv+ rha+ argH- 2

a Markers listed are those inherited from the Hfr.

alleles of WR2080 was examined in a matingwith the E. coli recipient WR3041, a Rha+derivative of WR3040. The inheritance patternof the E. coli rha-, metB-, and argH- allelesamong 100 ilv+-selected WR3041 exconjugants,shown in Table 4 differs significantly from thatseen in the haploid Hfr WR2017 x WR3040cross (compare Table 3). Although unselectedinheritance of the E. coli rha- marker ofWR2080 by 51 VWR3041 exconjugants was simi-lar to the 56% inheritance of the rha+ marker ofWR2017, there was a noticeable reduction in theinheritance of the E. coli metB- marker ofWR2080, compared with the VWR2017 cross, andthe WR2080 E. coli argH- marker was inheritedby only 5 exconjugants, as opposed to 41 whichreceived argH+ unselected from WR2017.

It should be pointed out here that WR3040and its derivative, WR3041, differ from WR3026and its derivative, WR3029, in their behavior asrecipients of Salmonella DNA. WhereasWR3026 and WR3029 are good acceptors ofSalmonella genes (recall the 42% unselected enbloc inheritance of the Salmonella rha+, metB+,and argH+ alleles by WR3029), WR3040 andWR3041 are not. As seen in the cross betweenWR2080 and W`R3040 (Table 5), in which inher-itance of the Salmonella rha+ allele can bescored, only 8 of 100 ilv+-selected WR3040exconjugants inherited this Salmonella marker.Inheritance of the E. coli metB- and argH-

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alleles in this cross was similar to that observedwith WR3041 (compare Table 4) with 29 excon-jugants expressing the E. coli metB- donorallele, and only 7 expressing the E. coli argH-donor allele. We believe that the low unselectedinheritance of the E. coli argH- allele ofWR2080 in these crosses is indicative of itsproximity to the inserted Salmonella geneticsegment (Fig. 2) whose inheritance by WR3041occurs at about the same low frequency.

In the mating of WR2080 with both WR3040and WR3041, the ilv--selected exconjugantsalso were examined for possible inheritance ofthe E. coli thiA- allele originally present in theWR3029 strain from which WR2080 was de-rived. None of the 200 exconjugants examinedwas observed to express this allele. In view ofthe fact that no Thi- segregants were everobserved from WR2080 or any of its predeces-sors, it might be presumed that, as the conse-quence of genetic recombination, the original E.coli thiA- allele no longer exists in WR2080.

DISCUSSIONWe conclude that the Salmonella chromo-

somal segment bearing the rha+, metB+, andargH+ alleles is inserted in the chromosome ofWR2080 near, and to the right of (see Fig. 2),

TABLE 4. Unselected inheritance of the recessive E.coli alleles of the partially diploid E. coli hybrid HFR

WR2080 among 100 ilv - selected E. coliWR3041 exconjugantsa

SummationHybrid class No. of marker No.

inheritance

ilv+ 48 rha- 51ilv+ rha- metB- 26 metB- 30ilv+ rha 21 argH- 5ilv+ rha- metB- argH- 4ilv+ argH- 1

aMarkers listed are those inherited from the Hfr.

TABLE 5. Unselected inheritance of dominantSalmonella and recessive E. coli alleles of the

partially diploid E. coli hybrid HFR WR2080 among100 ilv+-selected E. coli WR3040 exconjugantsa

SummationHybrid class No. of marker No.

inheritance

ilv+ 63 metB- 29ilv+ metB- 22 argH- 7ilv+ rha+ 8ilv+ metB- argH- 7

aMarkers listed are those inherited from the Hfr.The rha+ marker is a Salmonella allele, the others areE. coli alleles.

ilv

75 76

rho+ metB+ argH+

\ /\ /\ /

rha metB argH thiAn n n %v n

77 78 79 80

FIG. 2. Approximate location of the chromosom-ally inserted Salmonella genetic segment in E. colihybrid WR2080 relative to the original sites of itsargH and thiA loci. Time units are those of Taylor andTrotter (13). Assuming identity in the lengths ofSalmonella and E. coli chromosomes in this area, the1.5-min later interrupted mating entry time of the E.coli ilv+ marker of WR2080 (which represents theadditional time required for entry of the Salmonellagenes) suggests that the Salmonella rha+ and argH+markers very nearly define the extremities of theinserted Salmonella DNA segment. Although de-picted here in the normal order, it is not knownwhether the Salmonella genes are inserted in thismanner or in the reverse order (see text).

the original site of the argH locus, thus creatinga duplication of this region within the hybridchromosome. This arrangement is indicatedboth by the interrupted mating entry times forthe Salmonella alleles near the 25 min mark,and by the abnormally low unselected inheri-tance of the E. coli argH- marker of WR2080 inE. coli recipients that exhibit a similar diffi-culty inheriting Salmonella genes. Also, it isconsistent with the observation that entry of theE. coli ilv+ allele of WR2080, which is situateddistal to the proposed insertion site, was de-layed 1.5 min, whereas entry of E. coli markerslocated proximal to the insertion site in this Hfroccurred at the expected time.Although a somewhat earlier entry time was

determined for the Salmonella rha+ marker(24.7 min) than for the Salmonella argH+marker (25.3 min), we do not think that thisdifference is great enough to be consideredindicative of an inverted gene order within theinserted segment. Our opinion is that thesedifferent time determinations probably repre-sent variations of a single time of entry which isthe same for all of the Salmonella markers. Inno instance during these studies was any of theSalmonella alleles inherited separately, and it isnot unreasonable to suppose that recovery ofhybrids expressing them might require entryand conservation of the entire Salmonella ge-netic segment. If this is indeed the case, and ifthe lengths of the Salmonella and E. colichromosomes are as we believe identical in thisregion (1.4 min between argH and rha), then theminimum estimate of the common entry timefor the three Salmonella markers would be 24.9

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6 JOHNSON ET AL.

min. Regardless of whether or not this interpre-tation of the data is correct, however, we do notbelieve that any conclusions can be drawn as tothe order of the inserted Salmonella genes.

In our previous study (7) of the partiallydiploid hybrids formed in this mating system,we observed that about 25% of those examinedconserved the Salmonella DNA extrachromoso-mally in the CCC configuration. Maintenanceof the added DNA in that configuration in-volved, in every instance, association with atleast some part of the sex factor F. The majorityof the partially diploid hybrids examined, how-ever, were seen to conserve the added Salmo-nella DNA in some manner which did notinvolve association with F, or assumption of theCCC configuration. Examination of one suchhybrid in the present study showed that itsSalmonella DNA is not maintained extrachro-mosomally, but is conserved instead as aninsertion within the hybrid chromosome. Al-though none of these observations rules outthe possibility that some hybrids might con-serve the Salmonella DNA extrachromosomallyin a form other than CCC, we think it morelikely that, within this mating system, allpartially diploid hybrids in which F is notinvolved in maintaining a CCC exogenote con-serve the added DNA by chromosomal insertion.

In an earlier study (9) involving S. typhosa asthe recipient of E. coli genes, we found that theadded E. coli DNA in some partially diploidhybrids was maintained extrachromosomally inthe CCC configuration. The diploid hybrids inwhich this DNA form was detected, however,constituted a minority of those examined; themanner of conservation of the added E. coliDNA in the majority of the hybrids was notdetermined. Subsequently, in a report concern-ing S. typhimurium as the recipient of E. coligenes, Mojica-a and Middleton (10), interpre-ting data from their transduction studies, con-cluded that some partially diploid hybrids main-tained the added genetic material extrachromo-somally, whereas others conserved it by chromo-somal integration (insertion). The partiallydiploid S. typhimurium hybrids which theyclassified as conserving the added E. coli DNAby insertion constituted over 70% of those ex-amined. In retrospect, we think it very likelythat the added E. coli DNA in those S. typhosa

hybrids in which we detected no CCC materialwas conserved by chromosomal insertion.From all considerations, it appears to us that

chromosomal insertion probably is the generalmechanism by which partially diploid hybridsare formed in both E. coli Hfr x Salmonella F-and Salmonella Hfr x E. coli F- mating sys-tems. Extrachromosomal conservation of addedDNA in the CCC configuration occurs, webelieve, only in those special circumstances inwhich F or possibly some other replicator,chances to be present and active in maintainingthat configuration. In the presently studied S.typhimurium x E. coli system, the only replica-tor observed to be active in this manner was F.

LITERATURE CITED1. Baron, L. S., P. Gemski, Jr., E. M. Johnson, and J. A.

Wohlhieter. 1968. Intergeneric bacterial matings. Bac-teriol. Rev. 32:362-369.

2. Bazaral, M., and D. R. Helinski. 1968. Circular DNAforms of colicinogenetic factors E,, E2 and E, from E.coli. J. Mol. Biol. 37:133-143.

3. Crawford, E. M., and R. F. Gesteland. 1964. The adsorp-tion of bacteriophage R-17. Virology 22:165-167.

4. Cuzin, F. 1965. Un bacteriophage specifique du typesexuel F- d'Escherichia coli K-12. C. R. Acad. Sci.260:6482-6485.,

5. de Haan, P. G., and J. D. Gross. 1962. Transfer delay andwithdrawal during conjugation in Escherichia coli.Genet. Res. 3:251-272.

6. Jacob, F., and E. L. Wollman. 1957. Analyse des groupesde liaison genetique de differentes souches donatricesd'Escherichia coli K-12. C. R. Acad. Sci.245:1840-1843.

7. Johnson, E. M., W. G. Craig, Jr., J. A. Wohlhieter, J. R.Lazere, R. M. Synenki, and L. S. Baron. 1973. Conser-vation of Salmonella typhimurium deoxyribonucleicacid in partially diploid hybrids of Escherichia coli. J.Bacteriol. 115:629-634.

8. Johnson, E. M., S. Falkow, and L. S. Baron. 1964.Recipient ability of Salmonella typhosa in geneticcrosses with Escherichia coli. J. Bacteriol. 87:54-60.

9. Leavitt, R. W., J. A. Wohlhieter, E. M. Johnson, G. E.Olson, and L. S. Baron. 1971. Isolation of circulardeoxyribonucleic acid from Salmonella typhosa hy-brids obtained from matings with Escherichia coli Hfrdonors. J. Bacteriol. 108:1357-1365.

10. Mojica-a, T., and R. B. Middleton. 1972. Salmonellatyphimurium-Escherichia coli hybrids for the trypto-phan region. Genetics 71:491-505.

11. Paranchych, W., and A. F. Graham. 1962. Isolation andproperties of an RNA-containing bacteriophage. J.Cell. Comp. Physiol. 60:199-208.

12. Sanderson, K. E. 1972. Linkage map of Salmonellatyphimurium, edition IV. Bacteriol. Rev. 36:558-586.

13. Taylor, A. L., and C. D. Trotter. 1972. Linkage map ofEscherichia coli strain K-12. Bacteriol. Rev.36:504-524.

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