Vol. 123, No. 1 Printed in U.S.A.
JOURNAL
OF BACrERIOLOGY, July 1975, p. 28-35 Copyright 0 1975 American Society for Microbiology
RP1 Properties and Fertility Inhibition Among P, N, W, and X Incompatibility Group Plasmids RONALD 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 properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range. Plasmids unable to coexist stably in the same host are considered to comprise an incompatibility group (4, 15). The P incompatibility group presently includes RP1 (9), R751 (13), R702, and R906 (received from N. Datta). These plasmids have a broad host range among the gram-negative bacteria (3, 6, 16; unpublished data). They are also lysed by the P plasmidspecific ribonucleic acid phage PRR1 (18). In addition to sharing of phage susceptibility, the P group of plasmids also shows entry exclusion within itself. Another surface component determined by P plasmids as well as the N and W incompatibility groups is the PRD1 phage receptor (17). Thus, in addition to drug resistance determinants, the P plasmids encode genetic information for incompatibility, pili specific for phage PRR1, entry exclusion determininants, and the receptor for the cell wall-adsorbing phage PRD1. The latter characteristic, however, is not unique to the P plasmids. Another characteristic associated with some plasmids is fertility inhibition, an interaction
among compatible plasmids whereby the donor frequency of one or both resident plasmids is diminished. We have tested some of the P plasmids with respect to this characteristic. Some R factors were originally designated fi+ based on their ability to inhibit conjugation by the Escherichia coli sex factor F (23). Most of these plasmids initially studied were F-like in their properties and encoded an F-like pilus (15). Later, a plasmid specifying I-like pili also was shown to inhibit F (8, 21). However, in this instance, the mechanism of F inhibition by the I-like plasmid R62 differed in its properties from that associated with F inhibition by F-like plasmids (14). Recently, in addition to the foregoing, two group X R factors have been shown to be fi+ with respect to an Fl group R factor, R386 (10). Furthermore, R46, a group N plasmid, has been shown to be susceptible to repression by a group X R factor, R6K (6). Thus, it is becoming increasingly apparent that fertility inhibition of one plasmid by another is not confined to plasmid groupings based on 28
VL VOL. 123, 1975 7RP1PROPERTIES AND FERTILITY INHIBITION
incompatibility relationships (5, 15) or sex pilus properties. We report here further examples of plasmid relationships based on fertility inhibition among plasmids unrelated on the basis of their incompatibility or pilus properties. In some of these instances, the plasmids share in common the genetic information for encoding of the PRD1 phage (17) receptor site. Thus, as suggested previously (17), these fertility inhibition relationships may provide additional evidence for the common evolution of plasmids seemingly unrelated by other criteria. This investigation was initiated in an attempt to gain a preliminary estimate of linkage relationships for RP1 plasmid determinants. For this, we used a derivative of RP1, designated RP1-S2, which was obtained by transductional shortening of RP1 using phage P22 (22). A recombinant between RP1-S2 and a W group drug resistance plasmid designated RWP1 also was subsequently studied to determine the extent of RP1-S2 gene incorporation. The results of these studies and the subsequent observation of fertility inhibition relationships among plasmid groups are described in this report. MATERIALS AND METHODS Bacterial strains. Bacteria used and the relevant properties of their plasmids are listed in Table 1. These plasmid-containing strains have been used by us previously for the determination of RP1 host range (16) and plasmid specificity of phages infecting P incompatibility group plasmids (17, 18). Media. Complex medium TN and minimal medium VBG were prepared as described previously (16). Antibiotic supplements are as described in the tables. Mating and testing of exconjugants. R+ strains subsequently used for quantitative estimates of transfer frequencies were constructed by mixed inoculation of saline-glucose buffer (NaCl, 0.85%; glucose, 0.36%; pH 7.0) with growth from TN agar containing the antibiotic selective for one of the pertinent resistance determinants specified by the plasmid. Donor and recipient bacterial suspensions were incubated at 37 C for 1 h, and an aliquot was then plated on the appropriately supplemented medium. Exconjugants were purified by serial clone isolation on TN agar containing the appropriate antibiotic. When doubles, i.e., bacteria containing two plasmids, were used as donors for mating experiments, they were grown as above on the medium containing the antibiotic appropriate to the maintenance of both drug resistance factors. Matings done to determine entry exclusion or fertility inhibition of one plasmid by another were done in TN broth medium. For this, TN broth medium was inoculated with growth from TN agar medium containing the selective antibiotic. On the average, these TN broth cultures were incubated for
29
TABLE 1. Plasmid incompatibility group and resistance determinants Plasmida
RP1 R751 R702 R906 RP1-S2c R388 RSa RWPld R15 R46 RN3 R6K
Incompatibility group
Resistance determinantsb
P P P P P W W W N N N X
CbR, TcR, NmR/KmR TpR SmR, TcR, KmR, SuR SmR, ApR, SuR CbR, TcR TpR, SuR SmR, CmR, SuR, KmR CbR, TpR SmR, ApR, TcR, SuR SmR, ApR, TcR, SuR SmR, ApR, TcR, SuR ApR, SmR
a All plasmids used in this study were maintained in E. coli J53 or E. coli CR34. ° The abbreviations designate resistance to the following antibiotics: ApR, ampicillin; CbR, carbenicillin; CmR, chloramphenicol; KmR, kanamycin; NmR, neomycin; SmR, streptomycin; SuR, sulfonilamide; TcR, tetracycline; TpR, trimethoprim. c Derived from RP1 in Salmonella typhimurium LT2 using phage P22. See reference 22. d Recombinant plasmid, R388 x RP1-S2.
approximately 2 h at 37 C with agitation. Inoculations were adjusted to result in approximately 108 cells per ml of TN broth after 2 h of growth at 37 C. For mating, donor and recipient cultures were mixed 1:1 and 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, and plated directly. If supplemented VBG agar medium was used for selection, mating mixtures were centrifuged at ambient temperature and cell pellets were suspended to volume in 0.01 M phosphate buffer (pH 7.0). Plates were incubated for 48 h at 37 C for quantitative estimates of mating frequencies.
RESULTS Previous attempts in our laboratory to determine linkage between RP1 determinants by using the Escherichia coli transducing phage P1 have been unsuccessful. The entire RP1 plasmid was always transduced. However, the availability of the RP1 derivative RP1-S2 (22) allowed for the investigation of linkage between RP1 determinants concerned with surface exclusion and incompatibility with respect to their affect on other P group plasmids. The entry exclusion and incompatibility properties of RP1 and its derivative, RP1-S2, in relation to the other P group plasmids are shown in Table 2. It is clear from these data that RP1 excludes the entry of the related plasmids R751, R702, and R906. However, with RP1-S2 as the resident
30
J. BACTERIOL.
OLSEN AND SHIPLEY
TABLE 2. Entry exclusion and incompatibility properties of RPI or RP1-S2 in relation to other P group plasmids Donor
Recipienta
P group R+ recipients/donorb
J53 (R751)
CR34 CR34 (RP1) CR34 (RP1-S2)
2 x 10-2 1 x 10-6 2 x 10-3
J53 (R702)
CR34 CR34 (RP1) CR34 (RP1-S2)
3 x 10-3 3 x 10- 5 3 x 10-'
J53 (R906)
CR34 CR34 (RP1) CR34 (RP1-S2)
4 x 10- 3 1 x 10-6 2 x 10-
a Nutritional selection against the donor was done on appropriately supplemented minimal medium used previously (16). Trimethoprim (100 gg/ml) or streptomycin (25 ug/ml) was included in minimal medium to select for the transfer of R751 or R702 and R906, respectively.
plasmid in recipient bacteria, this exclusion, as indicated by a low frequency of exconjugant formation, is not apparent. Thus, RP1-S2 has lost genetic information specifying the entry exclusion properties common to P group plasmids in addition to the deletion of genes specifying P plasmid pili and the cell surface receptor for phage PRD1 noted previously (22). It is interesting to note that both entry exclusion and pilus genes,are associated with the transfer region of F and F-like plasmids and that the same linkage relationship apparently is obtained with RP1. From this we conclude that RP1 transfer region genes are linked to determinants for neomycin/kanamycin resistance. However, when exconjugants from the above matings are purified by serial restreaking on medium not selective for the P group plasmid originally present in the recipient, it is observed upon subsequent testing that they have lost RP1 or RP1-S2. Therefore, RP1-S2 has retained genetic information determining susceptibility to incompatibility by a related plasmid. We designate this phenotypic trait sinp. Thus, the matings (Table 2) with CR34(RP1-S2) recipients resulted in the detection of RP1-S2 susceptibility to incompatibility caused by entering P group plasmids. We next verified the ability of RP1-S2 to cure a resident P plasmid by transducing RP1-S2 into bacteria containing R702 or R906. For this, P1 phage was grown on E. coli J53(RP1-S2) and harvested as reported previously for P22 transduction (22). These transducing phage lysates transduced RP1-S2 into either
J53(R702) or to J53(R906) at a frequency of approximately 2 x 10- 6 per viable phage. When the transductants selected for carbenicillin or tetracycline resistance were purified on medium selective for RP1-S2 and individual colonies were tested, all were observed to have lost the P plasmid (R702 or R906) originally present in the recipient bacteria (20/20 tested). We have designated this trait incp, analogous to the phenotype designated for F-like plasmids (7). INCp (capital letters used for a gene product) then, is presumably a P plasmid gene product responsible for the loss of related plasmids containing the sinp locus maintained in bacteria grown under nonselected conditions with respect to the lost plasmid. Consequently, these studies have indicated that RP1-S2 has retained the P plasmid genes concerned with incompatibility as well as genes specifying resistance to carbenicillin and tetracycline. Accordingly, these characteristics constitute a linkage group in the RP1 plasmid. Properties of a P-W group recombinant plasmid. We reported the construction of a recombinant plasmid formed by the addition to the W group plasmid R388 (22) of carbenicillin resistance specified by the P group plasmid RP1. The recombinant plasmid, designated RWP1, was tested and found to belong to the W incompatibility group. We also determined whether RWP1 contained genetic information specifying incompatibility with P group plasmids (Table 3). The results show that the donor frequencies of the P plasmids are not affected by the presence of RWP1 in the recipient bacteria. This is also the finding for recipients containing R388, the parent of RWP1 (unpublished data). When exconjugants from the matings shown in Table 3 were purified by serial colony isolation on the agar medium containing an 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 carbenicillin resistance into R388 to form RWP1 resulted in TABLE 3. Transfer of P plasmids to CR34 (R WPl)a Donor
Recipient
J53 (R702) J53 (R906) J53 (RP1)
CR34 (RWP1)
P group R+
recipients/donor
1 x 10-9 3 x 10-4 1 x 10-3
a Selection for the transfer of RP1 was on minimal medium containing 500 Atg of carbenicillin per ml. Selection for the transfer of R702 and R906 was as described in Table 2.
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RP1 PROPERTIES AND FERTILITY INHIBITION
the loss of the P plasmid incompatibility function(s). Similarly, in the reverse situation, when the recipient bacteria contained the P plasmids used in Table 3 and the donor contained RWP1, exconjugants stably maintained both RWP1 and the P plasmid under nonselective conditions of growth. Fertility inhibition studies. We next tested RWP1 for the expression of genes affecting P plasmid fertility. We considered that genes possibly affecting P plasmid fertility may be linked to carbenicillin resistance in RP1 and hence, in some instances, would be retained when recombination with R388 occurred. This determination could not be done reproducibly in bacteria containing two P plasmids, perhaps reflecting the stringent incompatibility occurring among P plasmids. However, RWP1 is compatible with P plasmids, and hence "doubles" (bacteria containing RWP1 and a P plasmid) could be tested for the effect of RWP1 on P plasmids or the reverse with regard to donor frequency. The results of these determinations (Table 4) clearly show inhibition of RWP1 transfer by bacteria containing either of the P plasmids RP1 or R702. This unexpected result resembles fertility inhibition of F-mediated conjugation by the presence of fi+ F-like plasmids (24) and also the later reports for several other plasmid combinations (10, 19, 25). Indeed, the P plasmid RP4 has been shown TABLE 4. Mating of RWPl-P plasmid doubles with J53SmR Donora
J53 (RWP1) J53 (RP1) J53 (RP1/RWP1) J53 (RWP1/RP1) J53 (R702) J53 (R702/RWP1) J53 (RWP1/R702) a In
R+selectedb recipient RWP1 RP1 RP1 RWP1 RP1 RWP1 R702 R702 RWP1 R702 RWP1
R+ recipients/ donor 5x 2x 3x 1x 3x 2x 2x 7x 2x 8x 9x
10-3 10-3
10-' 10-6 10-4 10-6 10-3 10-4 10-5 10-4 10-6
the case of doubles, the first plasmid listed was added to bacterial recipients containing the second plasmid listed. For this strain construction, R+ CR34 donors were used. "TN agar containing 500 Ag of streptomycin and 100 ug of trimethoprim per ml was used to select for the acquisition of RWP1. For the selection of either RP1 or R702, 20 jg of tetracycline per ml was included in TN agar in addition to 500 ug of streptomycin per ml. J53SmR is chromosomally resistant to 500 jig of streptomycin per ml.
31
susceptible to fertility inhibition by the group Ia R factor R64 (6). A slight but reproducible lowering of transfer frequency for RP1 is also noted for these doubles. This may reflect a reciprocal effect on fertility or, alternatively, a nonspecific lowering of transfer frequency for RP1 caused by the mere presence of another plasmid. Further examples of this relationshp will be cited later. To determine whether RP1 inhibition RWP1 transfer reflected the presence of P plasmid genes linked to carbenicillin resistance or alternatively reflected a property of the progenitor plasmid R388, we next did the matings shown in Table 5. It is evident from the data that RP1 inhibits the transfer of W group plasmids R388 or RSa. Accordingly, the susceptibility of RWP1 transfer to RP1 inhibition shown in Table 4 is believed to be a W incompatibility group property. These data also show a diminution of RP1 transfer associated, in this case, with the presence in donor cells of R388. This effect for the other W group plasmid tested, RSa, is less pronounced and within the variation of transfer frequency occurring on repeated trials of these
matings. P and W group plasmids have in common the genetic information for the specification of the receptor for the plasmid-dependent phage PRD1 (17). This is known to be a cell wall function. The cognate observation of Willets (24) that the synthesis of an entry exclusion protein factor(s) was linked to fertility inhibition in the fi+ system prompted us to consider that RP1 might be inhibiting transfer region functions in group W plasmids analogous to the F-like plasmids and F since both P plasmids and W plasmids possess functionally equivalent information specifying a cell surface component. This model would propose that the W TABLE 5. Effect of RPJ on Wgroup plasmid transfer toJ53SmR Donor
J53 (RP1) J53 (R388) J53 (RP1/R388) J53 (RSa) J53 (RP1/RSa)
R+selecteda recipient RP1 R388 RP1 R388 RSa RP1 RSa
R+ recipients/ donor 1x 2x 4x 3x 2x 7x 1x
Mg
10-3 10-3 10-4 10-6
10-' 10-4 10-7
a TN agar containing 500 of streptomycin and 500 Ag carbenicillin per ml was used for the selection of RP1 transfer. For R388 transfer, 100 Mg of trimethoprim was substituted for carbenicillin. For RSa transfer, 30 ug of chloramphenicol per ml was used.
32
OLSEN AND SHIPLEY
J. BACTERIOL.
plasmid transfer region responds to regulation by a P plasmid gene product. To test this proposal, using another group of plasmids unrelated to P or W groups by incompatibility criteria, we constructed bacterial strains containing both RP1 and N group plasmids. The N group plasmids also specify RPD1 phage sensitivity and thus might be expected to contain a portion of their transfer region functionally related to W and P group plasmids. The results of these determinations are shown in Table 6. In these determinations, with N group plasmids, only RN3 apparently interacted with RP1. Furthermore, unlike RP1 inhibition of the W group, the transfer of RP1 was markedly inhibited in this case. Therefore, the result provides another instance of fertility inhibition effects by plasmids unrelated on the basis of their incompatibility group. The significance of whether a P plasmid causes inhibition of another plasmid or is inhibited by another will be discussed later. The previous report of Pinney and Smith (19) has shown the inhibition of an N group plasmid by the group X R factor R6K. Although in our previous report this plasmid did not encode information for PRD1 phage susceptibility, we considered that it may, however, possess genetic information related to the regulation of transfer function expression common to the P, W, and N plasmid groups. Accordingly, we next tested the effect of R6K on the W group plasmid R388 (Table 7). The results clearly show inhibition of R388 transfer by R6K. This observation, we believe, provides additional evidence for the common origin of transfer functions among the group
TABLE 6. Effect of RPJ Donor
J53 (RP1) J53 (R15) J53 (RP1/R15) J53 (R46) J53 (RP1/R46)
J53 (RN3) J53 (RP1/RN3)
on N group
R+
recipient
selected
RP1 R15 RP1
R15 R46 RP1 R46 RN3 RP1 RN3
transfer to CR34a R+
recipients/ donor"
2 x 10'2 x 10-' 4 x 10-s 1 x 10-4 4 x 10-" 5 x 10-4 2 x 105x
10-4
2
10-" 10-4
6
x x
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 in supplemented minimal medium to select for RN3, R46, or R15 transfer. The ampicillin resistance determinant of these plasmids does not specify resistance to carbenicillin at 500 ,g/ml. a
TABLE 7. R6K (group X) inhibition of R388 transfera Donor
J53 (R6K) J53 (R388) J53 (R388/R6K-1)c
J53(R388/R6K-2)c
R+ recipient
R+ recipients/
R6K R388 R388 R6K R388 R6K
2 x 10-4 1 x 10-a 3 x 10-5 7 x 10-a 3 x 10-" 1 x 10-4
selected"
donor
a CR34 was used as the recipient, and nutritional selection against the donor was done on supplemented minimal medium. 'Minimal medium containing ampicillin (50 Ag/ ml) was used for the selection of R6K. The same medium containing 100 jig of trimethoprim per ml was used for the selection of R388. cDoubles derived from two different matings and
purifications.
N and now W plasmid groups, since representatives of both groups are affected by R6K. Since previous observations in this study had indicated a relationship among the P, W, and N group plasmids extending beyond specification of the RPD1 phage receptor to include interaction of gene products affecting fertility associated with transfer functions, we completed this survey by determining the effect of the X group plasmid R6K on P group plasmid RP1 (Table 8). For this combination of plasmids, the data indicate that the fertility inhibition effect may be reciprocal, i.e., the transfer of both RP1 and R6K is inhibited. The inhibition of RP1 transfer, in this instance, resembles that seen in Tables 4 and 5 for RWP1 or R388, respectively. These examples of partial reciprocal fertility inhibition may reflect the reconstruction by complementation of a fertility regulation system from gene products encoded by both plasmids. However, RP1 is less affected by this reconstituted system than the other plasmid present, RWP1, R388, or R6K. With respect to the effect of R6K on RP1 transfer, however, this result provides further indication of common evolution of plasmid functions controlling fertility in the absence of genetic information for the specification of the PRD1 phage receptor. Plasmid-dependent phage sensitivity. The P group of plasmids specifies pili that allow infection by phage PRR1 (18). One might expect, then, that partial inhibition of fertility as observed for the effect of RWP1, R388, or R6K on RP1 might be accompanied by a decrease in sensitivity to PRR1 phage. Reproducible quantitative estimates of PRR1 adsorption by P plasmid pili, then, would indicate the degree of repression of the P plasmid transfer region due
RP1 PROPERTIES AND FERTILITY INHIBITION
VOL. 123, 1975
TABiz 8. Reciprocal inhibition of RPJ and R6K transferP Donor
J53 (RP1) J53 (R6K) J53 (RP1/R6K-1)c J53 (RPl/R6K-2)c
R+selected" recipient RP1
R6K RP1 R6K RP1 R6K
R+ recipients/ donor 2x 2x 2x 6x 2x 7x
10-3 10-4 10-4 10-6 10-4 10-1
a CR34 was the recipient, and nutritional selection against the donor was done on supplemented minimal medium. b Minimal medium containing 25 ,g of tetracycline per ml was used to select for the transfer of RP1. The same medium containing 25 .ug of streptomycin per ml was used to select for the transfer of R6K. C Doubles derived from two different matings and purifications.
to the presence of another plasmid. However, these quantitative estimations are hindered with the P plasmids, perhaps reflecting the fragility of pili or their uncommon occurrence. Therefore, to gain some preliminary estimate of compatible plasmid effects on PRR1 phage sensitivity, we compared the plating characteristics of PRR1 phage when spotted onto a solid medium surface inoculated bacterial lawns prepared from cells containing only RP1 or RP1 and the other plasmids studied in combination with RP1 cited in this report. In E. coli cells containing RP1, phage PRR1 normally shows small, slightly turbid plaques when tested in this way. However, in E. coli containing RP1 and either RN3, RWP1, R388, or R6K doubles, both the number of plaques and their clarity were diminished corresponding to the degree of fertility inhibition of RP1 reported in Tables 4, 5, 6, and 8. This result was not observed for other plasmid combinations in this report. Accordingly, these qualitative estimates of changes in the expression of a transfer region determinant, we feel, support our interpretations with regard to possible reciprocal fertility inhibition in doubles, although the diminution of RP1 fertility in some instances is only slightly greater than variations in transfer frequency observed on repeated trials for donor cells containing only RP1 or RP1 and another plasmid showing no fertility effects.
DISCUSSION The relatedness of P incompatibility group plasmids to either group W or group N plasmids on the basis of their fertility inhibition interactions may have been observed previously. Data
33
in the report of Coetzee et al. (4) show that the fertility of a P group R factor, RP4, and a W group R factor, RSa, is inhibited by an N group plasmid, R390. Although these authors did not report the donor frequency for RP4 or RSa singly maintained in donor bacteria, the frequencies reported for these plasmids in donor bacteria also containing R390 are low when compared with those in this report for RSa or the plasmid RP1. The model of Willetts (24) and Finnegan and Willetts (7) for the activity of F transfer inhibitor is useful in the consideration of results reported here. Put simply, these authors view fertility inhibition effects as resulting from the regulation of plasmid transfer region functions. This regulation is reported to involve the synthesis of a regulatory gene product, FIN, produced by the fin gene which interacts with another plasmid gene product designated tra P product (7). This FIN/tra P gene product complex, in turn, inhibits the expression of another transfer region gene, tra J, which is required for the expression of the other transfer region determinants including genes required for deoxyribonucleic acid mobilization, pilus synthesis, and entry exclusion. It follows from these considerations, then, that derepressed plasmids exhibiting high transfer, abundant pilus formation, or stringent entry exclusion may have mutations in genes specifying FIN, tra P product, or tra J product, making any one of these components unresponsive to another. It also follows from these considerations that the fertility system may be reconstructed by complementation occurring in bacteria containing derepressed plasmids that differ in the identity of the mutant cistron responsible for their derepressed transfer properties. The simplest example of this complementation causing fertility inhibition would be a combination of plasmids coexistent in the same bacterial cell showing repressed transfer for both plasmids. An example resembling this possibility is seen in this report for the behavior of R6K/RP1 doubles. The more definitive examples of fertility inhibition reported here, however, show inhibition of only one donor strain. This situation could result from an insensitive tra J-like gene product for the noninhibited plasmid analogous to the F-like plasmids that inhibit the expression of F functions. The data in Table 6 indicate that the behavior of RN3 may reflect this situation. In any case, plasmids derepressed by virtue of having functionally equivalent mutations in, for example, their fin or tra J loci would not be expected to complement each other, resulting in reciprocal inhibi-
34
OLSEN AND SHIPLEY
tion of their fertility. This could be the case with the N group plasmids R15 or R46 with respect to RP1. In the case of R46, an alternate explanation, a tra J-like mutation resulting in insensitivity to the FIN/tra P complex, is not likely in view of the prior report demonstrating the inhibition of R46 by the X group plasmid R6K (19). Accordingly, based on this surmise, it is possible that the mutations resulting in the derepression of R46 and RP1 are functionally equivalent and thus reflect the production of either a mutant fin gene product or a mutant tra P gene product. The fertility inhibition studies of others (7, 8, 14, 19, 21, 25) and ours may further facilitate understanding of the evolution of plasmids carrying drug resistance determinants when considered with other aspects of plasmid physiology. First, the identity of drug resistance determinants per se is apparently trivial to fundamental distinctions among plasmids. Numerous reports cite recombination occurring among plasmids resulting in the exchange or addition of drug resistance determinants accompanied, in some instances, by deletion of progenitor plasmid determinants (i.e., see references 1, 11, 12). Thus, recombination allows for the exchange of drug resistance information among plasmids while otherwise maintaining progenitor incompatibility and/or pilus properties. Second, the sub-classification of F-like or I-like plasmids based on the sharing of pili with identical phage specificities, although compatible when simultaneously present in a host bacterium, indicates that within two wellstudied groups a pool of genetic information for genes concerned with incompatibility functions as well as a gene pool for pilus properties may exist. It follows from this that plasmids occur with distinctive properties as a result of particular combinations of these pools. For this consideration, a gene pool would be defined as the minimum number of genes located contiguously on plasmid deoxyribonucleic acid that would result in the expression of a phenotypic trait. Third, fertility inhibition observed for particular combinations of plasmids when coexistent in a bacterium suggests common parentage of plasmids unrelated by previous criteria. This particular consideration focuses attention on the transfer functions comprising plasmids. Our previous observation that the PRD1 phage receptor gene(s) was common to plasmid groups unrelated on the basis of incompatibility or pilus properties is consistent with the sharing of genetic traits regulating fertility at the level of transfer functions, presuming the PRD1 receptor is a transfer
J. BACTERIOL.
region gene. Our deduction that the PRD1 receptor gene(s) is part of the transfer region is supported by the unpublished observation that spontaneous mutants of RP1 ii¶sensitive to phage PRR1 or phage PRD1 are also transfer defective. Presumably these are polar mutations in the RP1 transfer region, and the synthesis of the PRD1 receptor is therefore inhibited along with the other transfer region genes. In summary, reports to date concerning plasmid physiology support the view that plasmid gene pools for drug resistance determinants; incompatibility functions, and transfer functions occasionally recombine leading to the formation of new plasmids with distinctive properties. Furthermore, the hypothetical transfer region gene pool may be divided into two subpools. One pool contains information for pilus composition and resulting phage specificities, the second subpool contains genetic loci concerned with cell surface properties and fertility. The occurrence of fi+ (with respect to F) plasmids bearing pili of the I incompatibility group of plasmids would be an example of the formation of a plasmid with distinctive properties drawn from gene pools for pilus and for fertility inhibition of transfer region determinants (25). ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-07533 from the National Institute of Allergy and Infectious Diseases. One of us, P.S., was supported by Public Health Service training grant 1-TO1-GM-02204 to the Department of Microbiology, The University of Michigan, from the National Institute of General Medical Sciences. LITERATURE CITED 1. Anderson, E. S. 1974. Recombination between unrelated
bacterial plasmids. Ann. Microbiol. 125A:251-259. 2. Anderson, E. S., and E. Natkin. 1972. Transduction of
3. 4.
5. 6. 7.
8. 9.
resistance determinants and R factors of the A transfer systems by phage PlkC. Mg. Gen. Genet. 114:261-265. Bryan, L. E., H. M. Van Den Elzen, and J. T. Tseng. 1972. Transferable drug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1:22-29. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552. Datta, N., and R. W. Hedges. 1971. Compatibility groups among fi- R factors. Nature (London) 234:222-223. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. Finnegan, D., and N. Willets. 1973. The site of action of F transfer inhibitor. Mol. Gen. Genet. 127:307-316. Grindley, J. N., and E. S. Anderson. 1971. I-like resistance factors with the fi+ character. Genet. Res. 17:267-271. Grinsted, J., R. Saunders, L. C. Ingram, R. B. Sykes, and M. H. Richmond. 1972. Properties of an R factor which originated in Pseudomonas aeruginosa J. Bacteriol. 108:1244-1249.
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10. Hedges, R. W., N. Datta, J. N. Coetzee, and S. Dennison. 1973. R factors from Proteus morganii. J. Gen. Microbiol. 77:249-259. 11. Hedges, R. W., and A. E. Jacob. 1974. Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132:31-40. 12. Ingram, L. C., J. 0. Anderson, J. E. Arrand, and M. H. Richmond. 1974. A probable example of R-factor recombination in the human gastrointestinal tract. J. Med. Microbiol. 7:251-257. 13. Jobanputra, R. S., and N. Datta. 1974. Trimethoprim R factors in enterobacteria from clinical specimens. J. Med. Microbiol. 7:169-177. 14. Meynell, E. 1973. Pseudo-fi+ I-like sex factor R62(I), selective for increased pilus synthesis. J. Bacteriol. 113:502-503. 15. Meynell, E., G. G. Meynell, and N. Datta. 1968. Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacteriol. Rev. 32: 55-83. 16. Olsen, R. H., and P. Shipley. 1973. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J. Bacteriol. 113:772-780. 17. Olsen, R. H., J. Siak, and R. Gray. 1974. Characteristics of PRD1, a plasmid-dependent broad host range DNA
35
bacteriophage. J. Virol. 14:689-699. 18. Olsen, R. H., and D. D. Thomas. 1973. Characteristics and purification of PRR1, an RNA phage specific for the broad host range Pseudomonas R1822 drug resistance plasmid. J. Virol. 12:1560-1567. 19. Pinney, R. H., and J. T. Smith. 1974. Fertility inhibition of an N group R factor by a group X R factor, R6K. J. Gen. Microbiol. 82:415-418. 20. Richmond, M. H., and R. B. Sykes. 1972. The chromosomal integration of a ,B-lactamase gene derived from P-type R factor RP1 in Escherichia coli. Genet. Res. 20:231-237. 21. Romero, E., and E. Meynell. 1969. Covert fi- R factors with the fi+ character. J. Bacteriol. 97:780-786. 22. Shipley, P., and R. H. Olsen. 1975. Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1. J. Bacteriol. 123:20-27. 23. Watanabe, R. 1963. infective heredity of multiple drug resistance in bacteria. Bacteriol. Rev. 27:87-115. 24. Willetts, N. 1972. The genetics of transmissible plasmids. Annu. Rev. Genet. 6:257-268. 25. Willetts, N., and W. Paranchych. 1974. Inhibition of Flac transfer by the Fin+ I-like plasmid R62. J. Bacteriol. 120:101-105.
JOURNAL
OF BACrERIOLOGY, July 1975, p. 28-35 Copyright 0 1975 American Society for Microbiology
RP1 Properties and Fertility Inhibition Among P, N, W, and X Incompatibility Group Plasmids RONALD 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 properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range. Plasmids unable to coexist stably in the same host are considered to comprise an incompatibility group (4, 15). The P incompatibility group presently includes RP1 (9), R751 (13), R702, and R906 (received from N. Datta). These plasmids have a broad host range among the gram-negative bacteria (3, 6, 16; unpublished data). They are also lysed by the P plasmidspecific ribonucleic acid phage PRR1 (18). In addition to sharing of phage susceptibility, the P group of plasmids also shows entry exclusion within itself. Another surface component determined by P plasmids as well as the N and W incompatibility groups is the PRD1 phage receptor (17). Thus, in addition to drug resistance determinants, the P plasmids encode genetic information for incompatibility, pili specific for phage PRR1, entry exclusion determininants, and the receptor for the cell wall-adsorbing phage PRD1. The latter characteristic, however, is not unique to the P plasmids. Another characteristic associated with some plasmids is fertility inhibition, an interaction
among compatible plasmids whereby the donor frequency of one or both resident plasmids is diminished. We have tested some of the P plasmids with respect to this characteristic. Some R factors were originally designated fi+ based on their ability to inhibit conjugation by the Escherichia coli sex factor F (23). Most of these plasmids initially studied were F-like in their properties and encoded an F-like pilus (15). Later, a plasmid specifying I-like pili also was shown to inhibit F (8, 21). However, in this instance, the mechanism of F inhibition by the I-like plasmid R62 differed in its properties from that associated with F inhibition by F-like plasmids (14). Recently, in addition to the foregoing, two group X R factors have been shown to be fi+ with respect to an Fl group R factor, R386 (10). Furthermore, R46, a group N plasmid, has been shown to be susceptible to repression by a group X R factor, R6K (6). Thus, it is becoming increasingly apparent that fertility inhibition of one plasmid by another is not confined to plasmid groupings based on 28
VL VOL. 123, 1975 7RP1PROPERTIES AND FERTILITY INHIBITION
incompatibility relationships (5, 15) or sex pilus properties. We report here further examples of plasmid relationships based on fertility inhibition among plasmids unrelated on the basis of their incompatibility or pilus properties. In some of these instances, the plasmids share in common the genetic information for encoding of the PRD1 phage (17) receptor site. Thus, as suggested previously (17), these fertility inhibition relationships may provide additional evidence for the common evolution of plasmids seemingly unrelated by other criteria. This investigation was initiated in an attempt to gain a preliminary estimate of linkage relationships for RP1 plasmid determinants. For this, we used a derivative of RP1, designated RP1-S2, which was obtained by transductional shortening of RP1 using phage P22 (22). A recombinant between RP1-S2 and a W group drug resistance plasmid designated RWP1 also was subsequently studied to determine the extent of RP1-S2 gene incorporation. The results of these studies and the subsequent observation of fertility inhibition relationships among plasmid groups are described in this report. MATERIALS AND METHODS Bacterial strains. Bacteria used and the relevant properties of their plasmids are listed in Table 1. These plasmid-containing strains have been used by us previously for the determination of RP1 host range (16) and plasmid specificity of phages infecting P incompatibility group plasmids (17, 18). Media. Complex medium TN and minimal medium VBG were prepared as described previously (16). Antibiotic supplements are as described in the tables. Mating and testing of exconjugants. R+ strains subsequently used for quantitative estimates of transfer frequencies were constructed by mixed inoculation of saline-glucose buffer (NaCl, 0.85%; glucose, 0.36%; pH 7.0) with growth from TN agar containing the antibiotic selective for one of the pertinent resistance determinants specified by the plasmid. Donor and recipient bacterial suspensions were incubated at 37 C for 1 h, and an aliquot was then plated on the appropriately supplemented medium. Exconjugants were purified by serial clone isolation on TN agar containing the appropriate antibiotic. When doubles, i.e., bacteria containing two plasmids, were used as donors for mating experiments, they were grown as above on the medium containing the antibiotic appropriate to the maintenance of both drug resistance factors. Matings done to determine entry exclusion or fertility inhibition of one plasmid by another were done in TN broth medium. For this, TN broth medium was inoculated with growth from TN agar medium containing the selective antibiotic. On the average, these TN broth cultures were incubated for
29
TABLE 1. Plasmid incompatibility group and resistance determinants Plasmida
RP1 R751 R702 R906 RP1-S2c R388 RSa RWPld R15 R46 RN3 R6K
Incompatibility group
Resistance determinantsb
P P P P P W W W N N N X
CbR, TcR, NmR/KmR TpR SmR, TcR, KmR, SuR SmR, ApR, SuR CbR, TcR TpR, SuR SmR, CmR, SuR, KmR CbR, TpR SmR, ApR, TcR, SuR SmR, ApR, TcR, SuR SmR, ApR, TcR, SuR ApR, SmR
a All plasmids used in this study were maintained in E. coli J53 or E. coli CR34. ° The abbreviations designate resistance to the following antibiotics: ApR, ampicillin; CbR, carbenicillin; CmR, chloramphenicol; KmR, kanamycin; NmR, neomycin; SmR, streptomycin; SuR, sulfonilamide; TcR, tetracycline; TpR, trimethoprim. c Derived from RP1 in Salmonella typhimurium LT2 using phage P22. See reference 22. d Recombinant plasmid, R388 x RP1-S2.
approximately 2 h at 37 C with agitation. Inoculations were adjusted to result in approximately 108 cells per ml of TN broth after 2 h of growth at 37 C. For mating, donor and recipient cultures were mixed 1:1 and 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, and plated directly. If supplemented VBG agar medium was used for selection, mating mixtures were centrifuged at ambient temperature and cell pellets were suspended to volume in 0.01 M phosphate buffer (pH 7.0). Plates were incubated for 48 h at 37 C for quantitative estimates of mating frequencies.
RESULTS Previous attempts in our laboratory to determine linkage between RP1 determinants by using the Escherichia coli transducing phage P1 have been unsuccessful. The entire RP1 plasmid was always transduced. However, the availability of the RP1 derivative RP1-S2 (22) allowed for the investigation of linkage between RP1 determinants concerned with surface exclusion and incompatibility with respect to their affect on other P group plasmids. The entry exclusion and incompatibility properties of RP1 and its derivative, RP1-S2, in relation to the other P group plasmids are shown in Table 2. It is clear from these data that RP1 excludes the entry of the related plasmids R751, R702, and R906. However, with RP1-S2 as the resident
30
J. BACTERIOL.
OLSEN AND SHIPLEY
TABLE 2. Entry exclusion and incompatibility properties of RPI or RP1-S2 in relation to other P group plasmids Donor
Recipienta
P group R+ recipients/donorb
J53 (R751)
CR34 CR34 (RP1) CR34 (RP1-S2)
2 x 10-2 1 x 10-6 2 x 10-3
J53 (R702)
CR34 CR34 (RP1) CR34 (RP1-S2)
3 x 10-3 3 x 10- 5 3 x 10-'
J53 (R906)
CR34 CR34 (RP1) CR34 (RP1-S2)
4 x 10- 3 1 x 10-6 2 x 10-
a Nutritional selection against the donor was done on appropriately supplemented minimal medium used previously (16). Trimethoprim (100 gg/ml) or streptomycin (25 ug/ml) was included in minimal medium to select for the transfer of R751 or R702 and R906, respectively.
plasmid in recipient bacteria, this exclusion, as indicated by a low frequency of exconjugant formation, is not apparent. Thus, RP1-S2 has lost genetic information specifying the entry exclusion properties common to P group plasmids in addition to the deletion of genes specifying P plasmid pili and the cell surface receptor for phage PRD1 noted previously (22). It is interesting to note that both entry exclusion and pilus genes,are associated with the transfer region of F and F-like plasmids and that the same linkage relationship apparently is obtained with RP1. From this we conclude that RP1 transfer region genes are linked to determinants for neomycin/kanamycin resistance. However, when exconjugants from the above matings are purified by serial restreaking on medium not selective for the P group plasmid originally present in the recipient, it is observed upon subsequent testing that they have lost RP1 or RP1-S2. Therefore, RP1-S2 has retained genetic information determining susceptibility to incompatibility by a related plasmid. We designate this phenotypic trait sinp. Thus, the matings (Table 2) with CR34(RP1-S2) recipients resulted in the detection of RP1-S2 susceptibility to incompatibility caused by entering P group plasmids. We next verified the ability of RP1-S2 to cure a resident P plasmid by transducing RP1-S2 into bacteria containing R702 or R906. For this, P1 phage was grown on E. coli J53(RP1-S2) and harvested as reported previously for P22 transduction (22). These transducing phage lysates transduced RP1-S2 into either
J53(R702) or to J53(R906) at a frequency of approximately 2 x 10- 6 per viable phage. When the transductants selected for carbenicillin or tetracycline resistance were purified on medium selective for RP1-S2 and individual colonies were tested, all were observed to have lost the P plasmid (R702 or R906) originally present in the recipient bacteria (20/20 tested). We have designated this trait incp, analogous to the phenotype designated for F-like plasmids (7). INCp (capital letters used for a gene product) then, is presumably a P plasmid gene product responsible for the loss of related plasmids containing the sinp locus maintained in bacteria grown under nonselected conditions with respect to the lost plasmid. Consequently, these studies have indicated that RP1-S2 has retained the P plasmid genes concerned with incompatibility as well as genes specifying resistance to carbenicillin and tetracycline. Accordingly, these characteristics constitute a linkage group in the RP1 plasmid. Properties of a P-W group recombinant plasmid. We reported the construction of a recombinant plasmid formed by the addition to the W group plasmid R388 (22) of carbenicillin resistance specified by the P group plasmid RP1. The recombinant plasmid, designated RWP1, was tested and found to belong to the W incompatibility group. We also determined whether RWP1 contained genetic information specifying incompatibility with P group plasmids (Table 3). The results show that the donor frequencies of the P plasmids are not affected by the presence of RWP1 in the recipient bacteria. This is also the finding for recipients containing R388, the parent of RWP1 (unpublished data). When exconjugants from the matings shown in Table 3 were purified by serial colony isolation on the agar medium containing an 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 carbenicillin resistance into R388 to form RWP1 resulted in TABLE 3. Transfer of P plasmids to CR34 (R WPl)a Donor
Recipient
J53 (R702) J53 (R906) J53 (RP1)
CR34 (RWP1)
P group R+
recipients/donor
1 x 10-9 3 x 10-4 1 x 10-3
a Selection for the transfer of RP1 was on minimal medium containing 500 Atg of carbenicillin per ml. Selection for the transfer of R702 and R906 was as described in Table 2.
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RP1 PROPERTIES AND FERTILITY INHIBITION
the loss of the P plasmid incompatibility function(s). Similarly, in the reverse situation, when the recipient bacteria contained the P plasmids used in Table 3 and the donor contained RWP1, exconjugants stably maintained both RWP1 and the P plasmid under nonselective conditions of growth. Fertility inhibition studies. We next tested RWP1 for the expression of genes affecting P plasmid fertility. We considered that genes possibly affecting P plasmid fertility may be linked to carbenicillin resistance in RP1 and hence, in some instances, would be retained when recombination with R388 occurred. This determination could not be done reproducibly in bacteria containing two P plasmids, perhaps reflecting the stringent incompatibility occurring among P plasmids. However, RWP1 is compatible with P plasmids, and hence "doubles" (bacteria containing RWP1 and a P plasmid) could be tested for the effect of RWP1 on P plasmids or the reverse with regard to donor frequency. The results of these determinations (Table 4) clearly show inhibition of RWP1 transfer by bacteria containing either of the P plasmids RP1 or R702. This unexpected result resembles fertility inhibition of F-mediated conjugation by the presence of fi+ F-like plasmids (24) and also the later reports for several other plasmid combinations (10, 19, 25). Indeed, the P plasmid RP4 has been shown TABLE 4. Mating of RWPl-P plasmid doubles with J53SmR Donora
J53 (RWP1) J53 (RP1) J53 (RP1/RWP1) J53 (RWP1/RP1) J53 (R702) J53 (R702/RWP1) J53 (RWP1/R702) a In
R+selectedb recipient RWP1 RP1 RP1 RWP1 RP1 RWP1 R702 R702 RWP1 R702 RWP1
R+ recipients/ donor 5x 2x 3x 1x 3x 2x 2x 7x 2x 8x 9x
10-3 10-3
10-' 10-6 10-4 10-6 10-3 10-4 10-5 10-4 10-6
the case of doubles, the first plasmid listed was added to bacterial recipients containing the second plasmid listed. For this strain construction, R+ CR34 donors were used. "TN agar containing 500 Ag of streptomycin and 100 ug of trimethoprim per ml was used to select for the acquisition of RWP1. For the selection of either RP1 or R702, 20 jg of tetracycline per ml was included in TN agar in addition to 500 ug of streptomycin per ml. J53SmR is chromosomally resistant to 500 jig of streptomycin per ml.
31
susceptible to fertility inhibition by the group Ia R factor R64 (6). A slight but reproducible lowering of transfer frequency for RP1 is also noted for these doubles. This may reflect a reciprocal effect on fertility or, alternatively, a nonspecific lowering of transfer frequency for RP1 caused by the mere presence of another plasmid. Further examples of this relationshp will be cited later. To determine whether RP1 inhibition RWP1 transfer reflected the presence of P plasmid genes linked to carbenicillin resistance or alternatively reflected a property of the progenitor plasmid R388, we next did the matings shown in Table 5. It is evident from the data that RP1 inhibits the transfer of W group plasmids R388 or RSa. Accordingly, the susceptibility of RWP1 transfer to RP1 inhibition shown in Table 4 is believed to be a W incompatibility group property. These data also show a diminution of RP1 transfer associated, in this case, with the presence in donor cells of R388. This effect for the other W group plasmid tested, RSa, is less pronounced and within the variation of transfer frequency occurring on repeated trials of these
matings. P and W group plasmids have in common the genetic information for the specification of the receptor for the plasmid-dependent phage PRD1 (17). This is known to be a cell wall function. The cognate observation of Willets (24) that the synthesis of an entry exclusion protein factor(s) was linked to fertility inhibition in the fi+ system prompted us to consider that RP1 might be inhibiting transfer region functions in group W plasmids analogous to the F-like plasmids and F since both P plasmids and W plasmids possess functionally equivalent information specifying a cell surface component. This model would propose that the W TABLE 5. Effect of RPJ on Wgroup plasmid transfer toJ53SmR Donor
J53 (RP1) J53 (R388) J53 (RP1/R388) J53 (RSa) J53 (RP1/RSa)
R+selecteda recipient RP1 R388 RP1 R388 RSa RP1 RSa
R+ recipients/ donor 1x 2x 4x 3x 2x 7x 1x
Mg
10-3 10-3 10-4 10-6
10-' 10-4 10-7
a TN agar containing 500 of streptomycin and 500 Ag carbenicillin per ml was used for the selection of RP1 transfer. For R388 transfer, 100 Mg of trimethoprim was substituted for carbenicillin. For RSa transfer, 30 ug of chloramphenicol per ml was used.
32
OLSEN AND SHIPLEY
J. BACTERIOL.
plasmid transfer region responds to regulation by a P plasmid gene product. To test this proposal, using another group of plasmids unrelated to P or W groups by incompatibility criteria, we constructed bacterial strains containing both RP1 and N group plasmids. The N group plasmids also specify RPD1 phage sensitivity and thus might be expected to contain a portion of their transfer region functionally related to W and P group plasmids. The results of these determinations are shown in Table 6. In these determinations, with N group plasmids, only RN3 apparently interacted with RP1. Furthermore, unlike RP1 inhibition of the W group, the transfer of RP1 was markedly inhibited in this case. Therefore, the result provides another instance of fertility inhibition effects by plasmids unrelated on the basis of their incompatibility group. The significance of whether a P plasmid causes inhibition of another plasmid or is inhibited by another will be discussed later. The previous report of Pinney and Smith (19) has shown the inhibition of an N group plasmid by the group X R factor R6K. Although in our previous report this plasmid did not encode information for PRD1 phage susceptibility, we considered that it may, however, possess genetic information related to the regulation of transfer function expression common to the P, W, and N plasmid groups. Accordingly, we next tested the effect of R6K on the W group plasmid R388 (Table 7). The results clearly show inhibition of R388 transfer by R6K. This observation, we believe, provides additional evidence for the common origin of transfer functions among the group
TABLE 6. Effect of RPJ Donor
J53 (RP1) J53 (R15) J53 (RP1/R15) J53 (R46) J53 (RP1/R46)
J53 (RN3) J53 (RP1/RN3)
on N group
R+
recipient
selected
RP1 R15 RP1
R15 R46 RP1 R46 RN3 RP1 RN3
transfer to CR34a R+
recipients/ donor"
2 x 10'2 x 10-' 4 x 10-s 1 x 10-4 4 x 10-" 5 x 10-4 2 x 105x
10-4
2
10-" 10-4
6
x x
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 in supplemented minimal medium to select for RN3, R46, or R15 transfer. The ampicillin resistance determinant of these plasmids does not specify resistance to carbenicillin at 500 ,g/ml. a
TABLE 7. R6K (group X) inhibition of R388 transfera Donor
J53 (R6K) J53 (R388) J53 (R388/R6K-1)c
J53(R388/R6K-2)c
R+ recipient
R+ recipients/
R6K R388 R388 R6K R388 R6K
2 x 10-4 1 x 10-a 3 x 10-5 7 x 10-a 3 x 10-" 1 x 10-4
selected"
donor
a CR34 was used as the recipient, and nutritional selection against the donor was done on supplemented minimal medium. 'Minimal medium containing ampicillin (50 Ag/ ml) was used for the selection of R6K. The same medium containing 100 jig of trimethoprim per ml was used for the selection of R388. cDoubles derived from two different matings and
purifications.
N and now W plasmid groups, since representatives of both groups are affected by R6K. Since previous observations in this study had indicated a relationship among the P, W, and N group plasmids extending beyond specification of the RPD1 phage receptor to include interaction of gene products affecting fertility associated with transfer functions, we completed this survey by determining the effect of the X group plasmid R6K on P group plasmid RP1 (Table 8). For this combination of plasmids, the data indicate that the fertility inhibition effect may be reciprocal, i.e., the transfer of both RP1 and R6K is inhibited. The inhibition of RP1 transfer, in this instance, resembles that seen in Tables 4 and 5 for RWP1 or R388, respectively. These examples of partial reciprocal fertility inhibition may reflect the reconstruction by complementation of a fertility regulation system from gene products encoded by both plasmids. However, RP1 is less affected by this reconstituted system than the other plasmid present, RWP1, R388, or R6K. With respect to the effect of R6K on RP1 transfer, however, this result provides further indication of common evolution of plasmid functions controlling fertility in the absence of genetic information for the specification of the PRD1 phage receptor. Plasmid-dependent phage sensitivity. The P group of plasmids specifies pili that allow infection by phage PRR1 (18). One might expect, then, that partial inhibition of fertility as observed for the effect of RWP1, R388, or R6K on RP1 might be accompanied by a decrease in sensitivity to PRR1 phage. Reproducible quantitative estimates of PRR1 adsorption by P plasmid pili, then, would indicate the degree of repression of the P plasmid transfer region due
RP1 PROPERTIES AND FERTILITY INHIBITION
VOL. 123, 1975
TABiz 8. Reciprocal inhibition of RPJ and R6K transferP Donor
J53 (RP1) J53 (R6K) J53 (RP1/R6K-1)c J53 (RPl/R6K-2)c
R+selected" recipient RP1
R6K RP1 R6K RP1 R6K
R+ recipients/ donor 2x 2x 2x 6x 2x 7x
10-3 10-4 10-4 10-6 10-4 10-1
a CR34 was the recipient, and nutritional selection against the donor was done on supplemented minimal medium. b Minimal medium containing 25 ,g of tetracycline per ml was used to select for the transfer of RP1. The same medium containing 25 .ug of streptomycin per ml was used to select for the transfer of R6K. C Doubles derived from two different matings and purifications.
to the presence of another plasmid. However, these quantitative estimations are hindered with the P plasmids, perhaps reflecting the fragility of pili or their uncommon occurrence. Therefore, to gain some preliminary estimate of compatible plasmid effects on PRR1 phage sensitivity, we compared the plating characteristics of PRR1 phage when spotted onto a solid medium surface inoculated bacterial lawns prepared from cells containing only RP1 or RP1 and the other plasmids studied in combination with RP1 cited in this report. In E. coli cells containing RP1, phage PRR1 normally shows small, slightly turbid plaques when tested in this way. However, in E. coli containing RP1 and either RN3, RWP1, R388, or R6K doubles, both the number of plaques and their clarity were diminished corresponding to the degree of fertility inhibition of RP1 reported in Tables 4, 5, 6, and 8. This result was not observed for other plasmid combinations in this report. Accordingly, these qualitative estimates of changes in the expression of a transfer region determinant, we feel, support our interpretations with regard to possible reciprocal fertility inhibition in doubles, although the diminution of RP1 fertility in some instances is only slightly greater than variations in transfer frequency observed on repeated trials for donor cells containing only RP1 or RP1 and another plasmid showing no fertility effects.
DISCUSSION The relatedness of P incompatibility group plasmids to either group W or group N plasmids on the basis of their fertility inhibition interactions may have been observed previously. Data
33
in the report of Coetzee et al. (4) show that the fertility of a P group R factor, RP4, and a W group R factor, RSa, is inhibited by an N group plasmid, R390. Although these authors did not report the donor frequency for RP4 or RSa singly maintained in donor bacteria, the frequencies reported for these plasmids in donor bacteria also containing R390 are low when compared with those in this report for RSa or the plasmid RP1. The model of Willetts (24) and Finnegan and Willetts (7) for the activity of F transfer inhibitor is useful in the consideration of results reported here. Put simply, these authors view fertility inhibition effects as resulting from the regulation of plasmid transfer region functions. This regulation is reported to involve the synthesis of a regulatory gene product, FIN, produced by the fin gene which interacts with another plasmid gene product designated tra P product (7). This FIN/tra P gene product complex, in turn, inhibits the expression of another transfer region gene, tra J, which is required for the expression of the other transfer region determinants including genes required for deoxyribonucleic acid mobilization, pilus synthesis, and entry exclusion. It follows from these considerations, then, that derepressed plasmids exhibiting high transfer, abundant pilus formation, or stringent entry exclusion may have mutations in genes specifying FIN, tra P product, or tra J product, making any one of these components unresponsive to another. It also follows from these considerations that the fertility system may be reconstructed by complementation occurring in bacteria containing derepressed plasmids that differ in the identity of the mutant cistron responsible for their derepressed transfer properties. The simplest example of this complementation causing fertility inhibition would be a combination of plasmids coexistent in the same bacterial cell showing repressed transfer for both plasmids. An example resembling this possibility is seen in this report for the behavior of R6K/RP1 doubles. The more definitive examples of fertility inhibition reported here, however, show inhibition of only one donor strain. This situation could result from an insensitive tra J-like gene product for the noninhibited plasmid analogous to the F-like plasmids that inhibit the expression of F functions. The data in Table 6 indicate that the behavior of RN3 may reflect this situation. In any case, plasmids derepressed by virtue of having functionally equivalent mutations in, for example, their fin or tra J loci would not be expected to complement each other, resulting in reciprocal inhibi-
34
OLSEN AND SHIPLEY
tion of their fertility. This could be the case with the N group plasmids R15 or R46 with respect to RP1. In the case of R46, an alternate explanation, a tra J-like mutation resulting in insensitivity to the FIN/tra P complex, is not likely in view of the prior report demonstrating the inhibition of R46 by the X group plasmid R6K (19). Accordingly, based on this surmise, it is possible that the mutations resulting in the derepression of R46 and RP1 are functionally equivalent and thus reflect the production of either a mutant fin gene product or a mutant tra P gene product. The fertility inhibition studies of others (7, 8, 14, 19, 21, 25) and ours may further facilitate understanding of the evolution of plasmids carrying drug resistance determinants when considered with other aspects of plasmid physiology. First, the identity of drug resistance determinants per se is apparently trivial to fundamental distinctions among plasmids. Numerous reports cite recombination occurring among plasmids resulting in the exchange or addition of drug resistance determinants accompanied, in some instances, by deletion of progenitor plasmid determinants (i.e., see references 1, 11, 12). Thus, recombination allows for the exchange of drug resistance information among plasmids while otherwise maintaining progenitor incompatibility and/or pilus properties. Second, the sub-classification of F-like or I-like plasmids based on the sharing of pili with identical phage specificities, although compatible when simultaneously present in a host bacterium, indicates that within two wellstudied groups a pool of genetic information for genes concerned with incompatibility functions as well as a gene pool for pilus properties may exist. It follows from this that plasmids occur with distinctive properties as a result of particular combinations of these pools. For this consideration, a gene pool would be defined as the minimum number of genes located contiguously on plasmid deoxyribonucleic acid that would result in the expression of a phenotypic trait. Third, fertility inhibition observed for particular combinations of plasmids when coexistent in a bacterium suggests common parentage of plasmids unrelated by previous criteria. This particular consideration focuses attention on the transfer functions comprising plasmids. Our previous observation that the PRD1 phage receptor gene(s) was common to plasmid groups unrelated on the basis of incompatibility or pilus properties is consistent with the sharing of genetic traits regulating fertility at the level of transfer functions, presuming the PRD1 receptor is a transfer
J. BACTERIOL.
region gene. Our deduction that the PRD1 receptor gene(s) is part of the transfer region is supported by the unpublished observation that spontaneous mutants of RP1 ii¶sensitive to phage PRR1 or phage PRD1 are also transfer defective. Presumably these are polar mutations in the RP1 transfer region, and the synthesis of the PRD1 receptor is therefore inhibited along with the other transfer region genes. In summary, reports to date concerning plasmid physiology support the view that plasmid gene pools for drug resistance determinants; incompatibility functions, and transfer functions occasionally recombine leading to the formation of new plasmids with distinctive properties. Furthermore, the hypothetical transfer region gene pool may be divided into two subpools. One pool contains information for pilus composition and resulting phage specificities, the second subpool contains genetic loci concerned with cell surface properties and fertility. The occurrence of fi+ (with respect to F) plasmids bearing pili of the I incompatibility group of plasmids would be an example of the formation of a plasmid with distinctive properties drawn from gene pools for pilus and for fertility inhibition of transfer region determinants (25). ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-07533 from the National Institute of Allergy and Infectious Diseases. One of us, P.S., was supported by Public Health Service training grant 1-TO1-GM-02204 to the Department of Microbiology, The University of Michigan, from the National Institute of General Medical Sciences. LITERATURE CITED 1. 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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resistance determinants and R factors of the A transfer systems by phage PlkC. Mg. Gen. Genet. 114:261-265. Bryan, L. E., H. M. Van Den Elzen, and J. T. Tseng. 1972. Transferable drug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1:22-29. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552. Datta, N., and R. W. Hedges. 1971. Compatibility groups among fi- R factors. Nature (London) 234:222-223. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. Finnegan, D., and N. Willets. 1973. The site of action of F transfer inhibitor. Mol. Gen. Genet. 127:307-316. Grindley, J. N., and E. S. Anderson. 1971. I-like resistance factors with the fi+ character. Genet. Res. 17:267-271. Grinsted, J., R. Saunders, L. C. Ingram, R. B. Sykes, and M. H. Richmond. 1972. Properties of an R factor which originated in Pseudomonas aeruginosa J. Bacteriol. 108:1244-1249.
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RP1 PROPERTIES AND FERTILITY INHIBITION
10. Hedges, R. W., N. Datta, J. N. Coetzee, and S. Dennison. 1973. R factors from Proteus morganii. J. Gen. Microbiol. 77:249-259. 11. Hedges, R. W., and A. E. Jacob. 1974. Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132:31-40. 12. Ingram, L. C., J. 0. Anderson, J. E. Arrand, and M. H. Richmond. 1974. A probable example of R-factor recombination in the human gastrointestinal tract. J. Med. Microbiol. 7:251-257. 13. Jobanputra, R. S., and N. Datta. 1974. Trimethoprim R factors in enterobacteria from clinical specimens. J. Med. Microbiol. 7:169-177. 14. Meynell, E. 1973. Pseudo-fi+ I-like sex factor R62(I), selective for increased pilus synthesis. J. Bacteriol. 113:502-503. 15. Meynell, E., G. G. Meynell, and N. Datta. 1968. Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacteriol. Rev. 32: 55-83. 16. Olsen, R. H., and P. Shipley. 1973. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J. Bacteriol. 113:772-780. 17. Olsen, R. H., J. Siak, and R. Gray. 1974. Characteristics of PRD1, a plasmid-dependent broad host range DNA
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bacteriophage. J. Virol. 14:689-699. 18. Olsen, R. H., and D. D. Thomas. 1973. Characteristics and purification of PRR1, an RNA phage specific for the broad host range Pseudomonas R1822 drug resistance plasmid. J. Virol. 12:1560-1567. 19. Pinney, R. H., and J. T. Smith. 1974. Fertility inhibition of an N group R factor by a group X R factor, R6K. J. Gen. Microbiol. 82:415-418. 20. Richmond, M. H., and R. B. Sykes. 1972. The chromosomal integration of a ,B-lactamase gene derived from P-type R factor RP1 in Escherichia coli. Genet. Res. 20:231-237. 21. Romero, E., and E. Meynell. 1969. Covert fi- R factors with the fi+ character. J. Bacteriol. 97:780-786. 22. Shipley, P., and R. H. Olsen. 1975. Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1. J. Bacteriol. 123:20-27. 23. Watanabe, R. 1963. infective heredity of multiple drug resistance in bacteria. Bacteriol. Rev. 27:87-115. 24. Willetts, N. 1972. The genetics of transmissible plasmids. Annu. Rev. Genet. 6:257-268. 25. Willetts, N., and W. Paranchych. 1974. Inhibition of Flac transfer by the Fin+ I-like plasmid R62. J. Bacteriol. 120:101-105.
