Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18

PLASMID

8, 164- 174 (1982)

Tn7 Insertion Mutations Affecting the Host Range of the Promiscuous IncP-1 Plasmid R18 P. COWAN' AND V. KRISHNAPILLAI' Department of Genetics, Monash Vniversity,

Clayton,

Victoria 3168, Australia

Received April 26, 1982 The genetic basis of plasmid host range has been investigated by Tn7 insertion mutagenesis of the promiscuous plasmid R I8 in Pseudomonas aeruginosa. Six mutants have been isolated on the basis of greatly reduced transferability into Escherichia coli C while retaining normal transferability within P. aeruginosa. Their physical mapping shows that two of them map at coordinate 11.72 rt 0.14 kb, in the region of the origin of plasmid replication (orW) and one at 18.0 + 0.3 kb, in the trans-acting gene essential for initiation of replication at oriV &LA). Three map at 48.4 + 0.5 kb in the region of the origin of plasmid transfer (oriT) and the site at which a single-strand nick is introduced in the plasmid DNA-protein relaxation complex (r/x). Consistent with the postulated defective replication of the oriV and trfA mutants was their inability to transform E. coli C or K12 while being able to transform P. aeruginosa. As expected the oriT/rlx mutants transformed both hosts as effectively as R18. Furthermore the ?$A mutant was readily curable by mitomycin C in a DNA polymerase I-proficient P. aeruginosa and spontaneously lost from a polymerasedeficient mutant of P. aeruginosa suggesting a role of this polymerase in the replication of R18. Extensive transfer tests from P. aeruginosa into a range of emetic bacteria, other Pseudomonas species and into other Gram-negative bacteria indicated a complex host range pattern for these mutants. It appears that both plasmid replication and conjugation genesare responsible for host range in addition to the involvement of host gene products.

bility (Stalker et al., 1981). In addition a truns-acting gene t&A has been identified The molecular genetics of the IncP (or P- which is essential for initiation of replication I) group of plasmids is under detailed study at oriV (Thomas, 198lb). Other important chiefly because of their wide host range or features include the identification of the three promiscuous property (for a review seeTho- unlinked regions controlling plasmid conjumas, 1981a). Some members of this group, gational transfer (Tra) (Lanka and Barth, RPl, RP4, RK2, R68, and R18, have been 1981) and the overlapping location of the shown to be extremely similar if not identical origin of plasmid transfer, oriT, and the (Burkardt et al., 1979; Stokes et al., 1981). DNA-protein relaxation complex nick site, The region surrounding the unidirectional rlx, at one end of Tral (Guiney and Helinski, origin of plasmid replication (oriv) of RK2 1979; Lanka and Barth, 198 1; Thomas, has been sequenced and found to include 1981a). A unique DNA primase (pri) has also eight 17-basepair direct repeats, a Pribnow been mapped to the other end of Tral (Lanka box postulated to function in the initiation and Barth, 1981). of an RNA transcript required for the initiOne of the most important questions to ation of replication, A/T- and G/C-rich se- be asked about these plasmids is the genetic quences, and a region affecting incompatiand physical basis of their promiscuity. We have chosen R 18 as the plasmid and P. aeru’ Present address: Department of Microbiology, Uniginosa as the initial host to study this quesversity of Melbourne, Parkville, Victoria 3052, Austion. In order to identify the physical location tralia. of mutations affecting plasmid host range we * To whom reprint requests should bc addressed. INTRODUCTION

0147-619X/82/050164-1 1$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

164

165

PLASMID HOST RANGE MUTANTS

have used insertion mutagenesis with the transposon Tn 7 to obtain host range mutants because the asymmetric sites in Tn7 for restriction enzymes such as Hind111and EcoRI would facilitate their physical mapping (Moore and Krishnapillai, 1982). With the availability of a physical and functional map of RK2 (Thomas, 198la) it was hoped to correlate the physical location of the insertion mutations with the known genetic functions. This paper reports the isolation of host range mutants and their physical and genetic characterization. MATERIALS

AND METHODS

Media and reagents. Nutrient agar (NA), minimal medium (MM), and nutrient broth

(NB) have been described (Krishnapillai, 1971). Antibiotics were used at the following concentrations unless specified differently. Carbenicillin (CB) (Beecham Research Laboratories), 500 pg/ml; kanamycin (KM) (Sigma Chemical Co.), 300 &ml; rifampicin as Rifadin (RIF) (Lepetit Pharmaceuticals Ltd.), 200 &ml; streptomycin (SM) (Sigma), 250 pg/ml; tetracycline (TC) (Sigma), 100 ,u&lml; trimethoprim (TP) (Sigma), 1000 pg/ ml. Mercuric chloride (HG), freshly made up, was used in NA at 12 &ml. Bacteria and plasmids. These are described in Table 1. Incubation temperatures for all enteric bacteria, P. aeruginosa, and P. stutzeri was 37°C; for P. putida, A. calcoaceticus, R. meliloti, and P. glycinea 28°C. Quantitativeplasmid transfer. Direct plate

TABLE I BACTERIALSTRAINSANDF'LASMIDS

Reference/source

Genotype/phenotype’

Bacterial strain/plasmid Strain P. aeruginosa PA0

PA0 1 PA05 PA08 PA039 PAQ490b GMBll2 P. putida PPN 1021 P. stutzeri UQM782 P. maltophilia UQM497 P. glycinea F 13 A. calcoaceticus C4 11 R. meliloti GR4Rif’ E. coli C W2438 E. coli K12 JP777 E. coli 259 K. pneumoniae UNF5023 S. typhimurium SL 1641 S jlexneri 25SM

Plasmid RI8 RPI-85

Prototroph cml-2 trp-54 rif-5 Son-1 met-28 ilv-202 str-I ilv-260 pur-108 str-5 argF leu-IO Tn7 Prototroph polA3 leu-402 str-400 Prototroph str met his met ilv str arg-3 his-l Prototroph rif

Prototroph thr leu thi lacy1 tonA met hisD2 hsdR1 rpsL4 proC90 hsdR nit str

gal r$hsdR

X-F

Cb Nm/Km Tc Tra Dps Phi(G1O1) Eex IncP- 1 Cb Hg Tc Tra Dps Phi(G1O1) Eex IncP-1

Isaac and Holloway ( 1968) Moore and Krishnapillai ( 1982) Isaac and Holloway (1968) Pemberton and Holloway ( 1972) Royle (1980) Lehrbach et ai. (1978) Dean (1982) B. W. Holloway B. W. Holloway J. V. Leary Towner ( 1978) Casadesusand Olivares (1979) E. Lederberg B. Davey N. Willetts Dixon et al. (1977) B. A. D. Stocker Chandler and Krishnapillai ( 1974) Stokes et al. (198 1) Stanisich et al. (1977)

a The gene symbols are according to Bachmann and Low ( 1980) except for fon = resistance to phage FI 16L. The plasmid symbols are according to Novick et al. ( 1976). ’ Tn7 inserted in the chromosome. ’ Spontaneous Rif-resistant mutant of GR4.

166

COWAN AND KRISHNAPILLAI

matings were done for Cb’ transfer. Overnight donor and recipient NB cultures were centrifuged and the cells resuspended in equal volumes of saline. One-tenth milliliter donor (or appropriate dilutions) and 0.1 ml recipient were plated together on selective medium. For Hg’, Km’, Tc’ transfer, matings were first done on NA to allow tranfer and expression of these genes: 0.1 ml each of the donor and recipient were plated together and incubated for 2-3 h/37”C (or 4 h/28”C for transfer into recipients whose growth optimum was 28°C). The cells were washed off in 1 ml saline and dilutions plated on selective medium. Isolation of host range mutants. Mutants were sought which were normally transferable and capable of replicating within one host, e.g., P. aeruginosa but not in an unrelated host such as in E. coli. They were obtained by insertion mutagenesis with Tn7 in P. aeruginosa. The source of Tn7 was the P. aeruginosa PA0 strain PA0490 which had Tn7 or a plasmid fragment containing Tn 7 inserted into the chromosome by transposition from RP4::Tn7 (Royle, 1980). R18 was transferred into PA0490 and PA0490(Rl8) was mated with PA05 for 2 hr at 37°C on NA by spreading together 0.1 ml of each. The cells were washed off in saline and dilutions spread on MM supplemented with tryptophan, TP, and SM which selects for PA05(R18::Tn7) transconjugants. The transposition frequency was about 5 X 10e6 per conjugal event. The use of a chromosomal source of Tn7 had the advantage that every R 18::Tn 7 scored arises from a separate genetic event and this plus the observed transposition frequency facilitated the scoring of a large number of independent Tn7 insertions into the R 18 genome. The PAOS(R 18::Tn7) transconjugants were then replica plated onto MM prespread with either P. aeruginosa PA01 or E. coli C W2438 in turn. The selective medium for PA0 1 was supplemented with CB at 1000 Kg/ml and for W2438 at 250 &ml. The transconjugants were scored for lack of transfer into E. coli C while retaining transfera-

bility into P. aeruginosa. This method of scoring the initial transconjugants for retransfer into another PA0 subline of P. aeruginosa but not into E. coli C had the advantage of avoiding mutants unable to conjugally transfer into either recipient (Tra-) which occur at about 1% frequency. The use of E. coli C had the advantage that because it is naturally restriction deficient, plasmid transfer was optimal, facilitating the identification of the desired mutants. Entry exclusion (Eex), incompatibility (Inc), and plasmid stability tests. Eex was measured by transferring RPl-85 into PA05 sublines carrying the host range mutants or RI 8 by selection for Hg’. The transfer frequencies into PA05 carrying the mutants was compared with recipients carrying R 18 and also compared with R-PAOS. Incompatibility tests were done exactly the same way except the Hg’ transconjugants were purified and tested for Km’. Fifty to one hundred transconjugants were tested. Plasmid stability was tested as described previously (Chandler and Krishnapillai, 1974). Plasmid DNA extraction, purtjication, restriction enzyme digestion, and agarose gel electrophoresis. These were as described (Stokes et al., 1981). Buffers for restriction endonuclease digests were as recommended by the manufacturers except for Hind111 and EcoRI which contained 50 mM NaCl, 6 mM MgC&, 0.1% (w/v) gelatine, 6 mM Tris (pH 7.5). X Hind111 fragments served as size standards in gels. Transformation. Plasmid transformation of P. aeruginosa recipients was essentially by the method of Sinclair and Morgan (1978) and E. coli recipients by the method of Cohen et al. (1972). RESULTS

Isolation of Tn7 Insertion Mutants of R18 Aflected in Host Range We screened about 68,000 P. aeruginosa PA05 (R18::Tn7) clones for nontransmissibility into E. coli C W2438 while retaining transmissibility into P. aeruginosa PA0 1.

PLASMID HOST RANGE MUTANTS TABLE 2 TRANSFER FREQUENCIES OF HOST RANGE MUTANTS FROMP. aeruginosa PA05 TO P. aenrginosa PA01 AND TO E. coli C W2438

Plasmid R18 pM0507 pM0508 pM0509 pM0510 pMO5 11 pM0512

xPA0 1 7x 4x 4x 5x 8x 5x 5x

lo-‘* 10-l 10-l 10-I 10-l 10-l 10-I

xW2438

Ratio”

1x 2x 3x 2x

7 200,ooo 133,000 250,000 80,000 250,000 500,ooo

10-l 1o-6 10-6 10-6

1 x 1o-5

2 x 10-6 1 x 10-6

Note. Transconjugant colony size with all the mutant plasmids except with pM0508, was the same as with R18 in PAOl. Colony size with pM0508 was smaller. (1Ratio of transfer frequency into PA0 1/transfer frequency into W2438. * Cb’ transconjugants/donor.

Despite some difficulty with the scoring of replica-plates because of partial destruction of carbenicillin by the density of the recipient cells (about 2 x lo* cells/plate) six rare (about one per lo4 transconjugants scored) host range mutants were identified. The mutants were nonsibs and were designated pM05075 12. The quantitative transfer frequencies of these mutants are shown in Table 2. It is clear that pM0507-512 have a greatly reduced transfer ability (SO,OOOto 500,000-fold) into E. coli C from P. aeruginosa PA05 while retaining wild-type level of transfer into P. aeruginosa PAOl. In order to test whether the low but measurable transfer of pM05075 12 into W2438 was actually a manifestation of the mutant phenotype rather than reversions to the wild type of the plasmid, four transconjugants from each cross into W2438 were purified, the plasmids transferred back into P. aeruginosa PA05 and retested for transfer into PA01 and W2438. The plasmids from nearly all the transconjugants still retained the mutant phenotype. In those rare exceptions where the mutant plasmid had reverted, the transconjugant still carried Tn 7 (as judged by antibiotic resistance). At this point it was not possible to conclude whether the mutants were unable to

167

transfer into the appropriate hosts (Table 2) because of mutations affecting conjugation or replication/maintenance functions. Therefore transformation experiments were carried out. It was supposed that mutants defective in conjugation but not in replication/maintenance would be transformable into the appropriate host whereas those proficient in conjugation but defective in replication/maintenance would not be transformable. The data (Table 3) show that the mutants are distinguishable on this basis. As expected pM0507-5 12 mutants are transformable into P. aeruginosa as well as the wild-type R18 but pM0507-509 are not transformable into either of the E. coli strains C or K 12. pM05 1O-512 transform into both E. coli strains as well as R18. Although the transformation frequency into E. coli C W2438 was poor compared to that into E. coli K12 it is comparable to the latter with respect to those mutants which are transformable into E. coli and those which are not. Thus two types of host range mutants of R 18 are identifiable genetically: those unable to replicate or maintain themselves in E. coli while they do so in P. aeruginosa (pM0507-509) and those that are defective in conjugation between P. aeruginosa and E. coli (Table 2) but proficient in replication or maintenance once introduced by transformation (pM05 10-512; Table 3). Physical Mapping of the Tn7-Induced Host Range Mutants The plasmid RI 8 has been shown by restriction and heteroduplex analysis to be very similar if not identical to the plasmids RP 1, RP4, R68, and RK2 (Burkardt et al., 1979; Stokes et al., 1981). The precise location of restriction endonuclease sites in these plasmids varies between publications. As one of our aims was to seeif the mutant phenotype of pM0507-5 12 was related to the insertion of Tn7 into known functional regions of R 18, we decided to use the composite physical and functional RK2/RP4/RPl map of Thomas ( 198la). The map of Tn7 used was

168

COWAN AND KRISHNAPILLAI TABLE 3 PLASMIDDNA TRANSFORMATION Plasmids

Recipient

R18

pM0507

PM0508

pM0509

pM05 10

pM05 11

pM05 12

1600”

loo0

2206

1000

1400

1200

1200

P. aeruginosa

PA01 E. coli C

W2438

24

0

0

0

14

14

20

2500

0

0

0

3000

2500

2000

E. coli K12

JP777

Note. Plasmid DNA was extracted and purified from P. aeruginosa PAOS. Transformations were done with 0.5 r,~cg DNA except for E. co/i C where 1 rqg!was used. The number of competent cells for P. aeruginosa was 4 X lo9 and that for E. coli was 4 X 10’. ’ Number of transformants/pg plasmid DNA. Numbers above 1000 are approximate. b Transformant colony size slightly smaller than with R18. In all other cases. transformants with the mutant plasmids were of the same size as with R18.

that of Hernalsteens et al. (1980). The sites of insertion and orientation of the Tn7 insertions in pM0507-5 12 were determined by restriction endonuclease fragment analysis. Tn7 has one EcoRI site and three Hind111 sites (Hemalsteens et al., 1980); R 18 has one site only for these enzymes (Stokes et al., 1981). This information was utilized in the mapping of the site of insertion of Tn7 in pM05 12. The EcoRI/HindIII double digest of pM05 12 showed that the transposon has inserted within the smaller of the two EcoRI/ Hind111fragments of R18, i.e., between 36.6 and 56.4 kb. The actual position of insertion was 48.4 kb, given by the smaller of the two EcoRI fragments of pM05 12 which measured the distance from the EcoRI site of the inserted transposon to the EcoRI site of the plasmid. The presence of this 12.5-kb fragment in the EcoRI/HindIII double digest of pM05 12 indicated that the orientation of the inserted transposon was such that the end of Tn 7 closer to its EcoRI site was the clockwise end. The accuracy of mapping of pM05 12 was governed by the scarcity of enzyme sites in this region. More accurate mapping is in progress using SstII sites. pM05 10 and pM05 11 were mapped in a similar fashion and found to have an identical insertion to pM05 12. It was not pos-

sible, however, to accurately map most of the remaining mutants with EcoRI and HindIII. We therefore decided to extend the map of Tn7 to include cleavage sites for BgfiI and PstI, and to accurately map the two BamHI sites previously reported (Datta et al., 1980). pM05 11 was used for the internal mapping because of the relatively large distance from the nearest BglII, &I, and BamHI sites to the site of insertion of Tn7 in this mutant. Digestion of pM05 11 with BamHI showed that the two sites for this enzyme in Tn7 were 0.86 kb apart, and a BamHI/HindIII double digest confirmed their reported location on either side of the Hind111 site furthest from the EcoRI site. Digestion of pM05 11 with BgfiI indicated two sites for this enzyme in Tn7 separatedby 0.84 kb; their position close to the end of Tn7 furthest from its EcoRI site was determined by BamHI/BglII and BglII/ Hind111double digests. Finally, pM05 11 was digested with BglII/Z?stI. The replacement of the 0.84-kb Tn7 BgnI fragment with two smaller fragments indicated a single PstI site between the two BgZII sites. The precise position of the PstI site was determined by analysis of the HindIII/PstI double digest of pM05 11. Figure 1 shows the restriction fragment map of Tn7 including the newly mapped sites. This map agrees closely with

169

PLASMID HOST RANGE MUTANTS

ggp 8s I

BaH Ba

H

I I

I

I

04~21 0.49 045 2.15 o-4004B2.14

H

E

2.10 I0.mI 4.35 -4%

FIG. 1. Restriction enzyme map of Tn7. Bg = BgfiI; P = PstI; Ba = BumHI; H = HindIII; E = EcoRI. The distances are in kilobases.

those derived by others (Datta et al., 1980; De Greve et al., I98 1) except for our inability to identify a second PstI site 1.13 kb to the right of the PstI site shown in Fig. 1 identified by De Greve et al., (1981). We have deliberately and carefully looked for this 1.13-kb fragment in our gels without success. We therefore conclude that it does not exist in our stock of Tn7 perhaps by mutational loss of the enzyme site. This is not surprising because of their different origins. The extended Tn7 map was used to map the remaining insertion mutants. pM0507 and pM0509, mapped using data from digests from BumHI, BglII, EcoRI and combinations of these enzymes, had an identical insertion at 11.72 kb. The insertion in pM0508 was mapped at 18.0 kb using data from EcoRI/&zfi and BgfiI/HindIII double digests. The orientation of Tn 7 in every mutant except pM0508 was found to be the same. This is consistent with previous observations on the orientation specificity of

Tn7 on plasmids RP4 and R9 l-5 (Barth and Grinter, 1977; Moore and Krishnapillai, 1982). Whether the opposite orientation observed in pM0508 was due to an inversion during or after transposition could not be determined by restriction fragment analysis, but remains a possibility. Figure 2 shows the location of the Tn 7 insertions in R 18 which affect host range.

Genetic Characteristicsof the Host Range Mutants R 18 is an IncP- 1 plasmid of P. aeruginosa conferring resistance to carbenicilhn, kanamycin, and tetracycline and expressing entry exclusion. In order to further characterize the host range mutants their antibiotic resistance profile, entry exclusion and incompatibility status were tested. All the mutants (pMO5075 12) retained CbKmlTc’, a result consistent with their map location (Fig. 2), and the RI 8 entry exclusion phenotype. That is, PA05

FIG.2. Location of Tn7 mutations afkcting host range of R18. This map is based on the physical and functional maps of RK2 (Thomas, 198la) and RP4 (Lanka and Barth, 1981). For gene symbols seetext. The coordinates are in kilobases. a and @refer to orientation of insertion of Tn7; a indicates that end closest to the EcoRI site of Tn7 is clockwise end, j3, reverse orientation. The arrow at oriTJrlx refers to the orientation of plasmid transfer. E = EcoRI.

170

COWAN AND KRISHNAPILLAI

recipients carrying the mutant plasmids excluded the homologous plasmid RPl-85 as strongly as RI 8 (reduction in transfer frequency was > lOOO-fold).All the mutants retained IncP- 1 incompatibility. For example, following selection for the transfer of RPl85 into recipients carrying the mutants only 0.4% of the transconjugants were still Km’, which is typical for recipients carrying R18. All the mutants were tested for stability and found to be 100% stable in PA05 after growth in carbenicillin-free media for 20-25 generations, a result identical to that for RI 8. In view of the possibility that host DNA polymerase functions might be involved in the replication/maintenance of plasmids such as R18, the stability of the mutants pM0507, 508, and 509 was tested in the P, aeruginosa poti- mutant GMB 112 (Lehrbach et al., 1976). pM0507 and pM0509 were 100% stable when tested after 30 generations of growth in carbenicillin-free media, a result identical to that with R 18. But pM0508 was rapidly lost from the polA- host. After 15 generations of growth in antibiotic-free media, 3 1% of GMBl12 (pM0508) cells had lost the plasmid. This instability of pM0508 in polA- cells was also correlated with curability by mitomycin C of poZA+ (=PA05) cells carrying pM0508. Twenty-nine percent curing was observed following growth of the

cells in 2.0 pg/ml mitomycin C, a concentration which had no effect on R 18. Transmissibility of Host Range Mutants in Interspecies and Intergeneric Crosses Although the host range mutants were initially selected on the basis of reduced transmissibility into E. coli C (Table 2) the extended host range behavior of these mutants was tested quantitatively into a number of other enteric bacteria (Table 4). A complex pattern is apparent. From P. aeruginosa donors mutants pM0507-5 12 are poorly transmissible into S, typhimurium and into the E. coli K12 strain 259 similar to their behavior into E. coli C (Table 2). However, only the replication-defective mutants (pM0507-509), mapping in oriV or tr$A (Fig. 2) are poorly transmissible into E. coli K12 strain JP777, K. pneumoniae, and S. jlexneri. When their transfer from P. aeruginosa was tested into a range of other Pseudomonas species and other genera of bacteria such asAcinetobacter and Rhizobium again a complex pattern is apparent (Table 5). Quite clearly all the mutants are transferable at wild-type frequency into P. stutzeri and into P. putida just as into another PA0 strain of P. aeruginosa. However, in P. putida the size of transconjugant colonies with pM0507, 508, and 509 are

TABLE 4 QUANTITATIVE

TRANSFER FREQUENCIES OF THE HOST RANGE MUTANTS BETWEEN P. aeruginosn AND ENTERIC BACTERIA

IN CROSSES

Plasmid in donor Recipient

R18

E. coli JP777 E. coli 259 K. pneumoniae UNF5023 S. typhimurium SL1641 S. flexneri 25SM

3 x 10-l 4 x 10-Z

5 x lo+ 3 x 10-6

1 x lo-’ 5 x 1om6

1 x loo

7 x 10-5

6 X lo-’

1 x 10-6

3 x 10-s

pM0507

<5 x lo-*

PM0508

pM05 10

pMO5 11

pM05 12

5 x 1o-4 2 x 1om6

3 x 10-l I x 1o-4

2 x 10-l 5 x 1o-5

3 x 10-l 1 x 1o-4

5 x 1om4

1 x 1o-4

5 x 10-l

4 x 10-l

6 X 10-l

1 x 1om5

2 x 1o-6

2 x lo-6

3 x lo-6

5 x 1o-6

8 X 1O-5

5 x 1o-5

8 x 1O-5

15 x 10-s

pM0509

<5 x 1o-8

Note. Frequencies were measured in plate mating crossesselected on MM supplemented with the appropriate growth factor of the recipient + CB (250 &ml). The donor host was coatrasekcted by the omission of its growth requirement. The donor was P. aeruginosu PA05. The frequencies are expressed as Cb’ transconjugsnts/donor.

171

PLASMID HOST RANGE MUTANTS TABLE 5 QUANTITATIVETRANSFERFREQUENCIES OF THE HOST RANGE MUTANTS BETWEEN P. aeruginosa AND GIXER BACTERIA Plasmid in donor Recipient

pM05 12

Rl8

pM0507

pM0508

pM0509

pM05 10

pM05 11

7 x 10-j

6 X 10-l

3 x 10-l

3 x 10-l

4 x 10-l

3 x 10-l

7 x 10-l

2 x 10-l

1 x lo-‘”

2 x lo-‘”

9 x lo-*”

2 x 10-r

1 x 10-l

1 x 10-l

6 X 1O-3

4 x 10-r

1 x lo-)

4 x 10-r

2 x 10-r

2 x lo-’

2 x 10-r

1 x 1o-4

6 x 1O-6

1 x lo-5

4 x 10-6

3 x 10-s

4 x 10-s

2 x 10-s

3 x 10-6

3 x 10-7

2 x lo-’

9 x 10-s

2 x 1o-6

N.T.6

N.T.’

1 x lo-3


x lo-’

3 x 10-6”

3 x 10-60

1 x lo-en

1 x 10-l

2 x 10-5

5 x lo-*

2 x lo-*

5 x lo-*

P. aeruginosa

PA08 P. putida

PPNlO21 P. stutzeri

UQM782 A. calcoaceticus

c411 R. meliloti

GR4Rif P. maltophilia

UQM497

1 x 10-3”

tl

P. glycinea

F13

1 x 10-r

2 x lo-6

Note. Frequencies were measured in plate mating crossesselected on MM supplemented with the appropriate growth factor(s) of the recipient + CB or TC. The donor was contraselected by the omission of its growth factor requirement(s). CB for P. aeruginosa and A. calcoaceticus recipients was at 500 &ml; P. putida and P. glycinea, 250 &ml; P. stutzeri 1000 &ml; R. meliloti, 50 pg./ml. TC for P. maltophilia recipients was at 20 &ml. The MM for crosseswith A. calcoaceticus had 0.1% sodium acetate instead of glucose. All donors except for crosses with R. meliloti were P. aeruginosa PA05. For R. meliloti crosses the donor was P. aeruginosa PA039. All frequencies except for crosseswith P. ma~tophiiia are expressed as Cb’ transconjugants/donor. In P. maltophilia crosses,the frequencies are expressed as Tc’ transconjugants/donor. ’ Transconjugants much smaller than transconjugants formed with R 18. ’ Not tested.

smaller than with the other transconjugants with P. glycinea only the oriV mutants are suggestingsome impairment with their trans- reduced in their transferability whereas all fer and/or replication of these mutants in P. the other mutants transfer with wild-type freputida. All the mutants are reduced in their quency. transferability into A. calcoaceticus but only DISCUSSION to about a lo-fold extent. All the replicationdeficient mutants (pM0507, 508, and 509) We have identified three functional rebut not those mapping in oriT/rlx (pM05 10) gions affecting the host range of the wide host are also impaired slightly in their transferabil- range plasmid RI 8 by Tn7 insertion mutaity into R. meliloti. Transfer of the oriV mu- genesis.The procedure was based on reduced tants pM0507 and 509 was not detectable plasmid transmissibility into E. coli from P. into P. maltophilia but the oriT/rlx mutants aeruginosa while maintaining normal transare transferable at a very reduced rate. The missibility within P. aeruginosa. The three trfA mutant transfers at wild-type frequen- classesof mutants identified map at kilobase cies. However, when transfer of any of the coordinates 11.72 -+0.14 (pM0507 and 509), mutants is detectable into P. maltophilia the 18.0 + 0.3 (pM0508), and at 48.4 f 0.5 colony size of the transconjugants is much (pM05 10-5 12). These regions have been smaller than with R18, implying that with identified by others with particular aspects all the mutants there is some impairment of the replication, conjugation, and incomwith their transfer and/or replication. Finally patibility functions of these plasmids (Tho-

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mas, 198la). The mutants pM0507 and 509 map either within or at the edge of oriV, the site at which vegetative plasmid replication commences (Stalker et al., 1981) and pM0508 maps within trfA, the trans-acting gene essential for the initiation (and possibly the regulation) of replication at oriV (Thomas, 1981b). Thus the Tn7 insertion mutations in these mutants are believed to lead to defects in their replication in E. coli but not in P. aeruginosa hosts. This was supported by their inability to transform the former but not the latter host and by their reduced conjugal transmissibility or reduction in size of transconjugants into other hosts as well, such as into other enteric bacteria, other Pseudomonas species, Acinetobacter and Rhizobium. In addition the trfA mutant pM0508 was unstable as judged by spontaneous plasmid loss in DNA polymerase Ideficient cells of P. aeruginosa or by curability with mitomycin C in polA+ cells of P. aeruginosa. The mutants pM05 10-5 12 map at the oriT/rlx region of Tral, one of the regions controlling conjugation (Guiney and Helinski, 1979; Thomas, 1981a; Lanka and Barth, 1981). oriT is believed to be the site at which a nick is introduced on the double-stranded plasmid DNA followed by the transfer of a single strand to the recipient during conjugation (by analogy with the IncFI, FII and I” plasmids; Vapnek et al., 1971; Vapnek & Rupp, 1970). rlx is the site where a nick is introduced into plasmid DNA-protein relaxation complex by treatment with Pronase or SDS and this site has been shown to either overlap or be very near oriT (Guiney and Helinski, 1979). Thus the Tn7 insertion mutations in these mutants appear to affect oriT and thus their transfer during conjugation. Since only their conjugation is affected they were transformable into E. coli (C and K12). This implies that E. coli C and other recipients such as E. coli K12 strain 259, S. typhimurium, P. maltophilia, and to a lesser extent A. calcoaceticus are unable to biochemically process the transferred single strand into its complement and recircularize

it into double-stranded supercoiled DNA (again by analogy with the In&II, PII, and I” plasmids). The fact that P. aeruginosa, P. putida, P. stutzeri, P. glycinea, R. meliloti, E. coli JP777, K. pneumoniae, and S.jlexneri are able to do so as judged by normal conjugal transfer into them, further implies that there are biochemical differences in the way plasmid DNA is handled by these different recipients. Recently a unique DNA primase @ri) has been identified in RP4 which has been postulated to have a role in plasmid host range and plasmid maintenance (Lanka and Barth, 1981). It has been suggestedthat the primase may be involved in priming the synthesis of the strand complementary to that transferred to the recipient during conjugation. It is unlikely that mutants pM05 lo5 12 have Tn 7 insertions in the primase structural gene on both physical and genetic grounds. Unlike the instability of the pri mutants in E. coli, pM05 10-512 are very stable in P. aeruginosa. pM05 10-512 map at 48.4 t 0.5 kb whereas pri maps at 37-42 kb and although pri affects transfer of RP4 from E. coli K12 into P. mirabilis or S. typhimurium and vice versa to only about 5to 25-fold, the transfer of pM05 10-5 12 from P. aeruginosa to E. coli C is diminished by 80,000-to 500,000-fold, into S. typhimurium by lOOO-to 3000-fold, and into E. coli K12 strain 259 by 400- to lOOO-fold(Tables 2 and 4). However, it is possible that Tn 7 insertions at the oriT/rlx region either affect the function of pri or pri and oriT/rlx are both required for the conversion of the singlestranded plasmid DNA transferred during conjugation into the double-stranded form in the recipient. None of the mutants isolated here mapped in the trfB region (54-56 kb), previously reported to affect the transferability of a Tn76 insertion mutant of RP4 from E. coli into P. aeruginosa but not into other enteric bacteria (Barth, 1979). As the role of trfB in the replication and maintenance of this type of plasmid is not known, its significance to plasmid host range in general needs to be assessed. The major conclusion to arise from this

PLASMID HOST RANGE MUTANTS

work is that a genetically complex basis exists for plasmid host range. oriV, t$A, and oriT/ rlx all affect it. That is both plasmid replication/maintenance and conjugation control plasmid host range. Furthermore both plasmid and host gene products appear to influence host range. One feature of the mutants isolated here is their normal behavior in the P. stutzeri host suggesting that, whatever the defect, this host appearsto suppressit. Future experiments will be directed to the determination of the precise roles of the various genes in the host range of plasmids such as R18 in a range of Gram-negative bacteria. For example, since the DNA sequence of ori V is known (Stalker et al., 1981), it should be possible to determine the exact location of the Tn7 insertion in mutants pM0507 and 509 by cloning and sequence analysis, facilitating the identification of the sequence recognized by the different hosts in the initiation and regulation of plasmid replication from

ori V. ACKNOWLEDGMENTS The research was supported by the Australian Research Grants Scheme and Monash University Special Research Fund. We thank Professor B. W. Holloway for his comments on the manuscript and Beecham Research Laboratories for their gifts of carbenicillin. We thank colleagues listed in Table 1 for their generosity in the provision of strains.

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BARTH,P. T. ( 1979).RP4 and R3OOBas wide host range plasmid cloning vehicles. In “Plasmids of Medical Environmental and Commercial Importance” (K. N. Timmis and A. Ptthler, eds.), pp. 399-410. Elsevier/ North-Holland, Amsterdam/New York. BURKARDT, H., RIESS,G., AND I%HLER, A. (1979). Relationship of group PI plasmids revealed by heteroduplex experiments RPI, RP4, R68 and RK2 are identical. J. Gen. Microbial. 114, 341-348. CASADESUS,J., AND OLIVARES,J. (1979). Rough and fine linkage map of the Rhizobium meliloti chromosome. Mol. Gen. Genet. 174, 203-209.

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NOVICK, R. P., CLOWES,R. C., COHEN,S. N., CURTISS, R. III, DATTA, N., AND FALKOW,S. (1976). Uniform nomenclature for bacterial plasmids: A proposal. Bacteriol. Rev. 40, 168-189.

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ROYLE, P. L. (1980). “Plasmids and mapping in Pseudomonas aeruginosa strain PAO.” Ph.D. thesis, Monash University, Australia. SINCLAIR, M. I., AND MORGAN, A. F. (1978). Transformation of Pseudomonas aeruginosa strain PA0 with bacteriophage and plasmid DNA. Ausf. J. Biol. Sci. 31, 679-688.

STALKER, D. M., THOMAS, C. M., AND HELINSKI, D. R. (I 98 1). Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol. Gen. Genet. 181, 8-12. STANISICH,V. A., BENNETT, P. M., AND RICHMOND, M. H. (1977). Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a non-conjugative plasmid in Pseudomonas aeruginosa. J. Bacterial. 129, 1227-1233.

STOKES,H. W., MOORE, R. J., AND KRISHNAPILLAI,V. (198 I). Complementation analysis in Pseudomonas aeruginosa of the transfer genesof the wide host range R plasmid R 18. Plasmid 5, 202-2 12. THOMAS, C. M. (1981a). Molecular genetics of broad host range plasmid RK2. Plasmid 5, 10-19. THOMAS,C. M. ( I98 1b). Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RPl. Plasmid 5, 277-29 1. TOWNER,K. J. (1978). Chromosome mapping in Acinetobacter calcoaceticus. J. Gen. Microbial. 104, 175180. VAPNEK,D., AND RUPP,W. D. (1970). Asymmetric segregation of the complementary sex-factor DNA strands during conjugation in E. coli. J. Mol. Biol. 53, 287303.

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