Vol. 134, No. 2
JOURNAL OF BACrERIOLOGY, June 1978, p. 765-770
0021-9193/78/0134-0765$02.00/0
Printed in U.S.A.
Copyright i 1978 American Society for Microbiology
Analogs of the dnaB gene of Escherichia coli K-12 Associated with Conjugative R Plasmids P. Y. WANG AND V. N. IYER* Department of Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6
Received for publication 19 January 1978
The dnaB266(Am) mutation in Escherichia coli K-12 is an amber mutation such that strains carrying this mutation are not viable in a sup' strain. With five different R plasmids, it has been possible to construct viable R+ derivatives of this amber mutant and show that the plasmids themselves do not carry amber suppressors. This is interpreted as evidence for the presence of dnaB analog genes associated with these plasmids. Plasmid-positive strains carrying these genes often showed some degree of cryosensitivity of DNA synthesis and colonyforming ability. These observations indicate that the presence of dnaB analog genes in association with R plasmids must be relevant to the plasmid state or to some aspect of conjugative ability. Bacterial plasmids are a category of genetic elements that consist of autonomous genetic units of duplex DNA that replicate in nature only within cells. They are maintained in a stable manner in such cells (14, 16). This situation may suggest that many of the genes and gene products that are concerned with the replication of the DNA of the bacterial chromosome would be available for and utilizable by bacterial plasmids. Plasmids, according to this reasoning, need to have associated with them only those replication genes that are plasmid specific or which determine plasmid autonomy in some way, relying for most essential replication functions on genes and gene products provided by the host. In the case of dnaB, an essential DNA replication gene of Escherichia coli (3, 7, 17), there is evidence which suggests that this may not be generally true. The genome of bacteriophage P1, which in E. coli K-12 lysogens is maintained as a plasmid (9), includes an analog of the dnaB gene (3, 17). Recent observations in this laboratory (24) have also indicated that a majority of wild-type, lowcopy-number, antibiotic-resistant plasmids (conjugative R plasmids), when introduced into thermosensitive DnaB(Ts) mutants of E. coli K-12, either suppressed or enhanced the thermosensitivity of DNA synthesis and of colony-forming ability. The pattern of suppression or of enhancement of thermnosensitivity that was observed with strains carrying different dnaB(Ts) mutations suggested interactions between the DnaB protein and some plasmid-specified product as a possible explanation for this phenomenon. One reasonable hypothesis to entertain is that, like P1, the genome of several R plasmids
includes analogs of the dnaB gene. If this were the case and if the analog genes were expressed, such R plasmids should be able to rescue otherwise nonviable strains carrying a dnaB amber mutation in the absence of an amber suppressor. The construction of such viable R+ strains thus provides a positive test for the presence of plasmid-associated dnaB analog genes. We describe such constructions for five different R plasmids and the properties of the derived R+ strains. The possible implications of these observations are also discussed. MATERIALS AND METHODS Bacterial strains, plasmids, and bacteriophages. Table 1 lists the strains constructed as part of this study and strains obtained from others. Table 1 does not list the primary donors of the R plasmids. In all cases, these were derivatives of strain J53 carrying the respective R plasmid. The R plasmids in these strains belong to four different incompatibility groups and have diverse origins that have been summarized before (4). Strain Q1550 carried the genome of bacteriophage P1 Cml bac-l as a lysogen. In this form, the bacteriophage genome exists as a plasmid (9). P1 Cml bac-l is a derivative of bacteriophage P1, and its derivation has been described previously (3). It was obtained from M. Yarmolinsky. The notation Cm refers to chloramphenicol resistance, and, in the phage, the gene conferring resistance is part of the phage genome. The symbol bac refers to a P1 mutation that results in constitutive production of the P1-specified DnaB analog or Ban protein. Bacteriophage P7-C1 48ts is a thermoinducible mutant of bacteriophage P7, a phage that confers ampicillin resistance and which was called previously + amp (22). Like prophage P1,
765
P7 prophage is maintained as a plasmid, and P1 and P7 are intracellularly incompatible with one another (8). The thermoinducible P7 mutant was obtained
766
WANG AND IYER
from J. Scott. In the rest of this paper, the simplified notations P1 and P7 are used for P1 Cml bac-l and P7-C1 48ts, respectively. It should be understood, however, that in the experiments described and unless specifically indicated to be otherwise, it is these two mutants of the phages that are used. The following bacteriophages carrying different amber mutations have also been used in this study: T4 amB22 (a gene 43 amber mutant and T4 amE509 (a gene 23 amber mutant), both of which were obtained from J. Drake. Media and chemicals. Difco nutrient agar was the complex medium used for bacterial strains. The minimal medium (broth and agar) was that of Davis and Mingioli (5), which was autoclaved without added glucose and to which the following filter-sterilized supplements were added as required: L-amino acids (40 ug/ml), thiamine (1 pg/ml), thymine (2 ug/ml). The sources of the various antibiotics and the concentrations at which they were used have been described previously (24). [methyl-3H]thymine was purchased from New England Nuclear Corp. Measurement of growth and of plating or plaquing efficiency. Growth of cultures at different temperatures was measured as the increase in absorbancy at 450 nm and as the increase in the number of colonies arising on nutrient agar plates incubated at the permissive temperature (usually 370C). Temperature shifts were made by diluting a culture in the exponential phase of growth into appropriately prewarmed or precooled medium such that the temperature change for the cell populations was instantaneous. Plating efficiency of bacteria was measured by spreading dilutions of cultures on the surface of nutrient agar plates that were maintained at the different temperatures both before and after plating. Plating efficiency of phage was measured in the same manner except for the use of appropriate soft agar overlays. Measurement of increase in DNA at different temperatures. Cultures growing in supplemented minimal broth were labeled in their DNA with [3H]thymine (10 pCi/2 pg per ml) for approximately four generations. Such a culture in the exponential phase of growth was diluted 20-fold into a similar prewarmed medium containing [3H]thymine at the same specific activity. Further DNA synthesis was followed at the second temperature by the accumulation of trichloroacetic acid-precipitable counts. Samples were collected on filter paper circles (Whatman 3MM) and processed as described by Lark et al. (12). DNA-DNA hybridization. High-titer P1 phage was prepared from agar plates as described by Rosner (19) and purified as described by Yun and Vapnek (28). The procedure described by Kourilsky et al. (11) was used for extracting P1 DNA and alkali denaturing and embedding it on cellulose nitrate filters (Sartorius, 0.456-m-porosity, 47-mm diameter). Microdisks of 5mm diameter were then punched out from the dried filters that had been charged previously with the P1 DNA. The DNA to be hybridized with the P1 DNA was isolated from 10-ml amounts of supplemented glucose minimal broth cultures to-which [3H]thymine had been added (10 ,uCi/2 pg per ml). The ceUs were washed by centrifugal peUeting and resuspension in buffer and lysed by the addition of 50 pig of lysozyme and 0.1% sodium sarkosinate. The lysate was incu-
J. BACTERIOL.
bated with 50 ,ug of pancreatic ribonuclease per ml for 30 min at 370C and 1 mg of Pronase per ml for 1 h at 37°C. The DNA from the lysate was then banded and purified in a cesium chloride gradient as described by Brunk and Leick (2), and the cesium chloride was removed by dialysis against 0.15 M sodium chloride-0.015 M sodium citrate. This 3H-labeled DNA was sheared by sonic treatment, denatured, and hybridized with the P1 DNA on the filters, these steps being as described by Louarn et al. (15). Although similar amounts of sheared labeled DNA were used in each case, the specific radioactivity of the DNA samples showed some variations. Some unspecific binding to the filters also occurred (as demonstrated, for example, by binding to filters charged with plasmidnegative DNA of strain Q125-see Table 3).
RESULTS Rescue of Q1560 [a strain carrying dnaB266(Am)J by the R plasmids. The mutation dnaB266(Am) is an amber mutation, and, in the presence of this mutation, a plasmid-negative strain is viable only if it also carries an amber suppressor such as supD, supE, or supF (3, 17). However, it has been shown that in the intracellular and stable presence of P1, a viable strain carrying dnaB266(Am) and the sup' allele could be constructed (3, 17). Strain Q1550 is such a strain, and in it, the Pl-specified Ban protein is produced constitutively (3). The five R plasmids R64-11, R144-3, R386, RP4, and R621a were introduced singly and by conjugation into Q1550. Resistance to tetracycline or kanamycin served as the marker for these R plasmids (Table 1), and chloramphenicol resistance served as the marker for P1. After purification, the transconjugants in each case were resistant stably and in the absence of selection to both antibiotics, all five R plasmids being compatible with P1. Further evidence that they carried both P1 and the R plasmid was obtained from the following observations: (i) they were efficient conjugal donors of the respective Rspecified resistance markers to a secondary recombination-deficient (recA) recipient, and (ii) they showed a reduced efficiency of lysogenization by P7, a phage that belongs to the same incompatibility group as P1 (8). From such strains that were inferred to carry P1 and R plasmid, strains that had lost P1 were sought by the following two procedures: (i) screening colonies for the spontaneous loss of chloramphenicol resistance and (ii) lysogenization with P7 at 300C followed by selection for chloramphenicolsensitive colonies. Such chloramphenicol-sensitive colonies, whether ampicillin resistant or not (i.e., obtained spontaneously or by P7 induction), were always tetracycline- or kanamycinresistant (the R plasmid-associated phenotype). Furthermore, the choice of the heat-inducible
V VOL. 4ANALOGS OF THE dnaB GENE OF E. COLI K-12 134, 1978
TABLE 1. E. coli K-12 strains Plasmid(ifs) presenRelevant genetic markers' P
767
Source and
ifanmd()'pentreference On chromosome On plasmid(s) (if any)
Strain
Q125 Nil trp thy thi lac mal str 3 Q125/P1 Cml P1 Cml bac-l trp thy thi lac mal str bac-l ban' This study bac-l Q1550 P1 Cml bac-l trp thy thi lac mal str dnaB266 bac-l ban' 3 PW1 P1 Cml bac-1; R64-11 trp thy thi lac mal str dnaB266 bac-l ban'; str sul tet ban' This study PW2 R64-11 trp thy thi lac mal str dnaB266 str sul tet ban This study PW3 Pl Cml bac-l; R144-3. trp thy thi lac mal str dnaB266 bac-l ban'; kan tet ban This study PW4 R144-3 trp thy thi lac mal str dnaB266 kan tet ban' This study PW5 P1 Cml bac-1; R386 tUp thy thi lac mal str dnaB266 bac-l ban'; tet ban' This study PW6 R386 tip thy thi lac mal str dnaB266 tet ban+ This study PW7 P1 Cml bac-1; RP4 trp thy thi lac mal str dnaB266 bac-l ban+; amp kan tet ban' This study PW8 RP4 trp thy thi lac mal str dnaB266 amp kan tet ban+ This study PW9 P1 Cml bac-1; R621a trp thy thi lac mal str dnaB266 bac-l ban+; tet ban+ This study PW1O R621a trp thy thi lac mal str dnaB266 tet ban+ This study J53 Nil pro met 4 CR34 RecA Nil thr eu th thi str supE recA 1 "For all the plasmid-carrying strains for which the source is shown as "this study," the plasmid was introduced by conjugation with donor J53 strains carrying the respective plasmid. The PW series of strains with even numbers are, as a rule, those that were selected for spontaneous loss of P1. 'Abbreviations of genetic markers: trp, tryptophan; thy, thymine; thi, thiamine; lac, lactose; mal, maltose; str, streptomycin resistance; dnaB266, a dnaB amber mutant; cat, chloramphemical acetyl transferase; bac, Ban analog constitutive; ban, dnaB analog; sul, sulfonamide; tet, tetracycline; kan, kanamycin; amp, ampicillin.
TABLE 2. Frequency of spontaneous or P7-induced loss of P1 from strains carrying R plasmids in addition to Pi Strain
Relevant chromosomal markers in strain
R plasmid in strain
Frequency of occurrence of clones that have lost P1
Spontaneous
P7 induced
Q125/P1 Cml bac-1 Q1550 PW1 PW3 PW5
dnaB+ sup' Nil
JOURNAL OF BACrERIOLOGY, June 1978, p. 765-770
0021-9193/78/0134-0765$02.00/0
Printed in U.S.A.
Copyright i 1978 American Society for Microbiology
Analogs of the dnaB gene of Escherichia coli K-12 Associated with Conjugative R Plasmids P. Y. WANG AND V. N. IYER* Department of Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6
Received for publication 19 January 1978
The dnaB266(Am) mutation in Escherichia coli K-12 is an amber mutation such that strains carrying this mutation are not viable in a sup' strain. With five different R plasmids, it has been possible to construct viable R+ derivatives of this amber mutant and show that the plasmids themselves do not carry amber suppressors. This is interpreted as evidence for the presence of dnaB analog genes associated with these plasmids. Plasmid-positive strains carrying these genes often showed some degree of cryosensitivity of DNA synthesis and colonyforming ability. These observations indicate that the presence of dnaB analog genes in association with R plasmids must be relevant to the plasmid state or to some aspect of conjugative ability. Bacterial plasmids are a category of genetic elements that consist of autonomous genetic units of duplex DNA that replicate in nature only within cells. They are maintained in a stable manner in such cells (14, 16). This situation may suggest that many of the genes and gene products that are concerned with the replication of the DNA of the bacterial chromosome would be available for and utilizable by bacterial plasmids. Plasmids, according to this reasoning, need to have associated with them only those replication genes that are plasmid specific or which determine plasmid autonomy in some way, relying for most essential replication functions on genes and gene products provided by the host. In the case of dnaB, an essential DNA replication gene of Escherichia coli (3, 7, 17), there is evidence which suggests that this may not be generally true. The genome of bacteriophage P1, which in E. coli K-12 lysogens is maintained as a plasmid (9), includes an analog of the dnaB gene (3, 17). Recent observations in this laboratory (24) have also indicated that a majority of wild-type, lowcopy-number, antibiotic-resistant plasmids (conjugative R plasmids), when introduced into thermosensitive DnaB(Ts) mutants of E. coli K-12, either suppressed or enhanced the thermosensitivity of DNA synthesis and of colony-forming ability. The pattern of suppression or of enhancement of thermnosensitivity that was observed with strains carrying different dnaB(Ts) mutations suggested interactions between the DnaB protein and some plasmid-specified product as a possible explanation for this phenomenon. One reasonable hypothesis to entertain is that, like P1, the genome of several R plasmids
includes analogs of the dnaB gene. If this were the case and if the analog genes were expressed, such R plasmids should be able to rescue otherwise nonviable strains carrying a dnaB amber mutation in the absence of an amber suppressor. The construction of such viable R+ strains thus provides a positive test for the presence of plasmid-associated dnaB analog genes. We describe such constructions for five different R plasmids and the properties of the derived R+ strains. The possible implications of these observations are also discussed. MATERIALS AND METHODS Bacterial strains, plasmids, and bacteriophages. Table 1 lists the strains constructed as part of this study and strains obtained from others. Table 1 does not list the primary donors of the R plasmids. In all cases, these were derivatives of strain J53 carrying the respective R plasmid. The R plasmids in these strains belong to four different incompatibility groups and have diverse origins that have been summarized before (4). Strain Q1550 carried the genome of bacteriophage P1 Cml bac-l as a lysogen. In this form, the bacteriophage genome exists as a plasmid (9). P1 Cml bac-l is a derivative of bacteriophage P1, and its derivation has been described previously (3). It was obtained from M. Yarmolinsky. The notation Cm refers to chloramphenicol resistance, and, in the phage, the gene conferring resistance is part of the phage genome. The symbol bac refers to a P1 mutation that results in constitutive production of the P1-specified DnaB analog or Ban protein. Bacteriophage P7-C1 48ts is a thermoinducible mutant of bacteriophage P7, a phage that confers ampicillin resistance and which was called previously + amp (22). Like prophage P1,
765
P7 prophage is maintained as a plasmid, and P1 and P7 are intracellularly incompatible with one another (8). The thermoinducible P7 mutant was obtained
766
WANG AND IYER
from J. Scott. In the rest of this paper, the simplified notations P1 and P7 are used for P1 Cml bac-l and P7-C1 48ts, respectively. It should be understood, however, that in the experiments described and unless specifically indicated to be otherwise, it is these two mutants of the phages that are used. The following bacteriophages carrying different amber mutations have also been used in this study: T4 amB22 (a gene 43 amber mutant and T4 amE509 (a gene 23 amber mutant), both of which were obtained from J. Drake. Media and chemicals. Difco nutrient agar was the complex medium used for bacterial strains. The minimal medium (broth and agar) was that of Davis and Mingioli (5), which was autoclaved without added glucose and to which the following filter-sterilized supplements were added as required: L-amino acids (40 ug/ml), thiamine (1 pg/ml), thymine (2 ug/ml). The sources of the various antibiotics and the concentrations at which they were used have been described previously (24). [methyl-3H]thymine was purchased from New England Nuclear Corp. Measurement of growth and of plating or plaquing efficiency. Growth of cultures at different temperatures was measured as the increase in absorbancy at 450 nm and as the increase in the number of colonies arising on nutrient agar plates incubated at the permissive temperature (usually 370C). Temperature shifts were made by diluting a culture in the exponential phase of growth into appropriately prewarmed or precooled medium such that the temperature change for the cell populations was instantaneous. Plating efficiency of bacteria was measured by spreading dilutions of cultures on the surface of nutrient agar plates that were maintained at the different temperatures both before and after plating. Plating efficiency of phage was measured in the same manner except for the use of appropriate soft agar overlays. Measurement of increase in DNA at different temperatures. Cultures growing in supplemented minimal broth were labeled in their DNA with [3H]thymine (10 pCi/2 pg per ml) for approximately four generations. Such a culture in the exponential phase of growth was diluted 20-fold into a similar prewarmed medium containing [3H]thymine at the same specific activity. Further DNA synthesis was followed at the second temperature by the accumulation of trichloroacetic acid-precipitable counts. Samples were collected on filter paper circles (Whatman 3MM) and processed as described by Lark et al. (12). DNA-DNA hybridization. High-titer P1 phage was prepared from agar plates as described by Rosner (19) and purified as described by Yun and Vapnek (28). The procedure described by Kourilsky et al. (11) was used for extracting P1 DNA and alkali denaturing and embedding it on cellulose nitrate filters (Sartorius, 0.456-m-porosity, 47-mm diameter). Microdisks of 5mm diameter were then punched out from the dried filters that had been charged previously with the P1 DNA. The DNA to be hybridized with the P1 DNA was isolated from 10-ml amounts of supplemented glucose minimal broth cultures to-which [3H]thymine had been added (10 ,uCi/2 pg per ml). The ceUs were washed by centrifugal peUeting and resuspension in buffer and lysed by the addition of 50 pig of lysozyme and 0.1% sodium sarkosinate. The lysate was incu-
J. BACTERIOL.
bated with 50 ,ug of pancreatic ribonuclease per ml for 30 min at 370C and 1 mg of Pronase per ml for 1 h at 37°C. The DNA from the lysate was then banded and purified in a cesium chloride gradient as described by Brunk and Leick (2), and the cesium chloride was removed by dialysis against 0.15 M sodium chloride-0.015 M sodium citrate. This 3H-labeled DNA was sheared by sonic treatment, denatured, and hybridized with the P1 DNA on the filters, these steps being as described by Louarn et al. (15). Although similar amounts of sheared labeled DNA were used in each case, the specific radioactivity of the DNA samples showed some variations. Some unspecific binding to the filters also occurred (as demonstrated, for example, by binding to filters charged with plasmidnegative DNA of strain Q125-see Table 3).
RESULTS Rescue of Q1560 [a strain carrying dnaB266(Am)J by the R plasmids. The mutation dnaB266(Am) is an amber mutation, and, in the presence of this mutation, a plasmid-negative strain is viable only if it also carries an amber suppressor such as supD, supE, or supF (3, 17). However, it has been shown that in the intracellular and stable presence of P1, a viable strain carrying dnaB266(Am) and the sup' allele could be constructed (3, 17). Strain Q1550 is such a strain, and in it, the Pl-specified Ban protein is produced constitutively (3). The five R plasmids R64-11, R144-3, R386, RP4, and R621a were introduced singly and by conjugation into Q1550. Resistance to tetracycline or kanamycin served as the marker for these R plasmids (Table 1), and chloramphenicol resistance served as the marker for P1. After purification, the transconjugants in each case were resistant stably and in the absence of selection to both antibiotics, all five R plasmids being compatible with P1. Further evidence that they carried both P1 and the R plasmid was obtained from the following observations: (i) they were efficient conjugal donors of the respective Rspecified resistance markers to a secondary recombination-deficient (recA) recipient, and (ii) they showed a reduced efficiency of lysogenization by P7, a phage that belongs to the same incompatibility group as P1 (8). From such strains that were inferred to carry P1 and R plasmid, strains that had lost P1 were sought by the following two procedures: (i) screening colonies for the spontaneous loss of chloramphenicol resistance and (ii) lysogenization with P7 at 300C followed by selection for chloramphenicolsensitive colonies. Such chloramphenicol-sensitive colonies, whether ampicillin resistant or not (i.e., obtained spontaneously or by P7 induction), were always tetracycline- or kanamycinresistant (the R plasmid-associated phenotype). Furthermore, the choice of the heat-inducible
V VOL. 4ANALOGS OF THE dnaB GENE OF E. COLI K-12 134, 1978
TABLE 1. E. coli K-12 strains Plasmid(ifs) presenRelevant genetic markers' P
767
Source and
ifanmd()'pentreference On chromosome On plasmid(s) (if any)
Strain
Q125 Nil trp thy thi lac mal str 3 Q125/P1 Cml P1 Cml bac-l trp thy thi lac mal str bac-l ban' This study bac-l Q1550 P1 Cml bac-l trp thy thi lac mal str dnaB266 bac-l ban' 3 PW1 P1 Cml bac-1; R64-11 trp thy thi lac mal str dnaB266 bac-l ban'; str sul tet ban' This study PW2 R64-11 trp thy thi lac mal str dnaB266 str sul tet ban This study PW3 Pl Cml bac-l; R144-3. trp thy thi lac mal str dnaB266 bac-l ban'; kan tet ban This study PW4 R144-3 trp thy thi lac mal str dnaB266 kan tet ban' This study PW5 P1 Cml bac-1; R386 tUp thy thi lac mal str dnaB266 bac-l ban'; tet ban' This study PW6 R386 tip thy thi lac mal str dnaB266 tet ban+ This study PW7 P1 Cml bac-1; RP4 trp thy thi lac mal str dnaB266 bac-l ban+; amp kan tet ban' This study PW8 RP4 trp thy thi lac mal str dnaB266 amp kan tet ban+ This study PW9 P1 Cml bac-1; R621a trp thy thi lac mal str dnaB266 bac-l ban+; tet ban+ This study PW1O R621a trp thy thi lac mal str dnaB266 tet ban+ This study J53 Nil pro met 4 CR34 RecA Nil thr eu th thi str supE recA 1 "For all the plasmid-carrying strains for which the source is shown as "this study," the plasmid was introduced by conjugation with donor J53 strains carrying the respective plasmid. The PW series of strains with even numbers are, as a rule, those that were selected for spontaneous loss of P1. 'Abbreviations of genetic markers: trp, tryptophan; thy, thymine; thi, thiamine; lac, lactose; mal, maltose; str, streptomycin resistance; dnaB266, a dnaB amber mutant; cat, chloramphemical acetyl transferase; bac, Ban analog constitutive; ban, dnaB analog; sul, sulfonamide; tet, tetracycline; kan, kanamycin; amp, ampicillin.
TABLE 2. Frequency of spontaneous or P7-induced loss of P1 from strains carrying R plasmids in addition to Pi Strain
Relevant chromosomal markers in strain
R plasmid in strain
Frequency of occurrence of clones that have lost P1
Spontaneous
P7 induced
Q125/P1 Cml bac-1 Q1550 PW1 PW3 PW5
dnaB+ sup' Nil
