JOURNAL
OF
Vol. 140, No. 3
BACTERIOLOGY, Dec. 1979, p. 809-816
0021-9193/79/12-0809/08$02.00/0
Identification of Cistrons Involved in Conjugal Transfer of Narrow-Host-Range R Plasmid R91-5 of Pseudomonas aeruginosa JUDITH M. CARRIGAN AND VIJI KRISHNAPILLAI* Department of Genetics, Monash University, Clayton, Victoria 3168, Australia
Received for publication 9 March 1979
The development of a transductional method for complementation tests between transfer-deficient mutants of the narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa has allowed the identification of cistrons involved in the conjugal transfer of this plasmid. Complementation tests performed between transfer-deficient mutants characterized phenotypically with respect to sensitivity to donor-specific phage, ability to inhibit the replication of phage G101, and expression of entry-exclusion has identified a minimum of 10 transfer cistrons. Although most mutagen-induced mutants were relatively heterogeneous and appeared to be affected in a single cistron only, a high proportion of mutants isolated after selection for donor-specific phage resistance had deletions but always included tra Y. Mutants selected directly on the basis of transfer deficiency which also became donor-specific phage resistant fell into all 10 cistrons, suggesting that many R91-5 transfer cistrons are concerned with the synthesis of sex pili and other surface structures necessary for conjugal transfer. Conversely, most retaining donor-specific phage sensitivity belonged to one cistron, whereas transfer-deficient mutants which had also lost the ability to inhibit the replication of phage G101 comprised four cistrons.
Since as yet only limited information is available about the genetic basis of conjugal transfer in Pseudomonas aeruginosa plasmids isolated from strains of this species (although a study of the transfer [tra] cistrons of the P. aeruginosa IncP-1 wide-host-range R plasmid RP4 has been initiated in Escherichia coli; 8), a detailed genetic analysis of the transfer genes of the narrowhost-range P. aeruginosa R (antibiotic resistance) plasmid R91-5 has been undertaken (12, 15, 16). We previously isolated and characterized transfer-deficient (Tra-) mutants of R91-5 (12). This paper presents the development of a complementation method (by transduction) for the genetic analysis of these mutants in P. aeruginosa and the identification of cistrons involved in the conjugal transfer of this plasmid. The prevalence of R plasmids transferable only within Pseudomonas species (21) together with the proposed relationship in transfer properties between R91-5 and the IncP-1 wide-hostrange R plasmid R18 (this plasmid very likely being identical to RP1 or RP4; 21) (12) were the two main reasons for the choice of R91-5 as a prototype for such a study. This postulated relationship in transfer properties between these two plasmids arose from the observation that, after derepression for transfer, R91-5 expresses 809
specific transfer-related phenotypes which are similar to, but distinguishable from, those exhibited by R18-namely, increased plating efficiency of the donor-specific phages PRD1, PR3, and PR4 (Dps); ability to inhibit the replication of phage G101 [Phi(G1O1)], which is probably analogous to the phage infection inhibition (Pif) phenotype expressed by the F plasmid of E. coli (3); and entry-exclusion (Eex) (12, 14-16, 21). Such a relationship is further supported by the recent finding that R18 is subject to fertility inhibition by R91-5 (IH. W. Stokes, personal communication). It was hoped that an analysis of the transfer genes of R91-5 would provide information as to why the majority of P. aeruginosa R plasmids exhibit a narrow host range (21) and ultimately, by comparison with the transfer genes of R18, contribute towards an understanding of the genetic basis of plasmid host range in P. aeruginosa. We have developed a similar system for the analysis of the transfer genes of R18 which will be the subject of a separate communication (H. W. Stokes, M. J. Lehrer, and V. Krishnapillai, manuscript in preparation). Study of the transfer genes of the F plasmid of E. coli has resulted in the identification of a minimum of 21 tra cistrons (the recently iden-
810
J. BACTERIOL.
CARRIGAN AND KRISHNAPILLAI
tified traY is included in this total; S. McIntire and N. S. Willetts, cited in 36; McIntire and Willetts, cited in 37), and it is estimated that at least another three cistrons remain to be identified (3, 4, 27, 36). All of these cistrons, with the exception of traM (4) and -N (27), are clustered according to function in a 34-kilobase region of plasmid DNA (3, 27), most of these cistrons being in one 30-kilobase-long operon controlled by traJ, a positive control gene, mapping just outside (3). Twelve tra cistrons, as well as the promoter-proximal part of traG, are necessary for F pilus synthesis and assembly, traJ also being needed for expression of entry-exclusion (3, 27). Of the eight tra cistrons not required for F pilus synthesis, two (one of which is the promoter-distal part of traG) are thought to be needed for stabilization of mating aggregates and/or transfer of single-stranded DNA to the recipient (3, 23), two (assuming protein 6c is the traY gene product) are needed for DNA transfer itself (3, 23; M. Achtman, R. Thompson, and B. Kusecek, cited in 34), two are required for the initiation of conjugal DNA transfer (23, 37), and two are involved in entry-exclusion (1). Most of the tra gene products, together with proteins for
which no cistrons are yet known, have been mapped and identified, many of these being found to be strongly associated with the cell envelope and individually regulated at the posttranscriptional level (1, 22, 33, 34, 37). Both similarities and differences have been detected between the tra regions of F and F-like plasmids (2). Yet, despite extensive homology at all levels, very little similarity has been detected between tra proteins encoded by F and R6-5 (2). MATERIALS AND METHODS Bacteria, bacteriophages, and R plasmids. The bacterial strains PAO5 (trp-54 rif-5 F116L resistant; 16) and PAO8 (met-28 ilv-202 str-1; 20) and the transducing, UV-inducible phage F116L (24) were used. (Host chromosomal gene designations are according to Bachmann et al. [7].) R plasmids used are described in Table 1. Media. The media were described previously (24). Carbenicillin (Cb, Beecham Research Laboratories) was used at a final concentration of 500 ,ug/ml in blood agar base (Oxoid), as was streptomycin (Sigma Chemical Co.). Rifampin as Rifadin (Rif; Lepetit Pharmaceuticals Ltd.) was used at a final concentration of 200 ,ug/ml in blood agar base. R plasmid transfer. R plasmid transfer was de-
TABLE 1. R plasmids used a R plasmid
Relevant characteristicsc
Mode of isola-
tionb
Cb
R91-5
EMS
+
+
+
+
Eex +
pMO200, 201, 204, 206 pMO207, 209, 210, 212, 216, 217, 220, 224, 226, 227, 229, 231,
EMS NTG
+
-
-
+
+
234, 236, 237
pMO300, 301, 302, 304, 310, 311, 312, 313, 315, 316, 325, 329, 330, 333, 338, 340, 344, 347, 352, 353, 355, 358, 359, 360, 364, 366, 381
Tra Dps Phi(G101)
HA
pMO382, 384, 386, 388
or
pMO214, 222, 232, 239 pMO349, 362, 378
NTG HA
+
-
+
+
+
pMO356
HA
+
_
-
+1-d
+
pMO303, 306, 307, 334
HA
+
-
-
-
+
pMO305, 337
HA
+
-
-
-
-
+ + - NTG pMO208, 238 + + - - + pMO350 + +/_d _ pMO357 + - pMO335 all a All this from are R91-5 Similarly, the of paper. (15), wild-type R plasmids described, with the exception R plasmids described, with the exception of pMO208, 238, 350, 357, and 335, which are CbO derivatives of pMO207, 237, 349, 356, and 334, respectively, were derived from R91-5. bEMS, Ethyl methane sulfonate; NTG, N-methyl-N'-nitro-N-nitrosoguanidine; HA, hydroxylamine; (r, donor-specific phage resistance. Plasmid symbols are according to Novick et aL (28). d HA-induced Phi(GlOl)+/- Tra- mutants were previously described as Phi(G101)+ (12). '
P. AERUGINOSA PLASMID tra CISTRONS
VOL. 140, 1979
scribed previously (12-14, 16). Transduction and phage assays. Transduction and phage asays were described previously (24). Mutagenesis of R' bacteria. Mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (at a final concentration of 50 ,tg/ml for 20 min) was performed as described previously (13). Complementation tests. Complementation tests, in which transfer from transductionally constructed R plasmid Tra- transient heterozygotes to a final recipient was used as a measure of complementation, were employed to identify R91-5 tra cistrons. Phage F116L lysates of R91-5 Tra- mutants were used to transduce the intermediate strain PAO8 containing another R915 Tra- mutant. Complementation between the Tramutants was then assayed by measuring the ability of this strain to transfer R91-5-conferred carbenicillin resistance to the Rifr final recipient PAO5 (an F116Lresistant strain was used to prevent transduction of the final recipient). Matings were performed by spreading 0.4 ml of transduction mixture and 0.1 ml of an overnight broth-grown culture of PAO5 on blood agar base and incubating at 370C for 1 h to allow expression of tra genes. At the end of this time, the mating mixture was washed off with 1 ml of saline and 0.3 ml was plated on blood agar base with carbenicillin and rifampin. These plates were then incubated at 37°C overnight, and the number of transconjugants was counted. These values were then corrected for the residual transfer level of the mutant in the intermediate recipient by performing a parallel experiment with phage F116L propagated on the R- initial donor strain and subtracting the number of transconjugants obtained from the corresponding values. If the residual transfer level from the intermediate recipient was too high to allow unambiguous scoring, an N-methyl-N'nitro-N-nitrosoguanidine-induced Cb5 derivative of the Tra- plasmid being tested was used in the intermediate recipient. The plasmid mutant in the initial donor was checked in a similar manner by performing a parallel experiment with the isogenic R- intermediate recipient strain. The corrected values were then expressed as a percentage of the number of transconjugants obtained by transducing the same intermediate recipient culture with phage F116L propagated on wild-type R91-5. In those instances where Cbr transconjugants were tested for retransfer, spot transfer tests (12) with PAO8 (selection medium: blood agar base with carbenicillin and streptomycin) were employed. RESULTS
The relatively low frequency of efficiently suppressed R91-5 Tra- mutants in P. aeruginosa (12) indicated that conjugational complementation tests using suppressible donors (6) as a means of constructing transient heterozygotes for the identification of cistrons involved in the conjugal transfer of R91-5 would be limited. As an alternative, it was decided to investigate the feasibility of using transduction for the construction of heterozygotes in complementation tests (35). Since phage F116L (24) transduces R91-5
811
at a frequency of approximately 10-' per plaqueforming unit (16), phage F116L-mediated transduction was tested for the formation of R91-5 Tra- transient heterozygotes from which the transfer of carbenicillin resistance to an F116Lresistant final recipient could be used as a measure of complementation. A similar protocol has been successfully used in P. aeruginosa to study regulation of transfer in mutants of R91, the transfer repressed parent of R91-5 (16). The suitability of phage F116L for use in transductional complementation tests was suggested from results in which PAO8 was transduced with phage F116L propagated on cells R+ for R91-5 (transduction frequency = 3.1 x 10-7 per plaque-forming unit), and transfer of R+ transductants to PAO5 was measured. After 1 h of expression of tra genes, 278 Cb' transconjugants per 0.3 ml of mating mixture were obtained, the rate of increase in Cbr progeny with time (>1,000 and too many to count after 2 and 3 h of expression, respectively) suggesting epidemic spread of the plasmid (26). Preliminary experiments between Tra- mutants demonstrated that, although this phenomenon facilitated the detection of complementing pairs of mutants, no significant increase in transconjugant numbers was observed when noncomplementing pairs of mutants were tested (data not shown). However, since 1 h of expression of tra genes usually allowed quantitative measures of complementation to be made, 1 h of expression was routinely allowed in transductional complementation tests. The number of Cbr progeny scored in mutant crosses ranged from 0 to >1,000/0.3 'ml of mating mixture. Therefore, to accommodate inherent differences in mutants with respect to complementation ability and to minimize day-to-day variation in experimental conditions, the corrected number of Cbr transconjugants obtained in each cross was expressed relative to R91-5 transfer from each mutant in the intermediate recipient when transduced with wild-type R91-5 plasmid. To obtain an indication of the number of cistrons involved in R91-5 transfer, a series of crosses was performed from which an initial assignment of tra cistrons was made. Reference mutants from each tra cistron so identified, chosen on the basis of low levels of residual transfer and efficient complementation, were then used either in the initial donor and/or intermediate recipient to screen further Tra- mutants. A minimum of 10 cistrons were identified in this manner and these were designated traZ, - Y, -X, - W -V, -U, -T, -S, -R, and -Q. The data from which these cistron assignments were made are summarized in Tables 2 and 3, where the percent
812
CARRIGAN AND KRISHNAPILLAI TABLE 2. Summary of the seven tra cistrons, Z through Ta Efficiency of complementation (%) traZb traY
Plasmid recipi-
traX
ent
traZ pMO302
J. BACTERIOL.
traW
traV
traU
traT
pMO206 pMO305 pMO222 pMO232 pMO239 pMO200 pMO220 pMO301 pMO226 pMO236 0 1 171 180 65 437 80 90 100 94
traY pMO337 35 47 0 0 0 29 32 46 35 lie 122 traX pMO200 195 57 81 20 0 49 55 23 40 traW 34 pMO307 16d 179 175 70 1,580 0 76 93 54 pMO316 15e 32 146 218 65 1,035 1 108 130 43 traV pMO329 55 165 119 171 84 868 40 1 86 53 traU pMO350f 145 29 91 122 71 149 55 91 0 87 19h traT pMO313 262 82 45 67 48 416 256 260 0 a The figures in this table are percent efficiencies of complementation. Although not shown, homologous mutant crosses resulted in no or negligible CbT clones after correction for residual transfer of the Tra- mutant being tested. Reciprocal complementation tests were also performed between pairs of mutants demonstrating transfer efficiencies -1% and resulted in nondetectable complementation. Plasmid donor. Reciprocal complementation test = 9%. dReciprocal complementation test = 50%. eReciprocal complementation test = 3%. f pMO350 is a Cb' derivative of pMO349. gh Reciprocal complementation test = 73%. Reciprocal complementation test = 10%. TABLE 3. Identification of traQ, -R, and -S' Efficiency of complementation (%) Plasmid recipient
traQ
traS
traR
pMO207b pMO237 pMO334 pMO356
traQ pMO208c traR pMO238d traS pMO335e
pMO357f
0 69
78 0
156 169
136 121
78 83 31 50 30
256 281 44 21 43
0 0 174 163 239
0 0 106 110 95
traZ pMO302 traY pMO337 traX pMO200 traW 22 221 106 32 pMO307 32 148 14" 185 pMO316 140 105 67 33 traV pMO329 43 206 97 35 tra U pMO350 324 253 167 157 traT pMO313 The figures in the table are percent efficiencies of complementation. Although not shown in all instances, homologous mutant crosses resulted in no or negligible Cb' clones after correction for residual transfer of the Tra- mutant being tested. b Plasmid donor. c pMO208 is a Cb' derivative of pMO207. d pMO238 is a Cb' derivative of pMO237. e pMO335 is a Cb' derivative of pM0334. f pMO357 is a Cb' derivative of pMO356. g Reciprocal complementation test = 76%. h pMO350 is a Cb' derivative of pMO349. a
efficiencies of complementation obtained between reference mutants from each of the cistrons are shown.
Most Tra- mutants tested showed reproducible and efficient complementation, although the range in efficiency, dependent upon mutant combinations, was quite large. The significance of low figures obtained in a few instances was sometimes difficult to interpret in terms of complementation versus non-complementation. However, transfer efficiencies 100%, this phenomenon often being observed when a particular mutant was tested in the initial donor. However, extremely high complementation efficiencies were not always observed when the same mutant was tested in the intermediate recipient (e.g., pMO200 versus pMO360 resulted in 114% efficiency of complementation when pMO200 was in the initial donor but only 12% when pMO200 was in the intermediate recipient). Although such asymmetry in complementation efficiencies, dependent upon whether a particular mutant was tested in the initial donor or intermediate recipient, usually affected only the relative
P. AERUGINOSA PLASMID tra CISTRONS
VOL. 140, 1979
efficiencies of complementation obtained, in some instances it resulted in extremely low or nondetectable complementation in one direction. The same phenomenon was also observed in studies of plasmid transfer in E. coli plasmid F (35) and the P. aeruginosa plasmid R18 (Stokes et al., manuscript in preparation). In such situations, where a positive result was obtained in one direction only, complementation was assumed to be occurring. As already mentioned, the cistron assignment of Tra- mutants was generally clear-cut and may be tabulated as shown in Table 4. However, there were a few examples where the behavior of a particular mutant, although consistent within its defined tra cistron, was anomalous in relation to mutants from other cistrons. This was found to be the case between certain mutants from traZ and pMO316 from traW. For example, although pMO347, which was determined to belong to traZ, complemented pMO307 from traW quite efficiently, it failed to complement pMO316. In all these crosses, it was assumed that the detection of CbO progeny in the majority of instances was the result of complementation, as TABLE 4. Summary of cistron assigment of Tramutantsa tra cis-
Mutants
tron
209, 224, 231, 300, 302, 303, 304, 305, 306, 311, 312, 325, 333, 338,b 340, 344, 347,b 355,b 358,c 366 Y pMO214, 222, 232, 239, 337, 362, 378 X pMO200 W pMO216," 220, 307, 315, 316, 353, 381 V pMO217,e 301, 329 U pMO204, 212, 226, 227, 229, 234, 349, 352, 360 T pMO210, 236, 313, 364 S pMO334, 356 R pMO237 pMO207 Q a Mutants selected on the basis of donor-specific phage resistance, as well as other mutants affected in more than one cistron, are not included in this table. b These mutants also demonstrated low complementation efficiencies, or nondetectable complementation, with pMO316 from traW. c pMO358 also demonstrated low complementation efficiencies with pMO307 from traW (13% in one direction, 10% in the other). dpMO216 did not complement pMO316 but demonstrated low complementation efficiencies with pMO307 (12% in one direction, 2% in the other). epMO217 also demonstrated low complementation efficiencies with pMO302 from traZ (19% in one direction, 0% in the other), pMO337 from traY (13% in one direction, 0% in the other), and pMO316 from traW (13% in one direction, 0%'o in the other). Z
pMO201, 20
813
noted in studies of plasmid transfer in F (35) and R18 (Stokes et al., manuscript in preparation) and not due to plasmid recombination. However, to check that this was in fact the case, eight Cbr transconjugants arising from each of four independent crosses were tested for retransfer to another recipient. In all instances tested, no retransfer was observed. Consequently, Cbr progeny arising from crosses were not routinely tested for retransfer on the assumption that recombination would be occurring only in rare instances, and as such would not bias the scoring of complementation. Although most mutagen-induced Tra- mutants tested appeared to give results consistent with a single mutation, there were exceptions. For example, both pMO330 from traZ and pMO359 from traV also appeared to belong to traX since both mutants demonstrated extremely low transfer efficiencies with pMO200. Similarly, pMO310 failed to complement pMO302 from traZ, pMO337 from traY, and both pMO307 and pMO316 from traW. With reference to pMO310, it was also interesting to note that this mutant appeared to carry a transdominant mutation, i.e., when pMO310 was tested in the intermediate recipient, not only was there no complementation detected between this mutant and any reference donors tested, but it also completely repressed the transfer of wildtype R91-5. In addition, of nine Tra- mutants isolated after selection for donor-specific phage resistance, four were found to be mutant in at least two cistrons, but always including traY (Table 5). pMO382 appeared to be deleted in cistrons traY and -T, pMO384 in cistrons traY, -X, -V, -U, -T, -S, -R, and -Q, and both pMO386 and pMO388 in traY, -X, -U, -T, -R, and -Q. Physical evidence on the basis of their DNA molecular weight and EcoRI restriction fragment pattern supports the conclusion that indeed these are physical deletions (R. J. Moore, personal communication).
DISCUSSION Phage F116L-mediated transductional complementation tests have proved to be a very useful tool for the identification of cistrons involved in the conjugal transfer of the narrowhost-range P. aeruginosa R plasmid R91-5. A minimum of 10 cistrons has been identified using this method. Despite the relatively low transduction frequency of R91-5 with phage F116L, the sensitivity of such a system is greatly enhanced by the avoidance of entry-exclusion (16) and the 100% transfer frequency of this plasmid (12, 15, 16), an added bonus being the phenomenon of epidemic spread (26). Although most Tra- mutants tested demonstrated good comple-
814
J. BACTERIOL.
CARRIGAN AND KRISHNAPILLAI
TABLE 5. Complementation tests between 4)' (donor-specfic phage resistance) Tra - mutants and reference mutants from traZ through Qa
or mutant pMO384 pMO386 pMO388
Efficiency of complementation (%) with reference mutant: traS' traRc traQc traWb traVb traTb traUc pMO209 pMO337 pMO200 pMO316 pMO329 pMO350d pMO210 pMO357' pMO238f pMO208 1 0 0 0 0 NDh' 3 92 0 51 6 1 3 0 0 3 443 211 15i 103
trazb
trayb
16
0
traXb
0
276
86
4
0
11
2
0
pMO206b pMO222b pMO200b pMO220b pMO301 pMO226b pMO236b pMO334b pMO237b pMO207b 0 0 233 78 80 101 134 65 96 120 pMO382c 'The figures in this table are percent efficiencies of complementation. Although not shown, homologous mutant crosses resulted in no or negligible CbO clones after correction for residual transfer of the Tra- mutant being tested. Reciprocal complementation tests performed between pMO384, 386, and 388 and reference mutants from traZ, - V, and -T, demonstrating transfer efficiencies '3%, resulted in nondetectable complementation. Similarly, reciprocal complementation tests performed between pMO382 and reference mutants from traY and -T also resulted in nondetectable complementation. bPlasmid donors. c Plasmid recipients. dpMO350 is a CbO derivative of pMO349. epMO357 is a Cb derivative of pMO356. fpMO238 is a Cb3 derivative of pMO237. R pMO208 is a CbW derivative of pMO207. 'ND, Not done. pMO384 did not complement pMO313 from traT. Reciprocal complementation test =538%. "Reciprocal complementation test = 144%.
mentation efficiencies, there was a large range in values dependent upon the mutant combinations considered. The significance of values >100% cannot at present be evaluated. However, this is not unique to R91-5, since it has also been observed with F plasmid transfer (35). Conversely, the significance of low figures obtained in a few instances was sometimes difficult to interpret in terms of complementation versus non-complementation. However, the assumption that
OF
Vol. 140, No. 3
BACTERIOLOGY, Dec. 1979, p. 809-816
0021-9193/79/12-0809/08$02.00/0
Identification of Cistrons Involved in Conjugal Transfer of Narrow-Host-Range R Plasmid R91-5 of Pseudomonas aeruginosa JUDITH M. CARRIGAN AND VIJI KRISHNAPILLAI* Department of Genetics, Monash University, Clayton, Victoria 3168, Australia
Received for publication 9 March 1979
The development of a transductional method for complementation tests between transfer-deficient mutants of the narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa has allowed the identification of cistrons involved in the conjugal transfer of this plasmid. Complementation tests performed between transfer-deficient mutants characterized phenotypically with respect to sensitivity to donor-specific phage, ability to inhibit the replication of phage G101, and expression of entry-exclusion has identified a minimum of 10 transfer cistrons. Although most mutagen-induced mutants were relatively heterogeneous and appeared to be affected in a single cistron only, a high proportion of mutants isolated after selection for donor-specific phage resistance had deletions but always included tra Y. Mutants selected directly on the basis of transfer deficiency which also became donor-specific phage resistant fell into all 10 cistrons, suggesting that many R91-5 transfer cistrons are concerned with the synthesis of sex pili and other surface structures necessary for conjugal transfer. Conversely, most retaining donor-specific phage sensitivity belonged to one cistron, whereas transfer-deficient mutants which had also lost the ability to inhibit the replication of phage G101 comprised four cistrons.
Since as yet only limited information is available about the genetic basis of conjugal transfer in Pseudomonas aeruginosa plasmids isolated from strains of this species (although a study of the transfer [tra] cistrons of the P. aeruginosa IncP-1 wide-host-range R plasmid RP4 has been initiated in Escherichia coli; 8), a detailed genetic analysis of the transfer genes of the narrowhost-range P. aeruginosa R (antibiotic resistance) plasmid R91-5 has been undertaken (12, 15, 16). We previously isolated and characterized transfer-deficient (Tra-) mutants of R91-5 (12). This paper presents the development of a complementation method (by transduction) for the genetic analysis of these mutants in P. aeruginosa and the identification of cistrons involved in the conjugal transfer of this plasmid. The prevalence of R plasmids transferable only within Pseudomonas species (21) together with the proposed relationship in transfer properties between R91-5 and the IncP-1 wide-hostrange R plasmid R18 (this plasmid very likely being identical to RP1 or RP4; 21) (12) were the two main reasons for the choice of R91-5 as a prototype for such a study. This postulated relationship in transfer properties between these two plasmids arose from the observation that, after derepression for transfer, R91-5 expresses 809
specific transfer-related phenotypes which are similar to, but distinguishable from, those exhibited by R18-namely, increased plating efficiency of the donor-specific phages PRD1, PR3, and PR4 (Dps); ability to inhibit the replication of phage G101 [Phi(G1O1)], which is probably analogous to the phage infection inhibition (Pif) phenotype expressed by the F plasmid of E. coli (3); and entry-exclusion (Eex) (12, 14-16, 21). Such a relationship is further supported by the recent finding that R18 is subject to fertility inhibition by R91-5 (IH. W. Stokes, personal communication). It was hoped that an analysis of the transfer genes of R91-5 would provide information as to why the majority of P. aeruginosa R plasmids exhibit a narrow host range (21) and ultimately, by comparison with the transfer genes of R18, contribute towards an understanding of the genetic basis of plasmid host range in P. aeruginosa. We have developed a similar system for the analysis of the transfer genes of R18 which will be the subject of a separate communication (H. W. Stokes, M. J. Lehrer, and V. Krishnapillai, manuscript in preparation). Study of the transfer genes of the F plasmid of E. coli has resulted in the identification of a minimum of 21 tra cistrons (the recently iden-
810
J. BACTERIOL.
CARRIGAN AND KRISHNAPILLAI
tified traY is included in this total; S. McIntire and N. S. Willetts, cited in 36; McIntire and Willetts, cited in 37), and it is estimated that at least another three cistrons remain to be identified (3, 4, 27, 36). All of these cistrons, with the exception of traM (4) and -N (27), are clustered according to function in a 34-kilobase region of plasmid DNA (3, 27), most of these cistrons being in one 30-kilobase-long operon controlled by traJ, a positive control gene, mapping just outside (3). Twelve tra cistrons, as well as the promoter-proximal part of traG, are necessary for F pilus synthesis and assembly, traJ also being needed for expression of entry-exclusion (3, 27). Of the eight tra cistrons not required for F pilus synthesis, two (one of which is the promoter-distal part of traG) are thought to be needed for stabilization of mating aggregates and/or transfer of single-stranded DNA to the recipient (3, 23), two (assuming protein 6c is the traY gene product) are needed for DNA transfer itself (3, 23; M. Achtman, R. Thompson, and B. Kusecek, cited in 34), two are required for the initiation of conjugal DNA transfer (23, 37), and two are involved in entry-exclusion (1). Most of the tra gene products, together with proteins for
which no cistrons are yet known, have been mapped and identified, many of these being found to be strongly associated with the cell envelope and individually regulated at the posttranscriptional level (1, 22, 33, 34, 37). Both similarities and differences have been detected between the tra regions of F and F-like plasmids (2). Yet, despite extensive homology at all levels, very little similarity has been detected between tra proteins encoded by F and R6-5 (2). MATERIALS AND METHODS Bacteria, bacteriophages, and R plasmids. The bacterial strains PAO5 (trp-54 rif-5 F116L resistant; 16) and PAO8 (met-28 ilv-202 str-1; 20) and the transducing, UV-inducible phage F116L (24) were used. (Host chromosomal gene designations are according to Bachmann et al. [7].) R plasmids used are described in Table 1. Media. The media were described previously (24). Carbenicillin (Cb, Beecham Research Laboratories) was used at a final concentration of 500 ,ug/ml in blood agar base (Oxoid), as was streptomycin (Sigma Chemical Co.). Rifampin as Rifadin (Rif; Lepetit Pharmaceuticals Ltd.) was used at a final concentration of 200 ,ug/ml in blood agar base. R plasmid transfer. R plasmid transfer was de-
TABLE 1. R plasmids used a R plasmid
Relevant characteristicsc
Mode of isola-
tionb
Cb
R91-5
EMS
+
+
+
+
Eex +
pMO200, 201, 204, 206 pMO207, 209, 210, 212, 216, 217, 220, 224, 226, 227, 229, 231,
EMS NTG
+
-
-
+
+
234, 236, 237
pMO300, 301, 302, 304, 310, 311, 312, 313, 315, 316, 325, 329, 330, 333, 338, 340, 344, 347, 352, 353, 355, 358, 359, 360, 364, 366, 381
Tra Dps Phi(G101)
HA
pMO382, 384, 386, 388
or
pMO214, 222, 232, 239 pMO349, 362, 378
NTG HA
+
-
+
+
+
pMO356
HA
+
_
-
+1-d
+
pMO303, 306, 307, 334
HA
+
-
-
-
+
pMO305, 337
HA
+
-
-
-
-
+ + - NTG pMO208, 238 + + - - + pMO350 + +/_d _ pMO357 + - pMO335 all a All this from are R91-5 Similarly, the of paper. (15), wild-type R plasmids described, with the exception R plasmids described, with the exception of pMO208, 238, 350, 357, and 335, which are CbO derivatives of pMO207, 237, 349, 356, and 334, respectively, were derived from R91-5. bEMS, Ethyl methane sulfonate; NTG, N-methyl-N'-nitro-N-nitrosoguanidine; HA, hydroxylamine; (r, donor-specific phage resistance. Plasmid symbols are according to Novick et aL (28). d HA-induced Phi(GlOl)+/- Tra- mutants were previously described as Phi(G101)+ (12). '
P. AERUGINOSA PLASMID tra CISTRONS
VOL. 140, 1979
scribed previously (12-14, 16). Transduction and phage assays. Transduction and phage asays were described previously (24). Mutagenesis of R' bacteria. Mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (at a final concentration of 50 ,tg/ml for 20 min) was performed as described previously (13). Complementation tests. Complementation tests, in which transfer from transductionally constructed R plasmid Tra- transient heterozygotes to a final recipient was used as a measure of complementation, were employed to identify R91-5 tra cistrons. Phage F116L lysates of R91-5 Tra- mutants were used to transduce the intermediate strain PAO8 containing another R915 Tra- mutant. Complementation between the Tramutants was then assayed by measuring the ability of this strain to transfer R91-5-conferred carbenicillin resistance to the Rifr final recipient PAO5 (an F116Lresistant strain was used to prevent transduction of the final recipient). Matings were performed by spreading 0.4 ml of transduction mixture and 0.1 ml of an overnight broth-grown culture of PAO5 on blood agar base and incubating at 370C for 1 h to allow expression of tra genes. At the end of this time, the mating mixture was washed off with 1 ml of saline and 0.3 ml was plated on blood agar base with carbenicillin and rifampin. These plates were then incubated at 37°C overnight, and the number of transconjugants was counted. These values were then corrected for the residual transfer level of the mutant in the intermediate recipient by performing a parallel experiment with phage F116L propagated on the R- initial donor strain and subtracting the number of transconjugants obtained from the corresponding values. If the residual transfer level from the intermediate recipient was too high to allow unambiguous scoring, an N-methyl-N'nitro-N-nitrosoguanidine-induced Cb5 derivative of the Tra- plasmid being tested was used in the intermediate recipient. The plasmid mutant in the initial donor was checked in a similar manner by performing a parallel experiment with the isogenic R- intermediate recipient strain. The corrected values were then expressed as a percentage of the number of transconjugants obtained by transducing the same intermediate recipient culture with phage F116L propagated on wild-type R91-5. In those instances where Cbr transconjugants were tested for retransfer, spot transfer tests (12) with PAO8 (selection medium: blood agar base with carbenicillin and streptomycin) were employed. RESULTS
The relatively low frequency of efficiently suppressed R91-5 Tra- mutants in P. aeruginosa (12) indicated that conjugational complementation tests using suppressible donors (6) as a means of constructing transient heterozygotes for the identification of cistrons involved in the conjugal transfer of R91-5 would be limited. As an alternative, it was decided to investigate the feasibility of using transduction for the construction of heterozygotes in complementation tests (35). Since phage F116L (24) transduces R91-5
811
at a frequency of approximately 10-' per plaqueforming unit (16), phage F116L-mediated transduction was tested for the formation of R91-5 Tra- transient heterozygotes from which the transfer of carbenicillin resistance to an F116Lresistant final recipient could be used as a measure of complementation. A similar protocol has been successfully used in P. aeruginosa to study regulation of transfer in mutants of R91, the transfer repressed parent of R91-5 (16). The suitability of phage F116L for use in transductional complementation tests was suggested from results in which PAO8 was transduced with phage F116L propagated on cells R+ for R91-5 (transduction frequency = 3.1 x 10-7 per plaque-forming unit), and transfer of R+ transductants to PAO5 was measured. After 1 h of expression of tra genes, 278 Cb' transconjugants per 0.3 ml of mating mixture were obtained, the rate of increase in Cbr progeny with time (>1,000 and too many to count after 2 and 3 h of expression, respectively) suggesting epidemic spread of the plasmid (26). Preliminary experiments between Tra- mutants demonstrated that, although this phenomenon facilitated the detection of complementing pairs of mutants, no significant increase in transconjugant numbers was observed when noncomplementing pairs of mutants were tested (data not shown). However, since 1 h of expression of tra genes usually allowed quantitative measures of complementation to be made, 1 h of expression was routinely allowed in transductional complementation tests. The number of Cbr progeny scored in mutant crosses ranged from 0 to >1,000/0.3 'ml of mating mixture. Therefore, to accommodate inherent differences in mutants with respect to complementation ability and to minimize day-to-day variation in experimental conditions, the corrected number of Cbr transconjugants obtained in each cross was expressed relative to R91-5 transfer from each mutant in the intermediate recipient when transduced with wild-type R91-5 plasmid. To obtain an indication of the number of cistrons involved in R91-5 transfer, a series of crosses was performed from which an initial assignment of tra cistrons was made. Reference mutants from each tra cistron so identified, chosen on the basis of low levels of residual transfer and efficient complementation, were then used either in the initial donor and/or intermediate recipient to screen further Tra- mutants. A minimum of 10 cistrons were identified in this manner and these were designated traZ, - Y, -X, - W -V, -U, -T, -S, -R, and -Q. The data from which these cistron assignments were made are summarized in Tables 2 and 3, where the percent
812
CARRIGAN AND KRISHNAPILLAI TABLE 2. Summary of the seven tra cistrons, Z through Ta Efficiency of complementation (%) traZb traY
Plasmid recipi-
traX
ent
traZ pMO302
J. BACTERIOL.
traW
traV
traU
traT
pMO206 pMO305 pMO222 pMO232 pMO239 pMO200 pMO220 pMO301 pMO226 pMO236 0 1 171 180 65 437 80 90 100 94
traY pMO337 35 47 0 0 0 29 32 46 35 lie 122 traX pMO200 195 57 81 20 0 49 55 23 40 traW 34 pMO307 16d 179 175 70 1,580 0 76 93 54 pMO316 15e 32 146 218 65 1,035 1 108 130 43 traV pMO329 55 165 119 171 84 868 40 1 86 53 traU pMO350f 145 29 91 122 71 149 55 91 0 87 19h traT pMO313 262 82 45 67 48 416 256 260 0 a The figures in this table are percent efficiencies of complementation. Although not shown, homologous mutant crosses resulted in no or negligible CbT clones after correction for residual transfer of the Tra- mutant being tested. Reciprocal complementation tests were also performed between pairs of mutants demonstrating transfer efficiencies -1% and resulted in nondetectable complementation. Plasmid donor. Reciprocal complementation test = 9%. dReciprocal complementation test = 50%. eReciprocal complementation test = 3%. f pMO350 is a Cb' derivative of pMO349. gh Reciprocal complementation test = 73%. Reciprocal complementation test = 10%. TABLE 3. Identification of traQ, -R, and -S' Efficiency of complementation (%) Plasmid recipient
traQ
traS
traR
pMO207b pMO237 pMO334 pMO356
traQ pMO208c traR pMO238d traS pMO335e
pMO357f
0 69
78 0
156 169
136 121
78 83 31 50 30
256 281 44 21 43
0 0 174 163 239
0 0 106 110 95
traZ pMO302 traY pMO337 traX pMO200 traW 22 221 106 32 pMO307 32 148 14" 185 pMO316 140 105 67 33 traV pMO329 43 206 97 35 tra U pMO350 324 253 167 157 traT pMO313 The figures in the table are percent efficiencies of complementation. Although not shown in all instances, homologous mutant crosses resulted in no or negligible Cb' clones after correction for residual transfer of the Tra- mutant being tested. b Plasmid donor. c pMO208 is a Cb' derivative of pMO207. d pMO238 is a Cb' derivative of pMO237. e pMO335 is a Cb' derivative of pM0334. f pMO357 is a Cb' derivative of pMO356. g Reciprocal complementation test = 76%. h pMO350 is a Cb' derivative of pMO349. a
efficiencies of complementation obtained between reference mutants from each of the cistrons are shown.
Most Tra- mutants tested showed reproducible and efficient complementation, although the range in efficiency, dependent upon mutant combinations, was quite large. The significance of low figures obtained in a few instances was sometimes difficult to interpret in terms of complementation versus non-complementation. However, transfer efficiencies 100%, this phenomenon often being observed when a particular mutant was tested in the initial donor. However, extremely high complementation efficiencies were not always observed when the same mutant was tested in the intermediate recipient (e.g., pMO200 versus pMO360 resulted in 114% efficiency of complementation when pMO200 was in the initial donor but only 12% when pMO200 was in the intermediate recipient). Although such asymmetry in complementation efficiencies, dependent upon whether a particular mutant was tested in the initial donor or intermediate recipient, usually affected only the relative
P. AERUGINOSA PLASMID tra CISTRONS
VOL. 140, 1979
efficiencies of complementation obtained, in some instances it resulted in extremely low or nondetectable complementation in one direction. The same phenomenon was also observed in studies of plasmid transfer in E. coli plasmid F (35) and the P. aeruginosa plasmid R18 (Stokes et al., manuscript in preparation). In such situations, where a positive result was obtained in one direction only, complementation was assumed to be occurring. As already mentioned, the cistron assignment of Tra- mutants was generally clear-cut and may be tabulated as shown in Table 4. However, there were a few examples where the behavior of a particular mutant, although consistent within its defined tra cistron, was anomalous in relation to mutants from other cistrons. This was found to be the case between certain mutants from traZ and pMO316 from traW. For example, although pMO347, which was determined to belong to traZ, complemented pMO307 from traW quite efficiently, it failed to complement pMO316. In all these crosses, it was assumed that the detection of CbO progeny in the majority of instances was the result of complementation, as TABLE 4. Summary of cistron assigment of Tramutantsa tra cis-
Mutants
tron
209, 224, 231, 300, 302, 303, 304, 305, 306, 311, 312, 325, 333, 338,b 340, 344, 347,b 355,b 358,c 366 Y pMO214, 222, 232, 239, 337, 362, 378 X pMO200 W pMO216," 220, 307, 315, 316, 353, 381 V pMO217,e 301, 329 U pMO204, 212, 226, 227, 229, 234, 349, 352, 360 T pMO210, 236, 313, 364 S pMO334, 356 R pMO237 pMO207 Q a Mutants selected on the basis of donor-specific phage resistance, as well as other mutants affected in more than one cistron, are not included in this table. b These mutants also demonstrated low complementation efficiencies, or nondetectable complementation, with pMO316 from traW. c pMO358 also demonstrated low complementation efficiencies with pMO307 from traW (13% in one direction, 10% in the other). dpMO216 did not complement pMO316 but demonstrated low complementation efficiencies with pMO307 (12% in one direction, 2% in the other). epMO217 also demonstrated low complementation efficiencies with pMO302 from traZ (19% in one direction, 0% in the other), pMO337 from traY (13% in one direction, 0% in the other), and pMO316 from traW (13% in one direction, 0%'o in the other). Z
pMO201, 20
813
noted in studies of plasmid transfer in F (35) and R18 (Stokes et al., manuscript in preparation) and not due to plasmid recombination. However, to check that this was in fact the case, eight Cbr transconjugants arising from each of four independent crosses were tested for retransfer to another recipient. In all instances tested, no retransfer was observed. Consequently, Cbr progeny arising from crosses were not routinely tested for retransfer on the assumption that recombination would be occurring only in rare instances, and as such would not bias the scoring of complementation. Although most mutagen-induced Tra- mutants tested appeared to give results consistent with a single mutation, there were exceptions. For example, both pMO330 from traZ and pMO359 from traV also appeared to belong to traX since both mutants demonstrated extremely low transfer efficiencies with pMO200. Similarly, pMO310 failed to complement pMO302 from traZ, pMO337 from traY, and both pMO307 and pMO316 from traW. With reference to pMO310, it was also interesting to note that this mutant appeared to carry a transdominant mutation, i.e., when pMO310 was tested in the intermediate recipient, not only was there no complementation detected between this mutant and any reference donors tested, but it also completely repressed the transfer of wildtype R91-5. In addition, of nine Tra- mutants isolated after selection for donor-specific phage resistance, four were found to be mutant in at least two cistrons, but always including traY (Table 5). pMO382 appeared to be deleted in cistrons traY and -T, pMO384 in cistrons traY, -X, -V, -U, -T, -S, -R, and -Q, and both pMO386 and pMO388 in traY, -X, -U, -T, -R, and -Q. Physical evidence on the basis of their DNA molecular weight and EcoRI restriction fragment pattern supports the conclusion that indeed these are physical deletions (R. J. Moore, personal communication).
DISCUSSION Phage F116L-mediated transductional complementation tests have proved to be a very useful tool for the identification of cistrons involved in the conjugal transfer of the narrowhost-range P. aeruginosa R plasmid R91-5. A minimum of 10 cistrons has been identified using this method. Despite the relatively low transduction frequency of R91-5 with phage F116L, the sensitivity of such a system is greatly enhanced by the avoidance of entry-exclusion (16) and the 100% transfer frequency of this plasmid (12, 15, 16), an added bonus being the phenomenon of epidemic spread (26). Although most Tra- mutants tested demonstrated good comple-
814
J. BACTERIOL.
CARRIGAN AND KRISHNAPILLAI
TABLE 5. Complementation tests between 4)' (donor-specfic phage resistance) Tra - mutants and reference mutants from traZ through Qa
or mutant pMO384 pMO386 pMO388
Efficiency of complementation (%) with reference mutant: traS' traRc traQc traWb traVb traTb traUc pMO209 pMO337 pMO200 pMO316 pMO329 pMO350d pMO210 pMO357' pMO238f pMO208 1 0 0 0 0 NDh' 3 92 0 51 6 1 3 0 0 3 443 211 15i 103
trazb
trayb
16
0
traXb
0
276
86
4
0
11
2
0
pMO206b pMO222b pMO200b pMO220b pMO301 pMO226b pMO236b pMO334b pMO237b pMO207b 0 0 233 78 80 101 134 65 96 120 pMO382c 'The figures in this table are percent efficiencies of complementation. Although not shown, homologous mutant crosses resulted in no or negligible CbO clones after correction for residual transfer of the Tra- mutant being tested. Reciprocal complementation tests performed between pMO384, 386, and 388 and reference mutants from traZ, - V, and -T, demonstrating transfer efficiencies '3%, resulted in nondetectable complementation. Similarly, reciprocal complementation tests performed between pMO382 and reference mutants from traY and -T also resulted in nondetectable complementation. bPlasmid donors. c Plasmid recipients. dpMO350 is a CbO derivative of pMO349. epMO357 is a Cb derivative of pMO356. fpMO238 is a Cb3 derivative of pMO237. R pMO208 is a CbW derivative of pMO207. 'ND, Not done. pMO384 did not complement pMO313 from traT. Reciprocal complementation test =538%. "Reciprocal complementation test = 144%.
mentation efficiencies, there was a large range in values dependent upon the mutant combinations considered. The significance of values >100% cannot at present be evaluated. However, this is not unique to R91-5, since it has also been observed with F plasmid transfer (35). Conversely, the significance of low figures obtained in a few instances was sometimes difficult to interpret in terms of complementation versus non-complementation. However, the assumption that
