JouRNAL OF BACTERIOLOGY, Sept. 1978, p. 911-919 0021-9193/78/0135-0911$02.OO/O Copyright © 1978 American Society for Microbiology
Vol. 135, No. 3 Printed in U.S.A.
Transfer-Deficient Mutants of the Narrow-Host-Range Plasnid R91-5 of Pseudomonas aeruginosa JUDITH M. CARRIGAN, ZENA M. HELMAN, AND VIJI KRISHNAPILLAI* Department of Genetics, Monash University, Clayton, Victoria, 3168, Australia Received for publication 27 June 1978
Three methods have been succeful in the isolation of transfer-deficient mutants of the natrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa: (i) selection for donor-specific phage resistance; (ii) direct screening after mutagenic treatment with either ethyl methane sulfonate or N-methyl-N'-nitro-Nnitrosoguanidine; (iii) in vitro mutagenesis of plasmid DNA by hydroxylamine followed by transformation and direct screening. The majority of transfer-deficient mutants were donor-specific phage resistant, supporting the view that sex pili and other surface components are essential for conjugal transfer (since the phages PRD1 and PR4 adsorb to these sites). Some of the transfer-deficient mutants were also unable to inhibit the replication of phage G101 or lost entry exclusion or both phenotypes. The ability to revert these pleiotropic mutants to wild type implicates the latter two functions in R91-5 transfer. Suppressor mutations in P. aeruginosa enabled the detection of suppressor-sensitive, transfer-deficient mutants. Such mutants should prove useful in conjugational complementation tests for the identification of the transfer cistrons of R91-5.
Although many conjugally transferable R (antibiotic resistance) plasmids have been isolated from strains of Pseudomonas aeruginosa (24, 25, 37), very little is known about the genetic basis of their transfer, with the exception perhaps of the wide-host-range R plasmid RP4 (8). Such knowledge is essential for understanding why R plasmids which are transferable only within Pseudomonas species are so prevalent and, perhaps of more importance, how plasmids of the IncP-1 group transfer themselves across generic barriers. This paper presents the beginning of the genetic analysis of the transfer genes of P. aeruginosa plasmid R91-5, a derepressed mutant (100% transfer frequency) of the narrow-hostrange P. aeruginosa R plasmid R91 (which transfers at frequencies of 10-3 to 10-4 per donor, 14). Unlike R91, R91-5 shows increased plating efficiency of the donor-specific phages PRD1, PR3, and PR4 (Dps) (15), ability to inhibit the replication of phage G101 [Phi(G101)] (15), and expression of entry exclusion (Eex) (15). This observation suggests that these functions are an integral part of the transfer system of R91-5 (15). These phenotypic properties are also exhibited by the wide-host-range R plasmid R18 (this plasmid very likely being identical to RP1 or R1822) (13-S15). However, there are differences between R91-5 and R18, perhaps the most important in terms of plasmid transfer being the distinctive sensitivity of IncP-1 plasmids to the 911
donor-specific phages PRR1 and Pf3 (25), the ability to distinguish between the phage Gol1 inhibition systems (15), and the exhibition of distinct entry exclusion systems (P. M. Chandler, Ph.D. thesis, Monash University, Clayton, Victoria, Australia, 1975) (Table 1). Thus, a genetic analysis of the transfer genes of R91-5 is likely to contribute to an understanding of the genetic basis of plasmid host range in P. aeruginosa. The transfer genes of the F plasmid of Escherichia coli have been extensively studied. Genetic analysis has identified 16 tra cistrons, clustered according to function, in a 34-kilobase region of the plasmid DNA. At least 12 of these cistrons are in one 30-kilobase-long operon which is controlled by traJ, a positive control gene, mapping just outside and counterclockwise of the tra operon (2). traJ is regulated by a repressor complex, the products of the finP and finO genes (26). Nine tra cistrons (traA, -L, -E, -K, -B, -C, -F, -H, and part of -G) are necessary for F-pilus synthesis and assembly (2). traG is also needed for stabilization of cellular contact between F-carrying donor cells and F- recipients (2, 6). traM (which is outside the tra operon), -D, and -I are required for DNA transfer (26). traS and -T code for entry exclusion, the traT gene product (an outer membrane protein) preventing stabilization of cellular contacts between donor celLs, whereas the traS gene product reduces DNA transfer within stable mating aggre-
912
CARRIGAN, HELMAN, AND KRISHNAPILLAI
J. BACTrERIOL.
TABLE 1. Comparison ofplasmids R91-5 and R18 R pla8mid Property
Reference R91-5
R18
Narrow IncP-10 Cb (Nm/Km) (Tc)b Sensitive
Wide IncP-1 Cb, Nm/Km, Tc Tolerant
13, 14 G. A. Jacobya 13, 14 13, 14
+ +
+
15
Sensitivity to donor-specific phages
PRD1, PR3, and PR4 sensitive; PRR1 and Pf3 re-
PRR1, Pf3, PRD1, PR3, and PR4 sensitive
25
Entry exclusion pattern
sistant IncP-10
Inc P-1
Host range Incompatibility group Antibiotic resistances Aeruginocin AR41 Interference with the plating of: G101 GlOlhI GlOlh2
Chandler, Ph.D. thesis + Inhibition by prophage B3 29; unpublished observations aG. A. Jacoby, in R. G. Doggett (ed.), Pseudomonas aeruginosa: Clinical Manifestations of Infection and Current Therapy, in press. b Although R91-5 confers only Cb resistance in P. aeruginosa, its transfer into E. coli results in the expression of neomycin (Nm) and tetracycline (Tc) resistances as well (14). Km, Kanamycin.
gates (1). Identification of 12 of the F tra cistron gene products indicates that most of these conjugation proteins are associated with the cell envelope and are regulated at the post-transcriptional leveL However, the identification of other proteins encoded by F for which no genes are yet known indicates that there are many more as yet unidentified tra cistrons and proteins (26). The beginning of our genetic analysis of R915 transfer has been to isolate and characterize transfer-deficient (Tra-) mutants of R91-5 with the aim of identifying transfer cistrons in a manner analogous to that already developed for the F plasmid of E. coli (4). For this purpose, suppressor-sensitive Tra- mutants were sought for use in complementation tests. MATERIALS AND METHODS Bacteria, bacteriophages, and R plasmids. Bacteria, phages, and plasmids are described in Table 2. Media. The media have been described previously (27). Carbenicillin (Cb; Beecham Research Laboratories) was used at a final concentration of 500 tLg/ml in both blood agar base (BAB; Oxoid) and minimal medium (MM; Difco agar) plus Vogel-Bonner salt medium. Streptomycin (Sm; Sigma Chemical Co.) was used at a final concentration of 500 ug/ml in BAB and 250 ,ug/ml in MM. Rifampin as Rifadin (Lepetit Pharmaceuticals Ltd.) was used at a final concentration of 200 ug/ml in BAB. Trimethoprim (Sigma Chemical Co.) was used at a final concentration of 1,000 ug/ml in MM, and mercuric chloride from a freshly made up stock solution was used at a final concentration of 1.5 ytg/ml in MM. TES buffer is 30 mM
tris(hydroxymethyl)aminomethane-5 mM ethylenediaminetetraacetate-50 mM NaCl (pH 8.0). R-plasmid transfer. The methods previously described (12,13,15) were used to determine quantitative transfer frequencies. For qualitative transfer tests, "spot" transfers and "patch" transfers were used. The former were performed by spotting 0.01-ml samples of overnight broth-grown donor cells into 0.1 ml of recipients prespread on selective medium. Patch transfers involved patching of donor clones onto BAB containing Cb, growing overnight at 37°C, and replica plating to recipients prespread on selective medium. Since preliminary experiments indicated that trimethoprim, Sm, and mercuric chloride resistances are not constitutively expressed, a period was allowed for the expression of these antibiotic resistances after plasmid transfer. Samples of 0.1 ml of donor at different dilutions and 0.1 ml of undiluted recipient cells were spread on BAB and incubated at 37°C for 3 h. At the end of this time, the mating mixture was washed off with 1 ml of saline, and 0.1 ml was spread on selective medium. Transduction and phage assays. Transduction and phage assays were described previously (27). Phage E79tv-2 is a transducing variant of the virulent phage E79 (21) obtained after N-methyl-N'-nitro-Nnitrosoguanidine (NTG) mutagenesis (A. F. Morgan, submitted for publication). When E79tv-2-mediated transductions were performed, a low (ca. 0.1) multiplicity of infection was used, and the transduction mixture was plated on selective medium with an equal volume of a 1/250 dilution of phage E79 antiserum (having an inactivation constant, K, of about 400/min). Mutagenesis of R+ bacteria. (i) In vivo. Mutagenesis with either ethyl methane sulfonate (EMS) or NTG (at a final concentration of 20,ug/ml for 10 min) was performed as described previously (12, 14).
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TABLE 2. Bacterial strains, bacteriophages, and R plasmids used Strain, phage, or plasmid
Bacterial strains PA01 PA05 PA08 PA025 PA055b PA056b PA01670 PT0231 PT0232c Phages E79tv-2 G101 G101h2
Source/ reference
Relevant characteristicsa
cml-2 prototroph t7p-54 rif-5 F116L' met-28 ilv-202 str-1 arg-10 leu-10 cml-2 cml-2 prototroph supB cml-2 prototroph sup-2 pur-136 leu-8 cml-3 rif-1 met-28 trp-6 lys-12 his-4 pro-82 ilv-226 rif-7 sup-1 met-28 trp-6 lys-12 his-4 pro-82 ilv-226 rif- 7 sup-1'
23 15 23 19 This paper This paper 12 41 This paper
Transducing variant of the virulent phage E79 Transducing, UV noninducible, plating inhibition by bacteria R+ for R18 and R91-5 Host range mutant of G101 with EOP = 1 on bacteria R+ for R18 and R91-5 Donor specific for R18 and R91-5 Donor specific for R18 and R91-5
21; A. F. Morgan 15, 21, 28 15
33 PRD1 38 PR4 Plasmids 14,15 Cb (Nm/Km) (Tc) IncP-10 R91-5 This paper Cb (Nm/Km) (Tc) Tp Sm IncP-10 pMO21 This paper Cb (Nm/Km) (Tc) Hg IncP-10 pM022 Host chromosomal gene designations are according to Bachmann et al. (6). Other designation: F116L', resistance to phage F116L. Plasmid symbols are according to Novick et al. (31). EOP, Efficiency of plating of phage. IncP-10, Incompatibility group in P. aeruginosa (Jacoby, in press). Parentheses indicate that the markers are not expressed in P. aeruginosa (14, 15): Nm, neomycin; Km, Kanamycin; Tc, tetracycline; Tp, Hg, mercuric chloride. trimethoprim; b The suppressor strains were cured of the plasmid pLM2 (30) by selection for donor-specific phage resistance. I PT0232 was obtained by selection at 43°C for a spontaneous temperature-sensitive revertant of PT0231. Loss of sup-1 was also correlated with reduced efficiency of plating of phages E79 sus-I and sus-2 (41).
(ii) In vitro. Samples of 0.9-ml DNA solutions were incubated with 0.1 ml of 4.4 M hydroxylamine (HA) in 25 mM ethylenediaminetetraacetate at 370C for 8 to 9 h. Each sample was transferred to dialysis tubing (proaked in TES buffer) and dialyzed against TES buffer at 40C overnight. Extraction ofplasmid DNA. The method of Freifelder (17), as modified by Sinclair and Morgan (Aust. J. Sci., in press), was used, with the following alterations. (i) Protease was added from a 20-mg/ml solution in distilled water (predigested at 370C for 60 min) to give a final concentration of 2 to 3 mg/ml. Incubation at 370C for 60 min was then allowed to reduce fBlactamase contamination of the DNA solutions. (ii) Sepharose 4B was substituted for Sepharose 2B (Pharmacia Fine Chemicals) since the DNA solutions were extremely viscous. Assay of DNA solutions. Samples, 0.5 ml (in replicate), were assayed by the diphenylamine method
(36).
Transformation of plasmid DNA. For transformation of plasmid DNA, the method of Sinclair and Morgan (in press) was used. Screening for Tra- mutants. (i) Selection for donor-specific phage resistance. Overnight independent cultures of PA08(R91-5) were spread on BAB supplemented with Cb. Samples (0.01 ml) of lol to 1010 plaque-forming units of PRDL or PR4 phage per ml were spotted on the R' lawns. After overnight incu-
bation at 370C, resistant clones were picked and screened for Tra activity via spot transfers to PA05 (selection medium, BAB with Cb and rifampin). Putative Tra- mutants were then tested quantitatively for transfer function, those having a transfer frequency of 10-' or less per donor being saved for further study. (ii) EMS and NTG mutagenesis. Overnight independent cultures of mutagenized PA01(R91-5) were diluted to give approximately 100 colonies per plate on BAB + Cb. After overnight growth at 370C, these clones were replica plated to BAB + Cb and Sm prespread with PA08. After overnight incubation at 370C, putative Tra- mutants were picked and screened quantitatively for transfer, those demonstrating a transfer frequency of 10-7 or less per donor being saved. (iii) In vitro mutagenesis of plasmid DNA by HA. PA08(R91-5) transformants were patch mated to PA01670 (0.1 ml of an overnight broth culture of PA01670 being prespread on BAB + Cb and rifampin). After overnight growth at 370C, putative Tramutants were tested quantitatively for transfer, those demonstrating a transfer frequency of io-7 or less per donor being saved. Characterization of mutants. (i) Dps. Mutants were cross-streaked against phage PRD1 or PR4 (1010 to 10" plaque-forming units/ml) and scored as resistant or sensitive. (ii) Phi(G1l1). Phi(G101) resistance was deter-
914
CARRIGAN, HELMAN, AND KRISHNAPILLAI
mined by measuring the efficiency of plating of phage G101 (28). (iii) Eex. Since R91-5 expresses only Cb resistance in P. aerugmwsa (13, 14), derivatives of R91-5 with transposons inserted into it were constructed and used to measure the Eex phenotypes of $R91-5 Tra- mutants. Use was made of transposons Tn501 (40), coding for resistance to mercuric chloride, and Tn7 (formerly TnC;7), coding for simultaneous resistance to trimethoprim and Sm. The Tn7 transposon derivative of R915 was constructed as described (7), using plasmid RP4::Tn7 as the transposon donor. The Tn501 transposon derivative of R91-5 was constructed as previously described (40), using plasmid pVSl as the donor. Eex phenotypes were determined by measuring the conjugal transfer of pM021 or pM022 into each of the Tra- mutants. Selection was imposed for simultaneous trimethoprim and Sm resistance transfer from PA01670(pM021) into PA01 hosts harboring the Tramutants (selection medium, MM with trimethoprim and Sm) or mercuric chloride resistance transfer from PA01670(pM022) into PA08 hosts containing the Tra- mutants (selection medium, appropriately supplemented MM with mercuric chloride). Tra- mutants were scored on the basis of Eex indexes calculated as the ratio of the transfer frequency into an isogenic Rversus R+ recipient. Although these ratios were subject to variation from experiment to experiment because of variation in transconjugant numbers, routine incorporation of controls allowed unambiguous designation of Eex phenotypes. (iv) Suppressor-sensitive Tra- mutants. Suppressor-sensitive Tra- mutants were screened for suppression of the Tra- phenotype by sup-I (thought to be an informational suppressor, 41), supB (an amber suppressor, 30), and sup-2 (also presumably an amber suppressor since it was isolated in a manner similar to that for isolation of supB; unpublished observations). Since the sup bacterial strains were fortuitously partially or totally resistant to adsorption of phage F116L (27), phages GlOlh2 (15) and E79tv-2 (Morgan, submitted) were used to transduce the Tra- mutants into the isogenic sup and sup' hosts. [GlOlh2 was used because R91-5 inhibits the replication of phage G101; i.e., it is Phi(G101)+ (15).] G101h2 mediates R-plasmid transduction at a frequency of approximately 10-8 per plaque-forming unit, the chromosomal marker transduction frequency being approximately 10-7 per plaque-forming unit. E79tv-2 mediates both R-plasmid and chromosome marker transduction at a frequency of approximately 10-7 per plaque-forming unit. Tra- mutants were transduced into the sup-i host using GlOlh2 transduction (selection medium, BAB with Cb and rifampin). One transductant per transduction was purified, and each transductional derivative was screened for conjugal transfer to PA08 (selection medium, BAB + Cb and Sm). The same protocol was foliowed for the detection of supB- and sup2-sensitive Tra- mutants, except selection for these mutants in the sup hosts after G101h2 or E79tv-2 transduction was imposed on MM with Cb. Isogenic sup' transductional derivatives of those Tra- mutants (obtained by selection for donor-specific phage resistance or after in vivo mutagenesis) demonstrating transfer from the sup hosts were constructed. These
J. BACTERIOL.
were also tested for transfer to determine that the transfer observed from the sup hosts was not attributable to a chromosomal mutation affecting plasmid transfer in the original host background. No sup' derivatives of suppressible HA-induced Tra- mutants were constructed since Tra- mutants obtained using this method must be plasmid coded because of the transformation step.
RESULTS Isolation of Tra- mutants. Since spontaneous mutants are rare (frequency of less than 10-1 tested clones), Tra- mutants were isolated after induction of resistance to donor-specific phages or mutagenesis. (i) Seventy-six percent of phage PRD1-resistant derivatives were concomitantly Tra-, and 86% of phage PR4-resistant mutants were also Tra-. More than 100 Tra- mutants, from 40 independent cultures, were isolated. (ii) EMS-induced Tra- mutants occurred at a frequency of approxunately 1 per 5,000 clones. Seven EMS-induced mutants were isolated. Treatment with NTG resulted in mutant frequencies as high as 2.5 x 10-2 per clone tested. From 21 independent cultures, 106 Tra- mutants were isolated. (iii) HA mutagenesis of R91-5 DNA usually resulted in a 100 to 1,000-fold reduction in transformation frequency, giving a transformation frequency of mutagenized DNA in the range from 1o-7 to 10-9 per viable competent cell (compared with frequencies of the order of 10-5 to lo-' for nonmutagenized DNA). Control experiments indicated thet the reduction in transformation frequency was not due to HA-induced damage to plasmi DNA. Therefore, the decline was probably due to mechanical shearing during pipetting (although tlhi, was kept to a minimum) or single-strand breaks caused by dialysis (17). Tra- mutants isolated after in vitro mutagenesis of plasmid DNA by HA occurred at frequencies ranging from 102 to iO-3 per transformed clone. Seventy-svven mutants were isolated. Characterizttion 9f mutants. In view of the proposed relationphip between R91-5 transfer and the Dps, Phi(G1l1), and Eex phenotypes, Tra- mutants were characterized with respect to these properties. A summary of the results is given in Tables 3 and 4. To determine whether point mutations were responsible for the simultaneous loss of the Phi(G1l1) and/or Eex phenotypes in mutageninduced Tra- mutants described in this study, Tra+ revertants of such mutants were obtained. Restoration of the Tra+ and Phi(G101)+ phenotypes were observed in a spontaneous Dps+ revertant of an NTG-induced Tra- mutant. Similarly, Dps+ revertants (occurring at a frequency
TRANSFER MUTANTS OF PSEUDOMQNAS PLASMID
VOL. 135, 1978
915
TABLE 3. Characterization ofphage-resistant Tra- mutants R91-5 phenotype frequencies
Class of mutant
No.
Phi
Dps
tested
+
PRD1 resistant
80
All PR4 resistant
67
PR4 resistant
90
All PRD1 resistant
80
+/13
Eexb
(G101) No. tested 10 Phi (G101)+
+
9
+/0
1
8 3 2 13 Phi (G101)+/4 10 Phi (G101)+ 0 6 6 2 2 10 Phi (G101)+/aThe efficiencies of plating for phage G101 were titrated relative to isogenic R+ and R- bacteria. Phi (G101)+, 10-3 to 10-4; Phi (G101)+/-, 5 x 10-2 to 5 x 10-'; Phi (G101)-, 1. b Control ratio of transfer to R- versus R+ recipients, -1,700, Eex+ ratio, a20; Eex+4 ratio: 5; Eexratio, 45. TABLz 4. Characterization of EMS, NTG, and HA-induced Trac mutants 10
R91-5 phenotype frequencies
mutant
No. tested
Dps + 2 21
-
Phi (Q1O1) + 7 0 58 48
_ _ __ + 7 33
_
Classes of Phi (G10) and Eex
phenotypes
Phi (G101)+ Eex+: 7 Phi (G101)+ Eex+: 29 Phi (G101)+ Eex-: 29 Phi (G101)- Eex4c: 4 Phi (G101)- Eex-: 44 74 71 77 3 6 75 2 Phi (G101)+ Eex+: 71 HAinduced Phi (G1O1)+ Eex-: 0 Phi (G101)- Eex4: 4 Phi (G1O1) Eex- 2 a The efficiencies of plating for phage G101 were titrated relative to isogenic R+ and R- bacteria. Phi (G101)+, 10-3 to 10-1; Phi (G101)-, 1. b Control transfer ratio R- versus R+ recipients, -500;, Eex+ ratio, P6; Eex- ratio,
Vol. 135, No. 3 Printed in U.S.A.
Transfer-Deficient Mutants of the Narrow-Host-Range Plasnid R91-5 of Pseudomonas aeruginosa JUDITH M. CARRIGAN, ZENA M. HELMAN, AND VIJI KRISHNAPILLAI* Department of Genetics, Monash University, Clayton, Victoria, 3168, Australia Received for publication 27 June 1978
Three methods have been succeful in the isolation of transfer-deficient mutants of the natrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa: (i) selection for donor-specific phage resistance; (ii) direct screening after mutagenic treatment with either ethyl methane sulfonate or N-methyl-N'-nitro-Nnitrosoguanidine; (iii) in vitro mutagenesis of plasmid DNA by hydroxylamine followed by transformation and direct screening. The majority of transfer-deficient mutants were donor-specific phage resistant, supporting the view that sex pili and other surface components are essential for conjugal transfer (since the phages PRD1 and PR4 adsorb to these sites). Some of the transfer-deficient mutants were also unable to inhibit the replication of phage G101 or lost entry exclusion or both phenotypes. The ability to revert these pleiotropic mutants to wild type implicates the latter two functions in R91-5 transfer. Suppressor mutations in P. aeruginosa enabled the detection of suppressor-sensitive, transfer-deficient mutants. Such mutants should prove useful in conjugational complementation tests for the identification of the transfer cistrons of R91-5.
Although many conjugally transferable R (antibiotic resistance) plasmids have been isolated from strains of Pseudomonas aeruginosa (24, 25, 37), very little is known about the genetic basis of their transfer, with the exception perhaps of the wide-host-range R plasmid RP4 (8). Such knowledge is essential for understanding why R plasmids which are transferable only within Pseudomonas species are so prevalent and, perhaps of more importance, how plasmids of the IncP-1 group transfer themselves across generic barriers. This paper presents the beginning of the genetic analysis of the transfer genes of P. aeruginosa plasmid R91-5, a derepressed mutant (100% transfer frequency) of the narrow-hostrange P. aeruginosa R plasmid R91 (which transfers at frequencies of 10-3 to 10-4 per donor, 14). Unlike R91, R91-5 shows increased plating efficiency of the donor-specific phages PRD1, PR3, and PR4 (Dps) (15), ability to inhibit the replication of phage G101 [Phi(G101)] (15), and expression of entry exclusion (Eex) (15). This observation suggests that these functions are an integral part of the transfer system of R91-5 (15). These phenotypic properties are also exhibited by the wide-host-range R plasmid R18 (this plasmid very likely being identical to RP1 or R1822) (13-S15). However, there are differences between R91-5 and R18, perhaps the most important in terms of plasmid transfer being the distinctive sensitivity of IncP-1 plasmids to the 911
donor-specific phages PRR1 and Pf3 (25), the ability to distinguish between the phage Gol1 inhibition systems (15), and the exhibition of distinct entry exclusion systems (P. M. Chandler, Ph.D. thesis, Monash University, Clayton, Victoria, Australia, 1975) (Table 1). Thus, a genetic analysis of the transfer genes of R91-5 is likely to contribute to an understanding of the genetic basis of plasmid host range in P. aeruginosa. The transfer genes of the F plasmid of Escherichia coli have been extensively studied. Genetic analysis has identified 16 tra cistrons, clustered according to function, in a 34-kilobase region of the plasmid DNA. At least 12 of these cistrons are in one 30-kilobase-long operon which is controlled by traJ, a positive control gene, mapping just outside and counterclockwise of the tra operon (2). traJ is regulated by a repressor complex, the products of the finP and finO genes (26). Nine tra cistrons (traA, -L, -E, -K, -B, -C, -F, -H, and part of -G) are necessary for F-pilus synthesis and assembly (2). traG is also needed for stabilization of cellular contact between F-carrying donor cells and F- recipients (2, 6). traM (which is outside the tra operon), -D, and -I are required for DNA transfer (26). traS and -T code for entry exclusion, the traT gene product (an outer membrane protein) preventing stabilization of cellular contacts between donor celLs, whereas the traS gene product reduces DNA transfer within stable mating aggre-
912
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TABLE 1. Comparison ofplasmids R91-5 and R18 R pla8mid Property
Reference R91-5
R18
Narrow IncP-10 Cb (Nm/Km) (Tc)b Sensitive
Wide IncP-1 Cb, Nm/Km, Tc Tolerant
13, 14 G. A. Jacobya 13, 14 13, 14
+ +
+
15
Sensitivity to donor-specific phages
PRD1, PR3, and PR4 sensitive; PRR1 and Pf3 re-
PRR1, Pf3, PRD1, PR3, and PR4 sensitive
25
Entry exclusion pattern
sistant IncP-10
Inc P-1
Host range Incompatibility group Antibiotic resistances Aeruginocin AR41 Interference with the plating of: G101 GlOlhI GlOlh2
Chandler, Ph.D. thesis + Inhibition by prophage B3 29; unpublished observations aG. A. Jacoby, in R. G. Doggett (ed.), Pseudomonas aeruginosa: Clinical Manifestations of Infection and Current Therapy, in press. b Although R91-5 confers only Cb resistance in P. aeruginosa, its transfer into E. coli results in the expression of neomycin (Nm) and tetracycline (Tc) resistances as well (14). Km, Kanamycin.
gates (1). Identification of 12 of the F tra cistron gene products indicates that most of these conjugation proteins are associated with the cell envelope and are regulated at the post-transcriptional leveL However, the identification of other proteins encoded by F for which no genes are yet known indicates that there are many more as yet unidentified tra cistrons and proteins (26). The beginning of our genetic analysis of R915 transfer has been to isolate and characterize transfer-deficient (Tra-) mutants of R91-5 with the aim of identifying transfer cistrons in a manner analogous to that already developed for the F plasmid of E. coli (4). For this purpose, suppressor-sensitive Tra- mutants were sought for use in complementation tests. MATERIALS AND METHODS Bacteria, bacteriophages, and R plasmids. Bacteria, phages, and plasmids are described in Table 2. Media. The media have been described previously (27). Carbenicillin (Cb; Beecham Research Laboratories) was used at a final concentration of 500 tLg/ml in both blood agar base (BAB; Oxoid) and minimal medium (MM; Difco agar) plus Vogel-Bonner salt medium. Streptomycin (Sm; Sigma Chemical Co.) was used at a final concentration of 500 ug/ml in BAB and 250 ,ug/ml in MM. Rifampin as Rifadin (Lepetit Pharmaceuticals Ltd.) was used at a final concentration of 200 ug/ml in BAB. Trimethoprim (Sigma Chemical Co.) was used at a final concentration of 1,000 ug/ml in MM, and mercuric chloride from a freshly made up stock solution was used at a final concentration of 1.5 ytg/ml in MM. TES buffer is 30 mM
tris(hydroxymethyl)aminomethane-5 mM ethylenediaminetetraacetate-50 mM NaCl (pH 8.0). R-plasmid transfer. The methods previously described (12,13,15) were used to determine quantitative transfer frequencies. For qualitative transfer tests, "spot" transfers and "patch" transfers were used. The former were performed by spotting 0.01-ml samples of overnight broth-grown donor cells into 0.1 ml of recipients prespread on selective medium. Patch transfers involved patching of donor clones onto BAB containing Cb, growing overnight at 37°C, and replica plating to recipients prespread on selective medium. Since preliminary experiments indicated that trimethoprim, Sm, and mercuric chloride resistances are not constitutively expressed, a period was allowed for the expression of these antibiotic resistances after plasmid transfer. Samples of 0.1 ml of donor at different dilutions and 0.1 ml of undiluted recipient cells were spread on BAB and incubated at 37°C for 3 h. At the end of this time, the mating mixture was washed off with 1 ml of saline, and 0.1 ml was spread on selective medium. Transduction and phage assays. Transduction and phage assays were described previously (27). Phage E79tv-2 is a transducing variant of the virulent phage E79 (21) obtained after N-methyl-N'-nitro-Nnitrosoguanidine (NTG) mutagenesis (A. F. Morgan, submitted for publication). When E79tv-2-mediated transductions were performed, a low (ca. 0.1) multiplicity of infection was used, and the transduction mixture was plated on selective medium with an equal volume of a 1/250 dilution of phage E79 antiserum (having an inactivation constant, K, of about 400/min). Mutagenesis of R+ bacteria. (i) In vivo. Mutagenesis with either ethyl methane sulfonate (EMS) or NTG (at a final concentration of 20,ug/ml for 10 min) was performed as described previously (12, 14).
TRANSFER MUTANTS OF PSEUDOMONAS PLASMID
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TABLE 2. Bacterial strains, bacteriophages, and R plasmids used Strain, phage, or plasmid
Bacterial strains PA01 PA05 PA08 PA025 PA055b PA056b PA01670 PT0231 PT0232c Phages E79tv-2 G101 G101h2
Source/ reference
Relevant characteristicsa
cml-2 prototroph t7p-54 rif-5 F116L' met-28 ilv-202 str-1 arg-10 leu-10 cml-2 cml-2 prototroph supB cml-2 prototroph sup-2 pur-136 leu-8 cml-3 rif-1 met-28 trp-6 lys-12 his-4 pro-82 ilv-226 rif-7 sup-1 met-28 trp-6 lys-12 his-4 pro-82 ilv-226 rif- 7 sup-1'
23 15 23 19 This paper This paper 12 41 This paper
Transducing variant of the virulent phage E79 Transducing, UV noninducible, plating inhibition by bacteria R+ for R18 and R91-5 Host range mutant of G101 with EOP = 1 on bacteria R+ for R18 and R91-5 Donor specific for R18 and R91-5 Donor specific for R18 and R91-5
21; A. F. Morgan 15, 21, 28 15
33 PRD1 38 PR4 Plasmids 14,15 Cb (Nm/Km) (Tc) IncP-10 R91-5 This paper Cb (Nm/Km) (Tc) Tp Sm IncP-10 pMO21 This paper Cb (Nm/Km) (Tc) Hg IncP-10 pM022 Host chromosomal gene designations are according to Bachmann et al. (6). Other designation: F116L', resistance to phage F116L. Plasmid symbols are according to Novick et al. (31). EOP, Efficiency of plating of phage. IncP-10, Incompatibility group in P. aeruginosa (Jacoby, in press). Parentheses indicate that the markers are not expressed in P. aeruginosa (14, 15): Nm, neomycin; Km, Kanamycin; Tc, tetracycline; Tp, Hg, mercuric chloride. trimethoprim; b The suppressor strains were cured of the plasmid pLM2 (30) by selection for donor-specific phage resistance. I PT0232 was obtained by selection at 43°C for a spontaneous temperature-sensitive revertant of PT0231. Loss of sup-1 was also correlated with reduced efficiency of plating of phages E79 sus-I and sus-2 (41).
(ii) In vitro. Samples of 0.9-ml DNA solutions were incubated with 0.1 ml of 4.4 M hydroxylamine (HA) in 25 mM ethylenediaminetetraacetate at 370C for 8 to 9 h. Each sample was transferred to dialysis tubing (proaked in TES buffer) and dialyzed against TES buffer at 40C overnight. Extraction ofplasmid DNA. The method of Freifelder (17), as modified by Sinclair and Morgan (Aust. J. Sci., in press), was used, with the following alterations. (i) Protease was added from a 20-mg/ml solution in distilled water (predigested at 370C for 60 min) to give a final concentration of 2 to 3 mg/ml. Incubation at 370C for 60 min was then allowed to reduce fBlactamase contamination of the DNA solutions. (ii) Sepharose 4B was substituted for Sepharose 2B (Pharmacia Fine Chemicals) since the DNA solutions were extremely viscous. Assay of DNA solutions. Samples, 0.5 ml (in replicate), were assayed by the diphenylamine method
(36).
Transformation of plasmid DNA. For transformation of plasmid DNA, the method of Sinclair and Morgan (in press) was used. Screening for Tra- mutants. (i) Selection for donor-specific phage resistance. Overnight independent cultures of PA08(R91-5) were spread on BAB supplemented with Cb. Samples (0.01 ml) of lol to 1010 plaque-forming units of PRDL or PR4 phage per ml were spotted on the R' lawns. After overnight incu-
bation at 370C, resistant clones were picked and screened for Tra activity via spot transfers to PA05 (selection medium, BAB with Cb and rifampin). Putative Tra- mutants were then tested quantitatively for transfer function, those having a transfer frequency of 10-' or less per donor being saved for further study. (ii) EMS and NTG mutagenesis. Overnight independent cultures of mutagenized PA01(R91-5) were diluted to give approximately 100 colonies per plate on BAB + Cb. After overnight growth at 370C, these clones were replica plated to BAB + Cb and Sm prespread with PA08. After overnight incubation at 370C, putative Tra- mutants were picked and screened quantitatively for transfer, those demonstrating a transfer frequency of 10-7 or less per donor being saved. (iii) In vitro mutagenesis of plasmid DNA by HA. PA08(R91-5) transformants were patch mated to PA01670 (0.1 ml of an overnight broth culture of PA01670 being prespread on BAB + Cb and rifampin). After overnight growth at 370C, putative Tramutants were tested quantitatively for transfer, those demonstrating a transfer frequency of io-7 or less per donor being saved. Characterization of mutants. (i) Dps. Mutants were cross-streaked against phage PRD1 or PR4 (1010 to 10" plaque-forming units/ml) and scored as resistant or sensitive. (ii) Phi(G1l1). Phi(G101) resistance was deter-
914
CARRIGAN, HELMAN, AND KRISHNAPILLAI
mined by measuring the efficiency of plating of phage G101 (28). (iii) Eex. Since R91-5 expresses only Cb resistance in P. aerugmwsa (13, 14), derivatives of R91-5 with transposons inserted into it were constructed and used to measure the Eex phenotypes of $R91-5 Tra- mutants. Use was made of transposons Tn501 (40), coding for resistance to mercuric chloride, and Tn7 (formerly TnC;7), coding for simultaneous resistance to trimethoprim and Sm. The Tn7 transposon derivative of R915 was constructed as described (7), using plasmid RP4::Tn7 as the transposon donor. The Tn501 transposon derivative of R91-5 was constructed as previously described (40), using plasmid pVSl as the donor. Eex phenotypes were determined by measuring the conjugal transfer of pM021 or pM022 into each of the Tra- mutants. Selection was imposed for simultaneous trimethoprim and Sm resistance transfer from PA01670(pM021) into PA01 hosts harboring the Tramutants (selection medium, MM with trimethoprim and Sm) or mercuric chloride resistance transfer from PA01670(pM022) into PA08 hosts containing the Tra- mutants (selection medium, appropriately supplemented MM with mercuric chloride). Tra- mutants were scored on the basis of Eex indexes calculated as the ratio of the transfer frequency into an isogenic Rversus R+ recipient. Although these ratios were subject to variation from experiment to experiment because of variation in transconjugant numbers, routine incorporation of controls allowed unambiguous designation of Eex phenotypes. (iv) Suppressor-sensitive Tra- mutants. Suppressor-sensitive Tra- mutants were screened for suppression of the Tra- phenotype by sup-I (thought to be an informational suppressor, 41), supB (an amber suppressor, 30), and sup-2 (also presumably an amber suppressor since it was isolated in a manner similar to that for isolation of supB; unpublished observations). Since the sup bacterial strains were fortuitously partially or totally resistant to adsorption of phage F116L (27), phages GlOlh2 (15) and E79tv-2 (Morgan, submitted) were used to transduce the Tra- mutants into the isogenic sup and sup' hosts. [GlOlh2 was used because R91-5 inhibits the replication of phage G101; i.e., it is Phi(G101)+ (15).] G101h2 mediates R-plasmid transduction at a frequency of approximately 10-8 per plaque-forming unit, the chromosomal marker transduction frequency being approximately 10-7 per plaque-forming unit. E79tv-2 mediates both R-plasmid and chromosome marker transduction at a frequency of approximately 10-7 per plaque-forming unit. Tra- mutants were transduced into the sup-i host using GlOlh2 transduction (selection medium, BAB with Cb and rifampin). One transductant per transduction was purified, and each transductional derivative was screened for conjugal transfer to PA08 (selection medium, BAB + Cb and Sm). The same protocol was foliowed for the detection of supB- and sup2-sensitive Tra- mutants, except selection for these mutants in the sup hosts after G101h2 or E79tv-2 transduction was imposed on MM with Cb. Isogenic sup' transductional derivatives of those Tra- mutants (obtained by selection for donor-specific phage resistance or after in vivo mutagenesis) demonstrating transfer from the sup hosts were constructed. These
J. BACTERIOL.
were also tested for transfer to determine that the transfer observed from the sup hosts was not attributable to a chromosomal mutation affecting plasmid transfer in the original host background. No sup' derivatives of suppressible HA-induced Tra- mutants were constructed since Tra- mutants obtained using this method must be plasmid coded because of the transformation step.
RESULTS Isolation of Tra- mutants. Since spontaneous mutants are rare (frequency of less than 10-1 tested clones), Tra- mutants were isolated after induction of resistance to donor-specific phages or mutagenesis. (i) Seventy-six percent of phage PRD1-resistant derivatives were concomitantly Tra-, and 86% of phage PR4-resistant mutants were also Tra-. More than 100 Tra- mutants, from 40 independent cultures, were isolated. (ii) EMS-induced Tra- mutants occurred at a frequency of approxunately 1 per 5,000 clones. Seven EMS-induced mutants were isolated. Treatment with NTG resulted in mutant frequencies as high as 2.5 x 10-2 per clone tested. From 21 independent cultures, 106 Tra- mutants were isolated. (iii) HA mutagenesis of R91-5 DNA usually resulted in a 100 to 1,000-fold reduction in transformation frequency, giving a transformation frequency of mutagenized DNA in the range from 1o-7 to 10-9 per viable competent cell (compared with frequencies of the order of 10-5 to lo-' for nonmutagenized DNA). Control experiments indicated thet the reduction in transformation frequency was not due to HA-induced damage to plasmi DNA. Therefore, the decline was probably due to mechanical shearing during pipetting (although tlhi, was kept to a minimum) or single-strand breaks caused by dialysis (17). Tra- mutants isolated after in vitro mutagenesis of plasmid DNA by HA occurred at frequencies ranging from 102 to iO-3 per transformed clone. Seventy-svven mutants were isolated. Characterizttion 9f mutants. In view of the proposed relationphip between R91-5 transfer and the Dps, Phi(G1l1), and Eex phenotypes, Tra- mutants were characterized with respect to these properties. A summary of the results is given in Tables 3 and 4. To determine whether point mutations were responsible for the simultaneous loss of the Phi(G1l1) and/or Eex phenotypes in mutageninduced Tra- mutants described in this study, Tra+ revertants of such mutants were obtained. Restoration of the Tra+ and Phi(G101)+ phenotypes were observed in a spontaneous Dps+ revertant of an NTG-induced Tra- mutant. Similarly, Dps+ revertants (occurring at a frequency
TRANSFER MUTANTS OF PSEUDOMQNAS PLASMID
VOL. 135, 1978
915
TABLE 3. Characterization ofphage-resistant Tra- mutants R91-5 phenotype frequencies
Class of mutant
No.
Phi
Dps
tested
+
PRD1 resistant
80
All PR4 resistant
67
PR4 resistant
90
All PRD1 resistant
80
+/13
Eexb
(G101) No. tested 10 Phi (G101)+
+
9
+/0
1
8 3 2 13 Phi (G101)+/4 10 Phi (G101)+ 0 6 6 2 2 10 Phi (G101)+/aThe efficiencies of plating for phage G101 were titrated relative to isogenic R+ and R- bacteria. Phi (G101)+, 10-3 to 10-4; Phi (G101)+/-, 5 x 10-2 to 5 x 10-'; Phi (G101)-, 1. b Control ratio of transfer to R- versus R+ recipients, -1,700, Eex+ ratio, a20; Eex+4 ratio: 5; Eexratio, 45. TABLz 4. Characterization of EMS, NTG, and HA-induced Trac mutants 10
R91-5 phenotype frequencies
mutant
No. tested
Dps + 2 21
-
Phi (Q1O1) + 7 0 58 48
_ _ __ + 7 33
_
Classes of Phi (G10) and Eex
phenotypes
Phi (G101)+ Eex+: 7 Phi (G101)+ Eex+: 29 Phi (G101)+ Eex-: 29 Phi (G101)- Eex4c: 4 Phi (G101)- Eex-: 44 74 71 77 3 6 75 2 Phi (G101)+ Eex+: 71 HAinduced Phi (G1O1)+ Eex-: 0 Phi (G101)- Eex4: 4 Phi (G1O1) Eex- 2 a The efficiencies of plating for phage G101 were titrated relative to isogenic R+ and R- bacteria. Phi (G101)+, 10-3 to 10-1; Phi (G101)-, 1. b Control transfer ratio R- versus R+ recipients, -500;, Eex+ ratio, P6; Eex- ratio,
