Vol. 131, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 405-412 Copyright X 1977 American Society for Microbiology
Printed in U.S.A.
Mutations to Temperature Sensitivity in R Plasmid pSC101 TAMOTSU HASHIMOTO-GOTOHI * AND MUTSUO SEKIGUCHI Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan Received for publication 17 February 1977
Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43°C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 430C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells harboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells harboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotropic phenotype, such as an increase of drug resistance level at 300C and a decrease in the number of plasmid genomes in a cell. Plasmids, autonomously replicating extrachromosomal genetic elements, are found in many bacteria. Current research suggests that replication of various plasmids in Escherichia coli may conform to two distinct modes, i.e., relaxed and stringent controls. Plasmid pSC101 is a tetracycline resistance R plasmid that is thought to code for six proteins (2) and to undergo stringent control (15). It can form a stable association with the host in spite of its relatively small number of copies per chromosome equivalent (1), and its replication is inhibited by chloramphenicol but not by the polA mutation (15). It has been shown, moreover, that replication of pSC101 depends on the normal function of host deoxyribonucleic acid (DNA) replication genes, such as dnaA (K. Hasunuma and M. Sekiguchi, Mol. Gen. Genet., in press). Two models for the control mechanism of autonomous DNA replication have been presented by Jacob et al. (8) and Pritchard et al. (14), i.e., the positive- and the negative-control models, respectively. Since temperature-sensitive mutants are useful probes for studies on the control mechanism of DNA replication, the
isolation of temperature-sensitive mutant plasmid has been attempted in many laboratories (4, 9, 11). We previously reported the isolation of temperature-sensitive pSC101 mutants by in vitro mutagenesis of plasmid DNA with hydroxylamine, and divided them into three classes based on their growth characteristics (6). (i) Cells harboring type I mutant plasmids (pHS1, pHS2, pHS4, pHS6, pHS7, and pHS17) fail to produce colonies on tetracycline-containing plates at both 30 and 420C once they have been grown at 420C in the absence of the drug. (ii) Cells harboring a type II mutant plasmid (pHS3) produce colonies on tetracycline-containing plates at 300C but not at 420C irrespective of preincubation temperature. (iii) Cells harboring type III mutant plasmids (pHS5, pHS8, and pHS16) cannot grow on a tetracycline-containing plate if they are preincubated at 420C without the drug, but they do produce colonies even at 420C when preincubated at 300C. The present investigation was undertaken to determine the nature of the three types of plasthe plasmid segre1 Present address: Laboratory of Molecular Genetics, mid mutations. We analyzed University of Osaka Medical School, Kita-ku, Osaka 530, gation kinetics and DNA replication of temperature-sensitive mutants and found that type I Japan. 405
406
HASHIMOTO-GOTOH AND SEKIGUCHI
J. BACTERIOL.
mutant plasmids are unable to replicate plas- rotation. After the addition of 2 ml of 5 M NaCl, the mid DNA at the restrictive temperature, lysate was kept at 0°C for 2 h and was then centriat 2°C to obtain a whereas the replication of type III mutant plas- fuged at 39,000 rpm for 30 minfluid. The cleared lynonviscous clear, supernatant mids is partially suppressed. The replication of sate was treated with an equal volume of the type II mutant plasmid is normal at high saturated with 0.01 M Tris-hydrochloride (pHphenol 7.0), temperature, and the mutant appears to have a and nucleic acids in the buffer layer were precipimutation in the tetracycline resistance gene. tated by the addition of 2 volumes of isopropanol. Furthermore, it was demonstrated that the rep- The precipitate was dissolved in 5 ml of 1 x SSC (0.15 lication of pSC101 and its mutants conforms M NaCl plus 0.015 M sodium citrate) containing 0.4% Sarkosyl, and then 5 ml of 5 M NaCl was well with the random-dilution model. added. After the mixture stood at 0°C for 90 min, ribonucleic acid was precipitated by cenribosomal MATERIALS AND METHODS Bacterial strains and plasmids. All of the strains of bacteria used in this study are derivatives of E. coli K-12. Strains Om84 and OmlO5 are thymine (2 ,g/ml)-requiring mutants selected with trimethoprim from strains KN108 [trpE(Am)9828 tyr(Am) supD] and KN123 [trpE(Am)9828 tyr(Am) his ilv Su-) (13), respectively, which were provided by H. Iyehara. Strains C600 (thr leu thi lacY tonA supE) and W3110T (thy Su-) were obtained from K. Matsubara. The plasmids, pSC101 and pHS1 through pHS17, used in this study have been described previously (6). Media. PBB liquid medium, used for ordinary cell growth, contained 10 g of polypeptone (Diago Eiyo Chemicals), 5 g of bonito extract (Katayama Chemicals), and 2.5 g of NaCl in 1 liter of water. PBB agar contained 10 g of polypeptone, 10 g of bonito extract, 2.5 g of NaCl, and 13 g of agar (Wako Pure Chemicals) in 1 liter. PB medium, used for radioactive thymine labeling of DNA, contained 10 g of polypeptone, 1 g of bonito extract, and 2.5 g of NaCl in 1 liter. The pH of each medium was adjusted to 7.0 with NaOH. Antibiotic-containing agar was prepared by adding tetracycline to autoclaved agar at 55°C. The plates were dried at 42°C for 40 min and used without delay. Determination of level of tetracycline resistance. Cells were grown overnight in PBB medium. The culture was diluted 100-fold, and 1-Al portions were streaked onto PBB plates containing tetracycline at various concentrations (0.8, 1.5, 3.0, 6.0, 12, 25, 50, or 100 MAg/ml). The plates were incubated overnight at 30 or 42°C, and the degree of growth was examined. The level of tetracycline resistance was expressed by maximum allowance concentration (MAC), at and below which cells exhibit confluent growth. Isolation of plasmid DNA and transformation. Om84 bacteria carrying plasmids were grown in 100 ml of PBB to a density of 3 x 109/ml, harvested, and washed with 0.02 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 8.0)-0.14 M NaCl. The pellet (wet weight, 0.3 g) was suspended in 4.5 ml of 0.04 M Tris-hydrochloride (pH 8.0)-0.025 M ethylenediaminetetraacetic acid and incubated with 0.13 mg of lysozyme per ml at 0°C for 25 min, and 4 ml of Sarkosyl (Geigy NL97) (0.8% solution in the same buffer) was added. The viscous lysate was kept at 25°C for 10 min with occasional gentle mixing by
trifugation at 4,000 rpm for 10 min. The nucleic acids remaining in the solution were precipitated with ethanol, dissolved in 1 ml of 0.2x SSC, and kept at 94°C for 5 min. After heating, the mixture was quickly cooled by shaking in an ice bath, mixed with 0.4 ml of 20x SSC, and filtered through a membrane filter (Millipore Corp.; type HA; pore size, 0.45 ,m). Portions (50 ,A) were used for CaCl2 transformation, with Su+ and Su- cells as recipients (6). Determination of the number of plasmid copies per chromosome equivalent. The preparation of whole-cell lysate and determination of plasmid copy number per chromosome were done as described by Matsubara et al. (12). Om84 bacteria harboring plasmids were grown to an optical density at 660 nm of 0.3 in 5 ml of PB at 30°C, labeled with 1.6 ,tCi of [3Hlthymine per ml for 90 min, harvested, and washed twice with 0.02 M Tris-hydrochloride (pH 8.0)-0.14 M NaCl. Cells were suspended in 3 ml of 0.04 M Tris-hydrochloride (pH 8.0)-0.02 M ethylenediaminetetraacetic acid and lysed by the successive addition of 0.3 ml of lysozyme (4-mg/ml solution in the same buffer), 3 ml of Sarkosyl (0.8%), and 0.3 ml of self-digested Pronase (2 mg/ml; Kaken Chemicals). To the lysate (7.6 ml), 8.0 g of CsCl and 0.8 ml of ethidium bromide (4.67 mg/ml) were added, and the mixture was subjected to equilibrium centrifugation at 32,000 rpm for 80 h at 20°C with a Hitachi RP65 rotor. The content of each tube was fractionated into about 35 fractions, and the trichloroacetic acid-insoluble radioactivity was counted by using a toluene-2,5-diphenyloxazole (4 g/liter)-2,2'-phenylene-bis-(5-phenyloxazole) (50 mg/liter) liquid scintillation system. The amount of plasmid DNA was determined from the profiles of centrifugation. The number of plasmids per chromosome was calculated by assuming that the molecular weights of the plasmid and the E. coli chromosome are 5.8 x 106 (3) and 3 x 109, respectively. Since this assay measures only covalently closed circular (CCC) DNA but not other forms of plasmid DNA (e.g., relaxation complex), the value should be taken as the minimum copy number. However, there is evidence that most of pSC101 DNA is present as covalently closed circular DNA (Hasunuma and Sekiguchi, in press). Determination of plasmid DNA synthesis. Bacteria were incubated overnight in PB in the presence of 3 Ag of tetracycline per ml. Cells were washed twice with fresh PB lacking tetracycline, diluted 1:20 in 5 ml of fresh PB, and then cultured for about 2 h to an optical density at 660 nm of 0.2 in the ab-
TEMPERATURE-SENSITIVE R PLASMIDS
VOL. 131, 1977 of tetracycline. Cells were prelabeled with [14C]thymine (4 ,uCi/ml) for 90 min at 30°C, washed with fresh medium, and suspended in 5 ml of PB. After incubation for 10 min at 30°C, followed by additional incubation for 5 min at 43°C, cells were labeled with [3H]thymine (8 ACi/ml) for 35 min at 43°C. At the end of the incubation period, 1 ml of saturated thymine solution and 0.15 ml of 2 M NaN3 were added, and the mixture was chilled immediately. Cells were washed twice with ice-cold 0.02 M Tris-hydrochloride (pH 8.0)-0.01 M ethylenediaminetetraacetic acid-0.05 M NaN3 and lysed by the procedure described above. The lysate was centrifuged at 39,000 rpm for 30 min at 2°C, with a Hitachi RP65 rotor, to remove the bulk of chromosomal DNA. The supernatant fluid (cleared lysate), adjusted to 6.7 ml, was mixed with 6.7 g of CsCl and 0.7 ml of propidium diiodide (3.4 mg/ml), and the sample was subjected to equilibrium ultracentrifugation (39,000 rpm for 55 h at 20°C in the same rotor). Fractions (150 Ml) were collected from the bottom of the centrifuge tube, and the radioactivity of cold trichloroacetic acid-precipitable materials in each fraction was determined. sence
RESULTS
Suppressor sensitivity of temperature-sensitive mutant plasmids. Because plasmid mutants had been isolated in E. coli carrying a temperature-sensitive suppressor I mutation, supD(Ts), we examined whether the temperature sensitivity was due to an amber or missense mutation. Plasmid DNAs were isolated from the original strains, partially purified, and then applied to CaCl2-treated Su+ cells (Om84 supD and C600 supE) and Su- cells (OmlO5 and W3110T). Transformants were selected on tetracycline (3 ,g/ml)-containing plates at 30°C, and 30 clones for each mutant were used for further tests. Mutant plasmid DNAs, except pHS3 DNA, showed an ability to transform both Su+ and Su- bacteria to antibiotic-resistant cells with almost equal efficiency. Since all of the clones examined, irrespective of Su+ or Su- hosts, produced colonies at 30°C but not at 42°C in the presence of tetracycline after preincubation without tetracycline at 42°C, these mutants seem to be temperature sensitive owing to plasmid missense mutations but not to supD(Ts). On the other hand, pHS3 DNA transfonned strains Om84 (supD) and C600 (supE) but not OmlO5 and W3110T. When Su- cells were treated with pHS3 DNA, only small, shriveled colonies, which turned out to contain tetracycline-sensitive cells, were formed on selective plates at 30°C. It is concluded that pHS3 has an amber mutation, whereas the other mutants have true temperature-sensitive mutations in the plasmid genome. Tetracycline resistance of bacteria harbor-
407
ing temperature-sensitive mutant plasmids. The effect of temperature on the tetracycline resistance of bacteria harboring plasmid mutants was determined (Table 1) on the basis of two criteria: MAC for confluent growth of bacteria and efficiency of plating at a fixed concentration of the drug. MACs of cells harboring type I or II mutants decreased with increasing temperature, whereas MACs of cells harboring wild-type plasmid pSC1I1 increased at high temperatures. MACs for type III mutants were not significantly affected by temperature. More-dramatic effects of temperature were seen when the efficiencies of plating on tetracycline-containing plates were compared. The efficiencies of plating of cells harboring type I or TABLE 1. Effect of temperature on the antibiotic resistance levels of Om84 bacteria harboring temperature-sensitive mutant plasmids Colony-forming MACE (zg/ml) ~ ability on TcPlasmid containing 30d C 42°C agarb Group I 6 3 1.5 x 10pHS1 1.4 x 10-O 6 3 pHS2 6 3 1.1 X 10-4 pHS4 3 1.5 x 10-4 6 pHS61 3 9.5 x io-` 6 pHS7C 3 9.4 x 10-6 pHS17
Group II pHS3d
Group III pHS5 pHS8 pHS16 Wild type pSC101
3
12 6-12 12 6
1.5
3.3 x 10-:-
12 6-12 12
0.91'
12
1.0
1.1
0.82'
JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 405-412 Copyright X 1977 American Society for Microbiology
Printed in U.S.A.
Mutations to Temperature Sensitivity in R Plasmid pSC101 TAMOTSU HASHIMOTO-GOTOHI * AND MUTSUO SEKIGUCHI Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan Received for publication 17 February 1977
Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43°C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 430C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells harboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells harboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotropic phenotype, such as an increase of drug resistance level at 300C and a decrease in the number of plasmid genomes in a cell. Plasmids, autonomously replicating extrachromosomal genetic elements, are found in many bacteria. Current research suggests that replication of various plasmids in Escherichia coli may conform to two distinct modes, i.e., relaxed and stringent controls. Plasmid pSC101 is a tetracycline resistance R plasmid that is thought to code for six proteins (2) and to undergo stringent control (15). It can form a stable association with the host in spite of its relatively small number of copies per chromosome equivalent (1), and its replication is inhibited by chloramphenicol but not by the polA mutation (15). It has been shown, moreover, that replication of pSC101 depends on the normal function of host deoxyribonucleic acid (DNA) replication genes, such as dnaA (K. Hasunuma and M. Sekiguchi, Mol. Gen. Genet., in press). Two models for the control mechanism of autonomous DNA replication have been presented by Jacob et al. (8) and Pritchard et al. (14), i.e., the positive- and the negative-control models, respectively. Since temperature-sensitive mutants are useful probes for studies on the control mechanism of DNA replication, the
isolation of temperature-sensitive mutant plasmid has been attempted in many laboratories (4, 9, 11). We previously reported the isolation of temperature-sensitive pSC101 mutants by in vitro mutagenesis of plasmid DNA with hydroxylamine, and divided them into three classes based on their growth characteristics (6). (i) Cells harboring type I mutant plasmids (pHS1, pHS2, pHS4, pHS6, pHS7, and pHS17) fail to produce colonies on tetracycline-containing plates at both 30 and 420C once they have been grown at 420C in the absence of the drug. (ii) Cells harboring a type II mutant plasmid (pHS3) produce colonies on tetracycline-containing plates at 300C but not at 420C irrespective of preincubation temperature. (iii) Cells harboring type III mutant plasmids (pHS5, pHS8, and pHS16) cannot grow on a tetracycline-containing plate if they are preincubated at 420C without the drug, but they do produce colonies even at 420C when preincubated at 300C. The present investigation was undertaken to determine the nature of the three types of plasthe plasmid segre1 Present address: Laboratory of Molecular Genetics, mid mutations. We analyzed University of Osaka Medical School, Kita-ku, Osaka 530, gation kinetics and DNA replication of temperature-sensitive mutants and found that type I Japan. 405
406
HASHIMOTO-GOTOH AND SEKIGUCHI
J. BACTERIOL.
mutant plasmids are unable to replicate plas- rotation. After the addition of 2 ml of 5 M NaCl, the mid DNA at the restrictive temperature, lysate was kept at 0°C for 2 h and was then centriat 2°C to obtain a whereas the replication of type III mutant plas- fuged at 39,000 rpm for 30 minfluid. The cleared lynonviscous clear, supernatant mids is partially suppressed. The replication of sate was treated with an equal volume of the type II mutant plasmid is normal at high saturated with 0.01 M Tris-hydrochloride (pHphenol 7.0), temperature, and the mutant appears to have a and nucleic acids in the buffer layer were precipimutation in the tetracycline resistance gene. tated by the addition of 2 volumes of isopropanol. Furthermore, it was demonstrated that the rep- The precipitate was dissolved in 5 ml of 1 x SSC (0.15 lication of pSC101 and its mutants conforms M NaCl plus 0.015 M sodium citrate) containing 0.4% Sarkosyl, and then 5 ml of 5 M NaCl was well with the random-dilution model. added. After the mixture stood at 0°C for 90 min, ribonucleic acid was precipitated by cenribosomal MATERIALS AND METHODS Bacterial strains and plasmids. All of the strains of bacteria used in this study are derivatives of E. coli K-12. Strains Om84 and OmlO5 are thymine (2 ,g/ml)-requiring mutants selected with trimethoprim from strains KN108 [trpE(Am)9828 tyr(Am) supD] and KN123 [trpE(Am)9828 tyr(Am) his ilv Su-) (13), respectively, which were provided by H. Iyehara. Strains C600 (thr leu thi lacY tonA supE) and W3110T (thy Su-) were obtained from K. Matsubara. The plasmids, pSC101 and pHS1 through pHS17, used in this study have been described previously (6). Media. PBB liquid medium, used for ordinary cell growth, contained 10 g of polypeptone (Diago Eiyo Chemicals), 5 g of bonito extract (Katayama Chemicals), and 2.5 g of NaCl in 1 liter of water. PBB agar contained 10 g of polypeptone, 10 g of bonito extract, 2.5 g of NaCl, and 13 g of agar (Wako Pure Chemicals) in 1 liter. PB medium, used for radioactive thymine labeling of DNA, contained 10 g of polypeptone, 1 g of bonito extract, and 2.5 g of NaCl in 1 liter. The pH of each medium was adjusted to 7.0 with NaOH. Antibiotic-containing agar was prepared by adding tetracycline to autoclaved agar at 55°C. The plates were dried at 42°C for 40 min and used without delay. Determination of level of tetracycline resistance. Cells were grown overnight in PBB medium. The culture was diluted 100-fold, and 1-Al portions were streaked onto PBB plates containing tetracycline at various concentrations (0.8, 1.5, 3.0, 6.0, 12, 25, 50, or 100 MAg/ml). The plates were incubated overnight at 30 or 42°C, and the degree of growth was examined. The level of tetracycline resistance was expressed by maximum allowance concentration (MAC), at and below which cells exhibit confluent growth. Isolation of plasmid DNA and transformation. Om84 bacteria carrying plasmids were grown in 100 ml of PBB to a density of 3 x 109/ml, harvested, and washed with 0.02 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 8.0)-0.14 M NaCl. The pellet (wet weight, 0.3 g) was suspended in 4.5 ml of 0.04 M Tris-hydrochloride (pH 8.0)-0.025 M ethylenediaminetetraacetic acid and incubated with 0.13 mg of lysozyme per ml at 0°C for 25 min, and 4 ml of Sarkosyl (Geigy NL97) (0.8% solution in the same buffer) was added. The viscous lysate was kept at 25°C for 10 min with occasional gentle mixing by
trifugation at 4,000 rpm for 10 min. The nucleic acids remaining in the solution were precipitated with ethanol, dissolved in 1 ml of 0.2x SSC, and kept at 94°C for 5 min. After heating, the mixture was quickly cooled by shaking in an ice bath, mixed with 0.4 ml of 20x SSC, and filtered through a membrane filter (Millipore Corp.; type HA; pore size, 0.45 ,m). Portions (50 ,A) were used for CaCl2 transformation, with Su+ and Su- cells as recipients (6). Determination of the number of plasmid copies per chromosome equivalent. The preparation of whole-cell lysate and determination of plasmid copy number per chromosome were done as described by Matsubara et al. (12). Om84 bacteria harboring plasmids were grown to an optical density at 660 nm of 0.3 in 5 ml of PB at 30°C, labeled with 1.6 ,tCi of [3Hlthymine per ml for 90 min, harvested, and washed twice with 0.02 M Tris-hydrochloride (pH 8.0)-0.14 M NaCl. Cells were suspended in 3 ml of 0.04 M Tris-hydrochloride (pH 8.0)-0.02 M ethylenediaminetetraacetic acid and lysed by the successive addition of 0.3 ml of lysozyme (4-mg/ml solution in the same buffer), 3 ml of Sarkosyl (0.8%), and 0.3 ml of self-digested Pronase (2 mg/ml; Kaken Chemicals). To the lysate (7.6 ml), 8.0 g of CsCl and 0.8 ml of ethidium bromide (4.67 mg/ml) were added, and the mixture was subjected to equilibrium centrifugation at 32,000 rpm for 80 h at 20°C with a Hitachi RP65 rotor. The content of each tube was fractionated into about 35 fractions, and the trichloroacetic acid-insoluble radioactivity was counted by using a toluene-2,5-diphenyloxazole (4 g/liter)-2,2'-phenylene-bis-(5-phenyloxazole) (50 mg/liter) liquid scintillation system. The amount of plasmid DNA was determined from the profiles of centrifugation. The number of plasmids per chromosome was calculated by assuming that the molecular weights of the plasmid and the E. coli chromosome are 5.8 x 106 (3) and 3 x 109, respectively. Since this assay measures only covalently closed circular (CCC) DNA but not other forms of plasmid DNA (e.g., relaxation complex), the value should be taken as the minimum copy number. However, there is evidence that most of pSC101 DNA is present as covalently closed circular DNA (Hasunuma and Sekiguchi, in press). Determination of plasmid DNA synthesis. Bacteria were incubated overnight in PB in the presence of 3 Ag of tetracycline per ml. Cells were washed twice with fresh PB lacking tetracycline, diluted 1:20 in 5 ml of fresh PB, and then cultured for about 2 h to an optical density at 660 nm of 0.2 in the ab-
TEMPERATURE-SENSITIVE R PLASMIDS
VOL. 131, 1977 of tetracycline. Cells were prelabeled with [14C]thymine (4 ,uCi/ml) for 90 min at 30°C, washed with fresh medium, and suspended in 5 ml of PB. After incubation for 10 min at 30°C, followed by additional incubation for 5 min at 43°C, cells were labeled with [3H]thymine (8 ACi/ml) for 35 min at 43°C. At the end of the incubation period, 1 ml of saturated thymine solution and 0.15 ml of 2 M NaN3 were added, and the mixture was chilled immediately. Cells were washed twice with ice-cold 0.02 M Tris-hydrochloride (pH 8.0)-0.01 M ethylenediaminetetraacetic acid-0.05 M NaN3 and lysed by the procedure described above. The lysate was centrifuged at 39,000 rpm for 30 min at 2°C, with a Hitachi RP65 rotor, to remove the bulk of chromosomal DNA. The supernatant fluid (cleared lysate), adjusted to 6.7 ml, was mixed with 6.7 g of CsCl and 0.7 ml of propidium diiodide (3.4 mg/ml), and the sample was subjected to equilibrium ultracentrifugation (39,000 rpm for 55 h at 20°C in the same rotor). Fractions (150 Ml) were collected from the bottom of the centrifuge tube, and the radioactivity of cold trichloroacetic acid-precipitable materials in each fraction was determined. sence
RESULTS
Suppressor sensitivity of temperature-sensitive mutant plasmids. Because plasmid mutants had been isolated in E. coli carrying a temperature-sensitive suppressor I mutation, supD(Ts), we examined whether the temperature sensitivity was due to an amber or missense mutation. Plasmid DNAs were isolated from the original strains, partially purified, and then applied to CaCl2-treated Su+ cells (Om84 supD and C600 supE) and Su- cells (OmlO5 and W3110T). Transformants were selected on tetracycline (3 ,g/ml)-containing plates at 30°C, and 30 clones for each mutant were used for further tests. Mutant plasmid DNAs, except pHS3 DNA, showed an ability to transform both Su+ and Su- bacteria to antibiotic-resistant cells with almost equal efficiency. Since all of the clones examined, irrespective of Su+ or Su- hosts, produced colonies at 30°C but not at 42°C in the presence of tetracycline after preincubation without tetracycline at 42°C, these mutants seem to be temperature sensitive owing to plasmid missense mutations but not to supD(Ts). On the other hand, pHS3 DNA transfonned strains Om84 (supD) and C600 (supE) but not OmlO5 and W3110T. When Su- cells were treated with pHS3 DNA, only small, shriveled colonies, which turned out to contain tetracycline-sensitive cells, were formed on selective plates at 30°C. It is concluded that pHS3 has an amber mutation, whereas the other mutants have true temperature-sensitive mutations in the plasmid genome. Tetracycline resistance of bacteria harbor-
407
ing temperature-sensitive mutant plasmids. The effect of temperature on the tetracycline resistance of bacteria harboring plasmid mutants was determined (Table 1) on the basis of two criteria: MAC for confluent growth of bacteria and efficiency of plating at a fixed concentration of the drug. MACs of cells harboring type I or II mutants decreased with increasing temperature, whereas MACs of cells harboring wild-type plasmid pSC1I1 increased at high temperatures. MACs for type III mutants were not significantly affected by temperature. More-dramatic effects of temperature were seen when the efficiencies of plating on tetracycline-containing plates were compared. The efficiencies of plating of cells harboring type I or TABLE 1. Effect of temperature on the antibiotic resistance levels of Om84 bacteria harboring temperature-sensitive mutant plasmids Colony-forming MACE (zg/ml) ~ ability on TcPlasmid containing 30d C 42°C agarb Group I 6 3 1.5 x 10pHS1 1.4 x 10-O 6 3 pHS2 6 3 1.1 X 10-4 pHS4 3 1.5 x 10-4 6 pHS61 3 9.5 x io-` 6 pHS7C 3 9.4 x 10-6 pHS17
Group II pHS3d
Group III pHS5 pHS8 pHS16 Wild type pSC101
3
12 6-12 12 6
1.5
3.3 x 10-:-
12 6-12 12
0.91'
12
1.0
1.1
0.82'
