JOURNAL OF BACrERIOLOGY, July 1975, p. 28-35Copyright 0 1975 American Society for Microbiology

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

RP1 Properties and Fertility Inhibition Among P, N, W, and XIncompatibility Group PlasmidsRONALD H. OLSEN* AND PATRICIA L. SHIPLEY

Department of Microbiology, University of Michigan Medical School, Ann Arbor, Michigan 48104

Received for publication 21 January 1975

Incompatibility group P plasmids demonstrate strong entry exclusion proper-ties. Stringent incompatibility is also observed in the absence of entry exclusion.These observations have been facilitated by the study of a nontransmissibleplasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2retains carbenicillin and tetracycline resistances as well as loci that cause eitherthe loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) inthe presence of a P plasmid. RP1-S2 can be mobilized by an incompatibilitygroup W plasmid, R388, and also freely forms recombinants with R388. P, N, andW incompatibility group plasmids all encode information for the receptor of thecell wall-adsorbing phage PRD1. Based on the premise that the location of thisreceptor is analogous to entry exclusion factors for F-like plasmids and hence aregulated transfer region determinant, we tested fertility inhibition relationshipsamong these plasmid groups. We detected both reciprocal and nonreciprocalfertility inhibition relationships for bacteria containing various combinations ofW, N, and P group plasmids. The nonreciprocal nature of some combinations, webelieve, reflects the identity of the point mutation reading to derepression of theplasmid in question. Reciprocal fertility inhibition, on the other hand, mayreflect the reconstruction of a fertility inhibition system through complementa-tion. An X incompatibility group plasmid, known to affect the fertility of an Ngroup plasmid, was also shown to inhibit P plasmid fertility. These observationsmay indicate a possible evolutionary relationship(s) of plasmids unrelated by thecriteria of incompatibility, pilus phage specificity, or plasmid host range.

Plasmids unable to coexist stably in the samehost are considered to comprise an incompati-bility group (4, 15). The P incompatibilitygroup presently includes RP1 (9), R751 (13),R702, and R906 (received from N. Datta). Theseplasmids have a broad host range among thegram-negative bacteria (3, 6, 16; unpublisheddata). They are also lysed by the P plasmid-specific ribonucleic acid phage PRR1 (18). Inaddition to sharing of phage susceptibility, theP group of plasmids also shows entry exclusionwithin itself. Another surface component deter-mined by P plasmids as well as the N and Wincompatibility groups is the PRD1 phage re-ceptor (17). Thus, in addition to drug resistancedeterminants, the P plasmids encode geneticinformation for incompatibility, pili specific forphage PRR1, entry exclusion determininants,and the receptor for the cell wall-adsorbingphage PRD1. The latter characteristic, how-ever, is not unique to the P plasmids.Another characteristic associated with some

plasmids is fertility inhibition, an interaction

among compatible plasmids whereby the donorfrequency of one or both resident plasmids isdiminished. We have tested some of the Pplasmids with respect to this characteristic.Some R factors were originally designated fi+based on their ability to inhibit conjugation bythe Escherichia coli sex factor F (23). Most ofthese plasmids initially studied were F-like intheir properties and encoded an F-like pilus(15). Later, a plasmid specifying I-like pili alsowas shown to inhibit F (8, 21). However, in thisinstance, the mechanism of F inhibition by theI-like plasmid R62 differed in its properties fromthat associated with F inhibition by F-likeplasmids (14). Recently, in addition to theforegoing, two group X R factors have beenshown to be fi+ with respect to an Fl group Rfactor, R386 (10). Furthermore, R46, a group Nplasmid, has been shown to be susceptible torepression by a group X R factor, R6K (6).Thus, it is becoming increasingly apparent thatfertility inhibition of one plasmid by another isnot confined to plasmid groupings based on

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VL7RP1PROPERTIES AND FERTILITY INHIBITION 29

incompatibility relationships (5, 15) or sex pilusproperties. We report here further examples ofplasmid relationships based on fertility inhibi-tion among plasmids unrelated on the basis oftheir incompatibility or pilus properties. Insome of these instances, the plasmids share incommon the genetic information for encoding ofthe PRD1 phage (17) receptor site. Thus, assuggested previously (17), these fertility inhibi-tion relationships may provide additional evi-dence for the common evolution of plasmidsseemingly unrelated by other criteria.This investigation was initiated in an attempt

to gain a preliminary estimate of linkage rela-tionships for RP1 plasmid determinants. Forthis, we used a derivative of RP1, designatedRP1-S2, which was obtained by transductionalshortening of RP1 using phage P22 (22). Arecombinant between RP1-S2 and a W groupdrug resistance plasmid designated RWP1 alsowas subsequently studied to determine theextent of RP1-S2 gene incorporation. The re-sults of these studies and the subsequent obser-vation of fertility inhibition relationshipsamong plasmid groups are described in thisreport.

MATERIALS AND METHODSBacterial strains. Bacteria used and the relevant

properties of their plasmids are listed in Table 1.These plasmid-containing strains have been used byus previously for the determination of RP1 host range(16) and plasmid specificity of phages infecting Pincompatibility group plasmids (17, 18).

Media. Complex medium TN and minimal me-dium VBG were prepared as described previously(16). Antibiotic supplements are as described in thetables.Mating and testing of exconjugants. R+ strains

subsequently used for quantitative estimates of trans-fer frequencies were constructed by mixed inoculationof saline-glucose buffer (NaCl, 0.85%; glucose, 0.36%;pH 7.0) with growth from TN agar containing theantibiotic selective for one of the pertinent resistancedeterminants specified by the plasmid. Donor andrecipient bacterial suspensions were incubated at37 C for 1 h, and an aliquot was then plated on theappropriately supplemented medium. Exconjugantswere purified by serial clone isolation on TN agarcontaining the appropriate antibiotic. When doubles,i.e., bacteria containing two plasmids, were used asdonors for mating experiments, they were grown asabove on the medium containing the antibiotic appro-priate to the maintenance of both drug resistancefactors. Matings done to determine entry exclusion orfertility inhibition of one plasmid by another weredone in TN broth medium. For this, TN brothmedium was inoculated with growth from TN agarmedium containing the selective antibiotic. On theaverage, these TN broth cultures were incubated for

TABLE 1. Plasmid incompatibility group andresistance determinants

Plasmida Incompatibility Resistancegroup determinantsb

RP1 P CbR, TcR, NmR/KmRR751 P TpRR702 P SmR, TcR, KmR, SuRR906 P SmR, ApR, SuRRP1-S2c P CbR, TcRR388 W TpR, SuRRSa W SmR, CmR, SuR, KmRRWPld W CbR, TpRR15 N SmR, ApR, TcR, SuRR46 N SmR, ApR, TcR, SuRRN3 N SmR, ApR, TcR, SuRR6K X ApR, SmR

a All plasmids used in this study were maintainedin E. coli J53 or E. coli CR34.

° The abbreviations designate resistance to thefollowing antibiotics: ApR, ampicillin; CbR, car-benicillin; CmR, chloramphenicol; KmR, kanamycin;NmR, neomycin; SmR, streptomycin; SuR, sulfonila-mide; TcR, tetracycline; TpR, trimethoprim.

c Derived from RP1 in Salmonella typhimuriumLT2 using phage P22. See reference 22.

d Recombinant plasmid, R388 x RP1-S2.

approximately 2 h at 37 C with agitation. Inoculationswere adjusted to result in approximately 108 cells perml of TN broth after 2 h of growth at 37 C. Formating, donor and recipient cultures were mixed 1:1and incubated under static conditions for 1 h at 37 C.If selection of exconjugants was on TN agar medium,these mating mixtures were mixed, diluted, andplated directly. If supplemented VBG agar mediumwas used for selection, mating mixtures were cen-trifuged at ambient temperature and cell pellets weresuspended to volume in 0.01 M phosphate buffer (pH7.0). Plates were incubated for 48 h at 37 C forquantitative estimates of mating frequencies.

RESULTSPrevious attempts in our laboratory to deter-

mine linkage between RP1 determinants byusing the Escherichia coli transducing phage P1have been unsuccessful. The entire RP1 plas-mid was always transduced. However, the avail-ability of the RP1 derivative RP1-S2 (22) al-lowed for the investigation of linkage betweenRP1 determinants concerned with surface ex-clusion and incompatibility with respect to theiraffect on other P group plasmids. The entryexclusion and incompatibility properties of RP1and its derivative, RP1-S2, in relation to theother P group plasmids are shown in Table 2. Itis clear from these data that RP1 excludes theentry of the related plasmids R751, R702, andR906. However, with RP1-S2 as the resident

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TABLE 2. Entry exclusion and incompatibilityproperties of RPI or RP1-S2 in relation to other P

group plasmids

Donor Recipienta P group R+recipients/donorb

J53 (R751) CR34 2 x 10-2CR34 (RP1) 1 x 10-6CR34 (RP1-S2) 2 x 10-3

J53 (R702) CR34 3 x 10-3CR34 (RP1) 3 x 10- 5

CR34 (RP1-S2) 3 x 10-'

J53 (R906) CR34 4 x 10- 3

CR34 (RP1) 1 x 10-6CR34 (RP1-S2) 2 x 10-

a Nutritional selection against the donor was doneon appropriately supplemented minimal mediumused previously (16).

Trimethoprim (100 gg/ml) or streptomycin (25ug/ml) was included in minimal medium to select forthe transfer of R751 or R702 and R906, respectively.

plasmid in recipient bacteria, this exclusion, asindicated by a low frequency of exconjugantformation, is not apparent. Thus, RP1-S2 haslost genetic information specifying the entryexclusion properties common to P group plas-mids in addition to the deletion of genes specify-ing P plasmid pili and the cell surface receptorfor phage PRD1 noted previously (22). It isinteresting to note that both entry exclusionand pilus genes,are associated with the transferregion of F and F-like plasmids and that thesame linkage relationship apparently is ob-tained with RP1. From this we conclude thatRP1 transfer region genes are linked to determi-nants for neomycin/kanamycin resistance.However, when exconjugants from the abovematings are purified by serial restreaking onmedium not selective for the P group plasmidoriginally present in the recipient, it is observedupon subsequent testing that they have lostRP1 or RP1-S2. Therefore, RP1-S2 has retainedgenetic information determining susceptibilityto incompatibility by a related plasmid. Wedesignate this phenotypic trait sinp. Thus, thematings (Table 2) with CR34(RP1-S2) recipi-ents resulted in the detection of RP1-S2 suscep-tibility to incompatibility caused by entering Pgroup plasmids. We next verified the ability ofRP1-S2 to cure a resident P plasmid by trans-ducing RP1-S2 into bacteria containing R702 orR906. For this, P1 phage was grown on E. coliJ53(RP1-S2) and harvested as reported previ-ously for P22 transduction (22). These transduc-ing phage lysates transduced RP1-S2 into either

J53(R702) or to J53(R906) at a frequency ofapproximately 2 x 10- 6 per viable phage. Whenthe transductants selected for carbenicillin or

tetracycline resistance were purified on mediumselective for RP1-S2 and individual colonieswere tested, all were observed to have lost the Pplasmid (R702 or R906) originally present in therecipient bacteria (20/20 tested). We have des-ignated this trait incp, analogous to the pheno-type designated for F-like plasmids (7). INCp(capital letters used for a gene product) then, ispresumably a P plasmid gene product responsi-ble for the loss of related plasmids containingthe sinp locus maintained in bacteria grown

under nonselected conditions with respect tothe lost plasmid. Consequently, these studieshave indicated that RP1-S2 has retained the Pplasmid genes concerned with incompatibilityas well as genes specifying resistance to car-

benicillin and tetracycline. Accordingly, thesecharacteristics constitute a linkage group in theRP1 plasmid.

Properties of a P-W group recombinantplasmid. We reported the construction of a

recombinant plasmid formed by the addition tothe W group plasmid R388 (22) of carbenicillinresistance specified by the P group plasmidRP1. The recombinant plasmid, designatedRWP1, was tested and found to belong to the Wincompatibility group. We also determinedwhether RWP1 contained genetic informationspecifying incompatibility with P group plas-mids (Table 3). The results show that the donorfrequencies of the P plasmids are not affectedby the presence of RWP1 in the recipientbacteria. This is also the finding for recipientscontaining R388, the parent of RWP1 (unpub-lished data). When exconjugants from the mat-ings shown in Table 3 were purified by serialcolony isolation on the agar medium containingan antibiotic selective for either the donor or

recipient plasmid, the nonselected plasmid was

maintained in all instances (20/20 tested).Thus, the incorporation of RP1 carbenicillinresistance into R388 to form RWP1 resulted in

TABLE 3. Transfer of P plasmids to CR34 (RWPl)a

Donor Recipient P group R+recipients/donor

J53 (R702) CR34 (RWP1) 1 x 10-9J53 (R906) 3 x 10-4J53 (RP1) 1 x 10-3

a Selection for the transfer of RP1 was on minimalmedium containing 500 Atg of carbenicillin per ml.Selection for the transfer of R702 and R906 was asdescribed in Table 2.

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the loss of the P plasmid incompatibilityfunction(s). Similarly, in the reverse situation,when the recipient bacteria contained the Pplasmids used in Table 3 and the donor con-tained RWP1, exconjugants stably maintainedboth RWP1 and the P plasmid under nonselec-tive conditions of growth.

Fertility inhibition studies. We next testedRWP1 for the expression of genes affecting Pplasmid fertility. We considered that genespossibly affecting P plasmid fertility may belinked to carbenicillin resistance in RP1 andhence, in some instances, would be retainedwhen recombination with R388 occurred. Thisdetermination could not be done reproduciblyin bacteria containing two P plasmids, perhapsreflecting the stringent incompatibility occur-ring among P plasmids. However, RWP1 iscompatible with P plasmids, and hence "dou-bles" (bacteria containing RWP1 and a P plas-mid) could be tested for the effect ofRWP1 on Pplasmids or the reverse with regard to donorfrequency. The results of these determinations(Table 4) clearly show inhibition of RWP1transfer by bacteria containing either of the Pplasmids RP1 or R702. This unexpected resultresembles fertility inhibition of F-mediatedconjugation by the presence of fi+ F-like plas-mids (24) and also the later reports for severalother plasmid combinations (10, 19, 25). In-deed, the P plasmid RP4 has been shown

TABLE 4. Mating of RWPl-P plasmid doubles withJ53SmR

Donora R+ recipient R+ recipients/selectedb donor

J53 (RWP1) RWP1 5 x 10-3J53 (RP1) RP1 2 x 10-3J53 (RP1/RWP1) RP1 3 x 10-'

RWP1 1 x 10-6J53 (RWP1/RP1) RP1 3 x 10-4

RWP1 2 x 10-6J53 (R702) R702 2 x 10-3J53 (R702/RWP1) R702 7 x 10-4

RWP1 2 x 10-5J53 (RWP1/R702) R702 8 x 10-4

RWP1 9x 10-6

a In the case of doubles, the first plasmid listed wasadded to bacterial recipients containing the secondplasmid listed. For this strain construction, R+ CR34donors were used."TN agar containing 500 Ag of streptomycin and

100 ug of trimethoprim per ml was used to select forthe acquisition of RWP1. For the selection of eitherRP1 or R702, 20 jg of tetracycline per ml was includedin TN agar in addition to 500 ug of streptomycin perml. J53SmR is chromosomally resistant to 500 jig ofstreptomycin per ml.

susceptible to fertility inhibition by the group IaR factor R64 (6). A slight but reproduciblelowering of transfer frequency for RP1 is alsonoted for these doubles. This may reflect areciprocal effect on fertility or, alternatively, anonspecific lowering of transfer frequency forRP1 caused by the mere presence of anotherplasmid. Further examples of this relationshpwill be cited later.To determine whether RP1 inhibition RWP1

transfer reflected the presence of P plasmidgenes linked to carbenicillin resistance or alter-natively reflected a property of the progenitorplasmid R388, we next did the matings shown inTable 5. It is evident from the data that RP1inhibits the transfer of W group plasmids R388or RSa. Accordingly, the susceptibility of RWP1transfer to RP1 inhibition shown in Table 4 isbelieved to be a W incompatibility group prop-erty. These data also show a diminution of RP1transfer associated, in this case, with the pres-ence in donor cells of R388. This effect for theother W group plasmid tested, RSa, is lesspronounced and within the variation of transferfrequency occurring on repeated trials of thesematings.P and W group plasmids have in common the

genetic information for the specification of thereceptor for the plasmid-dependent phagePRD1 (17). This is known to be a cell wallfunction. The cognate observation of Willets(24) that the synthesis of an entry exclusionprotein factor(s) was linked to fertility inhibi-tion in the fi+ system prompted us to considerthat RP1 might be inhibiting transfer regionfunctions in group W plasmids analogous to theF-like plasmids and F since both P plasmidsand W plasmids possess functionally equivalentinformation specifying a cell surface compo-nent. This model would propose that the W

TABLE 5. Effect of RPJ on Wgroup plasmid transfertoJ53SmR

Donor R+ recipient R+ recipients/selecteda donor

J53 (RP1) RP1 1 x 10-3J53 (R388) R388 2 x 10-3J53 (RP1/R388) RP1 4 x 10-4

R388 3 x 10-6J53 (RSa) RSa 2 x 10-'J53 (RP1/RSa) RP1 7 x 10-4

RSa 1 x 10-7a TN agar containing 500 Mg of streptomycin and

500 Ag carbenicillin per ml was used for the selectionof RP1 transfer. For R388 transfer, 100 Mg of trimetho-prim was substituted for carbenicillin. For RSa trans-fer, 30 ug of chloramphenicol per ml was used.

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group plasmid transfer region responds to regu-lation by a P plasmid gene product. To test thisproposal, using another group of plasmids un-related to P or W groups by incompatibilitycriteria, we constructed bacterial strains con-taining both RP1 and N group plasmids. The Ngroup plasmids also specify RPD1 phage sensi-tivity and thus might be expected to contain aportion of their transfer region functionallyrelated to W and P group plasmids. The resultsof these determinations are shown in Table 6. Inthese determinations, with N group plasmids,only RN3 apparently interacted with RP1. Fur-thermore, unlike RP1 inhibition of the W group,the transfer of RP1 was markedly inhibited inthis case. Therefore, the result provides anotherinstance of fertility inhibition effects by plas-mids unrelated on the basis of their incompati-bility group. The significance of whether a Pplasmid causes inhibition of another plasmid oris inhibited by another will be discussed later.The previous report of Pinney and Smith (19)

has shown the inhibition of an N group plasmidby the group X R factor R6K. Although in ourprevious report this plasmid did not encodeinformation for PRD1 phage susceptibility, weconsidered that it may, however, possess geneticinformation related to the regulation of transferfunction expression common to the P, W, and Nplasmid groups. Accordingly, we next tested theeffect of R6K on the W group plasmid R388(Table 7). The results clearly show inhibition ofR388 transfer by R6K. This observation, webelieve, provides additional evidence for thecommon origin of transfer functions among the

TABLE 6. Effect of RPJ onN group transfer to CR34a

Donor R+ recipient R+ recipients/selected donor"

J53 (RP1) RP1 2 x 10'-J53 (R15) R15 2 x 10-'J53 (RP1/R15) RP1 4 x 10-s

R15 1 x 10-4J53 (R46) R46 4 x 10-"J53 (RP1/R46) RP1 5 x 10-4

R46 2 x 10-J53 (RN3) RN3 5 x 10-4J53 (RP1/RN3) RP1 2 x 10-"

RN3 6 x 10-4a Nutritional selection against the donor was done

on supplemented minimal medium.Selection for RP1 transfer was as described in

Table 3. Streptomycin (25 gg/ml) was included insupplemented minimal medium to select for RN3,R46, or R15 transfer. The ampicillin resistance deter-minant of these plasmids does not specify resistanceto carbenicillin at 500 ,g/ml.

TABLE 7. R6K (group X) inhibition of R388 transfera

Donor R+ recipient R+ recipients/selected" donor

J53 (R6K) R6K 2 x 10-4J53 (R388) R388 1 x 10-aJ53 (R388/R6K-1)c R388 3 x 10-5

R6K 7 x 10-aJ53(R388/R6K-2)c R388 3 x 10-"

R6K 1 x 10-4

a CR34 was used as the recipient, and nutritionalselection against the donor was done on supplementedminimal medium.

'Minimal medium containing ampicillin (50 Ag/ml) was used for the selection of R6K. The samemedium containing 100 jig of trimethoprim per mlwas used for the selection of R388.

cDoubles derived from two different matings andpurifications.

N and now W plasmid groups, since representa-tives of both groups are affected by R6K.

Since previous observations in this study hadindicated a relationship among the P, W, and Ngroup plasmids extending beyond specificationof the RPD1 phage receptor to include interac-tion of gene products affecting fertility associ-ated with transfer functions, we completed thissurvey by determining the effect of the X groupplasmid R6K on P group plasmid RP1 (Table8). For this combination of plasmids, the dataindicate that the fertility inhibition effect maybe reciprocal, i.e., the transfer of both RP1 andR6K is inhibited. The inhibition of RP1 trans-fer, in this instance, resembles that seen inTables 4 and 5 for RWP1 or R388, respectively.These examples of partial reciprocal fertilityinhibition may reflect the reconstruction bycomplementation of a fertility regulation sys-tem from gene products encoded by both plas-mids. However, RP1 is less affected by thisreconstituted system than the other plasmidpresent, RWP1, R388, or R6K. With respect tothe effect of R6K on RP1 transfer, however, thisresult provides further indication of commonevolution of plasmid functions controlling fertil-ity in the absence of genetic information for thespecification of the PRD1 phage receptor.

Plasmid-dependent phage sensitivity. TheP group of plasmids specifies pili that allowinfection by phage PRR1 (18). One might ex-pect, then, that partial inhibition of fertility asobserved for the effect of RWP1, R388, or R6Kon RP1 might be accompanied by a decrease insensitivity to PRR1 phage. Reproducible quan-titative estimates of PRR1 adsorption by Pplasmid pili, then, would indicate the degree ofrepression of the P plasmid transfer region due

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TABiz 8. Reciprocal inhibition of RPJ and R6KtransferP

Donor R+ recipient R+ recipients/selected" donor

J53 (RP1) RP1 2 x 10-3J53 (R6K) R6K 2 x 10-4J53 (RP1/R6K-1)c RP1 2 x 10-4

R6K 6 x 10-6J53 (RPl/R6K-2)c RP1 2 x 10-4

R6K 7x 10-1

a CR34 was the recipient, and nutritional selectionagainst the donor was done on supplemented minimalmedium.

b Minimal medium containing 25 ,g of tetracyclineper ml was used to select for the transfer of RP1. Thesame medium containing 25 .ug of streptomycin per mlwas used to select for the transfer of R6K.

CDoubles derived from two different matings andpurifications.

to the presence of another plasmid. However,these quantitative estimations are hinderedwith the P plasmids, perhaps reflecting thefragility of pili or their uncommon occurrence.Therefore, to gain some preliminary estimate ofcompatible plasmid effects on PRR1 phagesensitivity, we compared the plating character-istics of PRR1 phage when spotted onto a solidmedium surface inoculated bacterial lawns pre-pared from cells containing only RP1 or RP1and the other plasmids studied in combinationwith RP1 cited in this report. In E. coli cellscontaining RP1, phage PRR1 normally showssmall, slightly turbid plaques when tested inthis way. However, in E. coli containing RP1and either RN3, RWP1, R388, or R6K doubles,both the number of plaques and their claritywere diminished corresponding to the degree offertility inhibition of RP1 reported in Tables 4,5, 6, and 8. This result was not observed forother plasmid combinations in this report. Ac-cordingly, these qualitative estimates ofchanges in the expression of a transfer regiondeterminant, we feel, support our interpreta-tions with regard to possible reciprocal fertilityinhibition in doubles, although the diminutionof RP1 fertility in some instances is only slightlygreater than variations in transfer frequencyobserved on repeated trials for donor cellscontaining only RP1 or RP1 and another plas-mid showing no fertility effects.

DISCUSSIONThe relatedness of P incompatibility group

plasmids to either group W or group N plasmidson the basis of their fertility inhibition interac-tions may have been observed previously. Data

in the report of Coetzee et al. (4) show that thefertility of a P group R factor, RP4, and a Wgroup R factor, RSa, is inhibited by an N groupplasmid, R390. Although these authors did notreport the donor frequency for RP4 or RSasingly maintained in donor bacteria, the fre-quencies reported for these plasmids in donorbacteria also containing R390 are low whencompared with those in this report for RSa orthe plasmid RP1.The model of Willetts (24) and Finnegan and

Willetts (7) for the activity of F transfer inhibi-tor is useful in the consideration of resultsreported here. Put simply, these authors viewfertility inhibition effects as resulting from theregulation of plasmid transfer region functions.This regulation is reported to involve the syn-thesis of a regulatory gene product, FIN, pro-duced by the fin gene which interacts withanother plasmid gene product designated tra Pproduct (7). This FIN/tra P gene product com-plex, in turn, inhibits the expression of anothertransfer region gene, tra J, which is required forthe expression of the other transfer region deter-minants including genes required for deoxyribo-nucleic acid mobilization, pilus synthesis, andentry exclusion. It follows from these considera-tions, then, that derepressed plasmids exhibit-ing high transfer, abundant pilus formation, orstringent entry exclusion may have mutationsin genes specifying FIN, tra P product, or tra Jproduct, making any one of these componentsunresponsive to another. It also follows fromthese considerations that the fertility systemmay be reconstructed by complementation oc-curring in bacteria containing derepressed plas-mids that differ in the identity of the mutantcistron responsible for their derepressed transferproperties. The simplest example of this com-plementation causing fertility inhibition wouldbe a combination of plasmids coexistent in thesame bacterial cell showing repressed transferfor both plasmids. An example resembling thispossibility is seen in this report for the behaviorof R6K/RP1 doubles. The more definitive exam-ples of fertility inhibition reported here, how-ever, show inhibition of only one donor strain.This situation could result from an insensitivetra J-like gene product for the noninhibitedplasmid analogous to the F-like plasmids thatinhibit the expression of F functions. The datain Table 6 indicate that the behavior of RN3may reflect this situation. In any case, plasmidsderepressed by virtue of having functionallyequivalent mutations in, for example, their finor tra J loci would not be expected to comple-ment each other, resulting in reciprocal inhibi-

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34 OLSEN AND SHIPLEY

tion of their fertility. This could be the case withthe N group plasmids R15 or R46 with respect toRP1. In the case of R46, an alternate explana-tion, a tra J-like mutation resulting in insensi-tivity to the FIN/tra P complex, is not likely inview of the prior report demonstrating theinhibition of R46 by the X group plasmid R6K(19). Accordingly, based on this surmise, it ispossible that the mutations resulting in thederepression of R46 and RP1 are functionallyequivalent and thus reflect the production ofeither a mutant fin gene product or a mutant traP gene product.The fertility inhibition studies of others

(7, 8, 14, 19, 21, 25) and ours may furtherfacilitate understanding of the evolution ofplasmids carrying drug resistance determinantswhen considered with other aspects of plasmidphysiology. First, the identity of drug re-sistance determinants per se is apparentlytrivial to fundamental distinctions among plas-mids. Numerous reports cite recombinationoccurring among plasmids resulting in the ex-change or addition of drug resistance determi-nants accompanied, in some instances, by dele-tion of progenitor plasmid determinants (i.e.,see references 1, 11, 12). Thus, recombinationallows for the exchange of drug resistance infor-mation among plasmids while otherwise main-taining progenitor incompatibility and/or pilusproperties. Second, the sub-classification ofF-like or I-like plasmids based on the sharing ofpili with identical phage specificities, althoughcompatible when simultaneously present in ahost bacterium, indicates that within two well-studied groups a pool of genetic information forgenes concerned with incompatibility functionsas well as a gene pool for pilus properties mayexist. It follows from this that plasmids occurwith distinctive properties as a result of particu-lar combinations of these pools. For this consid-eration, a gene pool would be defined as theminimum number of genes located contigu-ously on plasmid deoxyribonucleic acid thatwould result in the expression of a phenotypictrait. Third, fertility inhibition observed forparticular combinations of plasmids when co-existent in a bacterium suggests common par-entage of plasmids unrelated by previous cri-teria. This particular consideration focuses at-tention on the transfer functions comprisingplasmids. Our previous observation that thePRD1 phage receptor gene(s) was common toplasmid groups unrelated on the basis of in-compatibility or pilus properties is consistentwith the sharing of genetic traits regulatingfertility at the level of transfer functions,presuming the PRD1 receptor is a transfer

region gene. Our deduction that the PRD1receptor gene(s) is part of the transfer regionis supported by the unpublished observationthat spontaneous mutants of RP1 ii¶sensitiveto phage PRR1 or phage PRD1 are also trans-fer defective. Presumably these are polar muta-tions in the RP1 transfer region, and thesynthesis of the PRD1 receptor is thereforeinhibited along with the other transfer regiongenes.

In summary, reports to date concerning plas-mid physiology support the view that plasmidgene pools for drug resistance determinants;incompatibility functions, and transfer func-tions occasionally recombine leading to theformation of new plasmids with distinctiveproperties. Furthermore, the hypotheticaltransfer region gene pool may be divided intotwo subpools. One pool contains information forpilus composition and resulting phage specifici-ties, the second subpool contains genetic lociconcerned with cell surface properties and fer-tility. The occurrence of fi+ (with respect to F)plasmids bearing pili of the I incompatibilitygroup of plasmids would be an example of theformation of a plasmid with distinctive proper-ties drawn from gene pools for pilus and forfertility inhibition of transfer region determi-nants (25).

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicegrant AI-07533 from the National Institute of Allergy andInfectious Diseases. One of us, P.S., was supported by PublicHealth Service training grant 1-TO1-GM-02204 to the Depart-ment of Microbiology, The University of Michigan, from theNational Institute of General Medical Sciences.

LITERATURE CITED1. Anderson, E. S. 1974. Recombination between unrelated

bacterial plasmids. Ann. Microbiol. 125A:251-259.2. Anderson, E. S., and E. Natkin. 1972. Transduction of

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