JOURNAL OF BACrERIOLOGY, Feb. 1978, p. 1042-1043 0021-9193/78/0133-1042$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 133, No. 2
Printed in U.S.A.
Plasmid Conferring Increased Sensitivity to Mercuric Chloride and Cobalt Chloride Found in Some Laboratory Strains of Escherichia coli K-12 P. Y. WANG,' J. RELF,' S. PALCHAUDIJURI,' AND V. N. IYER' Department of Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6,1 and Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 482012 Received for publication 3 October 1977
Some lines of the widely used strain CR34 of Escherichia coli K-12 carry a plasmid. The plasmid has a mass of 12 ± 2 x 106 megadaltons and is maintained at a low copy number per cell (1 to 2), and this number is not amplified by growth of cells in the presence of chloramphenicol. The plasmid is designated as pCU3. The presence of pCU3 confers on the strain increased sensitivity to mercuric chloride and cobalt chloride. The plasmid has also been observed to fuse or recombine with the plasmid R64-11.
Temperature-sensitive mutations affecting DNA synthesis have been isolated from several strains of Escherichia coli K-12. During a study (8) of plasmid-induced suppression of this temperature sensitivity, the total DNA from supposedly plasmid-negative strains was radioactively labeled and examined after cesium chloride-ethidium bromide equilibrium gradient centrifugation (2). Unexpectedly, this revealed the presence of supercoiled DNA in some of the dnaB mutants (those mutant strains bearing the prefix "E", such as strains E107 and E279 (4, 9). These dnaB mutants had been isolated from a strain labeled CR34 (4, 9). We obtained and examined this particular CR34 strain (called CR34G to distinguish it from CR34 strains originating from other laboratories), and it was also found to contain supercoiled DNA. CR34 is a designation used for strains of E. coli K-12 that are widely used in several laboratories (1), and one purpose of this communication is to draw attention to the existence of this plasmid in some CR34 strains. Fortunately, we have not found supercoiled DNA in CR34 strains originating from three other sources (F. Bonhoeffer, S. Bressler, and P. Hanawalt). The presence of supercoiled DNA in CR34G was not associated with resistance to any of a number of antibiotics and other chemotherapeutics tested (ampicillin, bacitracin, carbenicillin, cephalothalin, chloramphenicol, cloxacillin, erythromycin, gentamycin, kanamycin, methicillin, nalidixic acid, nitrofurantoin, oxacillin, sulfamethanol, streptomycin, tetracycline, trimethoprim). However, we observed that CR34G and strains derived from it showed increased sensitivity to mercuric chloride (Fig. 1) and co-
balt chloride (data not shown). By growing CR34G in the presence of acridine orange (5) and screening 500 colonies for decreased sensitivity to mercuric chloride, a strain was obtained that had normal levels of resistance to mercuric chloride (comparable to CR34 strains originating from other laboratories). DNA isolated from this strain no longer contained a supercoiled DNA component. Thus, hypersensitivity to the metal salts is correlated with the presence of the plasmid. This plasmid is designated as pCU3. The introduction and stable maintenance of plasmid R64-11 by a CR34 strain (CR34B) obtained from F. Bonhoeffer to give CR34B(R64-11) did not result in increased sensitivity to the metal salts. From 1.0-liter Penassay broth cultures of the three strains, CR34G, CR34B(R64-11),. and CR34G(R64-11), supercoiled DNA was isolated and examined by electron microscopy (7). The contour lengths of 30 open circular molecules of each size class were measured. pCU3 is smaller than R64-11 and has a mass of 12 ± 2 x 10' megadaltons (Mdal). The mass of plasmid DNA isolated from CR34B(R64-11) was 64 ± 5 Mdal, but strain CR34G(R64-11) had the above two size classes of plasmid DNA and in addition a third size class with an average molecular weight of 75 + 5 Mdal, which is approximately the sum of the sizes of plasmid R64-11 and plasmid pCU3. This suggests that pCU3 is able to fuse or recombine with R64-11 with the loss of little, if any, DNA. For making measurements of the length of plasmid DNA molecules, we have selected electron micrographs showing open circular molecules. In the case of plasmid DNA preparations such as those from CR34G(R64-11), in which three size classes of
1042
NOTES
VOL. 133, 1978
pCU3 was maintained at a low copy number, and other experiments (data not shown) have indicated that it is not amplified by growth of the strain in the presence of chloramphenicol. Apart from the increased sensitivity to the two metal salts and its ability to fuse or recombine with R64-11, no other phenotype has been found to be associated with pCU3. In staphylococci, the ability of some naturally occurring plasmids to increase the sensitivity of bacteria to metal salts has been reported (6). In the plasmid R100 of enteric bacteria, a plasmid that has been more intensely studied than most R plasmids, a locus (mer) conferring mercuric resistance has been recognized and mapped approximately (3). Mercuric-hypersensitive variants of R100 have been isolated recently, and it is thought that the mercury hypersensitivity of these strains reflects a mercury uptake function in the absence of the usual mercury-detoxifying enzyme (S. Silver, T. Foster, and R. Rownd, personal communication). It is therefore possible that the region on pCU3 defining mercuric sensitivity may be structurally and functionally related to that part of the mer region of R100 that determines mercuric uptake.
-2\
z w
0
x w
a. 0
-4
2
3
CONCENTRATION OF HgCI2
4
5
(x103M)
FIG. 1. Survival of strain CR34B (0) and strain CR34G (0) on Penassay agar containing different concentrations of HgC42. Dilutions of Penassay broth cultures in the late exponential phase ofgrowth were spread on Penassay agar plates containing varying concentrations of HgCl2 and incubated for 2 days at 37°C. The differential sensitivity of the strains to HgCl2 and CoCl2 could also be readily recognized by replicating colonies directly onto nutrient agar plates containing HgCl2 or CoCl2 in the range of 1Cr-
to
1043
lt5 M.
open circular molecules are observed and measured, the proportion of the three measured classes may not accurately reflect the in vivo proportion of these molecules. For example, the smallest molecules may be nicked less frequently so that fewer of them assume the open circular form; on the other hand, the largest molecules may tend to be nicked more than once and become linear. With this important reservation stated, we are able to report that in preparations of plasmid DNA from CR34G(R64-11), the proportion of the three size classes of molecules was 4:5:5. This suggests that fusion or recombination between R64-11 and pCU3 probably occurs frequently within the cells. In CR34G,
We thank F. Bonhoeffer, S. Bresler, H. Goldfine, and P. Hanawalt for the CR34 strains used. V.N.I. is grateful to the Medical Research Council of Canada for support of this research.
LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Bazaral, M., and D. Helinski. 1968. Circular DNA forms of colicinogenic factors El, E2 and E3 from E. coli. J.
Mol. Biol. 36:185-194. 3. Dempsey, W. B., and N. S. Willets. 1976. Plasmid cointegrates of prophage lambda and R factor R100. J. Bacteriol. 126:166-176. 4. Gross, J. D. 1972. DNA replication in bacteria. Curr. Top. Microbiol. Immunol. 57:39-74. 5. Hirota, Y. 1960. The effect of acridine dyes on mating type factors in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 46:57-61. 6. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J. Bacteriol. 95:1335-1342. 7. Palchaudhuri, S., E. Bell, and M. R. J. Salton. 1975.
Electron microscopy of plasmid deoxyribonucleic acid from Neiseria gonorrhoeae. Infect. Immun. 11:1141-1146. 8. Wang, P. Y., and V. N. Iyer. 1977. Suppression and enhancement of temperature sensitivity of dnaB mutations of Escherichia coli K-12 by conjugative plasmids. Plasmid 1:19-33. 9. Wechsler, J., and J. D. Gross. 1971. Escherichia coli mutants temperature sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284.
Vol. 133, No. 2
Printed in U.S.A.
Plasmid Conferring Increased Sensitivity to Mercuric Chloride and Cobalt Chloride Found in Some Laboratory Strains of Escherichia coli K-12 P. Y. WANG,' J. RELF,' S. PALCHAUDIJURI,' AND V. N. IYER' Department of Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6,1 and Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 482012 Received for publication 3 October 1977
Some lines of the widely used strain CR34 of Escherichia coli K-12 carry a plasmid. The plasmid has a mass of 12 ± 2 x 106 megadaltons and is maintained at a low copy number per cell (1 to 2), and this number is not amplified by growth of cells in the presence of chloramphenicol. The plasmid is designated as pCU3. The presence of pCU3 confers on the strain increased sensitivity to mercuric chloride and cobalt chloride. The plasmid has also been observed to fuse or recombine with the plasmid R64-11.
Temperature-sensitive mutations affecting DNA synthesis have been isolated from several strains of Escherichia coli K-12. During a study (8) of plasmid-induced suppression of this temperature sensitivity, the total DNA from supposedly plasmid-negative strains was radioactively labeled and examined after cesium chloride-ethidium bromide equilibrium gradient centrifugation (2). Unexpectedly, this revealed the presence of supercoiled DNA in some of the dnaB mutants (those mutant strains bearing the prefix "E", such as strains E107 and E279 (4, 9). These dnaB mutants had been isolated from a strain labeled CR34 (4, 9). We obtained and examined this particular CR34 strain (called CR34G to distinguish it from CR34 strains originating from other laboratories), and it was also found to contain supercoiled DNA. CR34 is a designation used for strains of E. coli K-12 that are widely used in several laboratories (1), and one purpose of this communication is to draw attention to the existence of this plasmid in some CR34 strains. Fortunately, we have not found supercoiled DNA in CR34 strains originating from three other sources (F. Bonhoeffer, S. Bressler, and P. Hanawalt). The presence of supercoiled DNA in CR34G was not associated with resistance to any of a number of antibiotics and other chemotherapeutics tested (ampicillin, bacitracin, carbenicillin, cephalothalin, chloramphenicol, cloxacillin, erythromycin, gentamycin, kanamycin, methicillin, nalidixic acid, nitrofurantoin, oxacillin, sulfamethanol, streptomycin, tetracycline, trimethoprim). However, we observed that CR34G and strains derived from it showed increased sensitivity to mercuric chloride (Fig. 1) and co-
balt chloride (data not shown). By growing CR34G in the presence of acridine orange (5) and screening 500 colonies for decreased sensitivity to mercuric chloride, a strain was obtained that had normal levels of resistance to mercuric chloride (comparable to CR34 strains originating from other laboratories). DNA isolated from this strain no longer contained a supercoiled DNA component. Thus, hypersensitivity to the metal salts is correlated with the presence of the plasmid. This plasmid is designated as pCU3. The introduction and stable maintenance of plasmid R64-11 by a CR34 strain (CR34B) obtained from F. Bonhoeffer to give CR34B(R64-11) did not result in increased sensitivity to the metal salts. From 1.0-liter Penassay broth cultures of the three strains, CR34G, CR34B(R64-11),. and CR34G(R64-11), supercoiled DNA was isolated and examined by electron microscopy (7). The contour lengths of 30 open circular molecules of each size class were measured. pCU3 is smaller than R64-11 and has a mass of 12 ± 2 x 10' megadaltons (Mdal). The mass of plasmid DNA isolated from CR34B(R64-11) was 64 ± 5 Mdal, but strain CR34G(R64-11) had the above two size classes of plasmid DNA and in addition a third size class with an average molecular weight of 75 + 5 Mdal, which is approximately the sum of the sizes of plasmid R64-11 and plasmid pCU3. This suggests that pCU3 is able to fuse or recombine with R64-11 with the loss of little, if any, DNA. For making measurements of the length of plasmid DNA molecules, we have selected electron micrographs showing open circular molecules. In the case of plasmid DNA preparations such as those from CR34G(R64-11), in which three size classes of
1042
NOTES
VOL. 133, 1978
pCU3 was maintained at a low copy number, and other experiments (data not shown) have indicated that it is not amplified by growth of the strain in the presence of chloramphenicol. Apart from the increased sensitivity to the two metal salts and its ability to fuse or recombine with R64-11, no other phenotype has been found to be associated with pCU3. In staphylococci, the ability of some naturally occurring plasmids to increase the sensitivity of bacteria to metal salts has been reported (6). In the plasmid R100 of enteric bacteria, a plasmid that has been more intensely studied than most R plasmids, a locus (mer) conferring mercuric resistance has been recognized and mapped approximately (3). Mercuric-hypersensitive variants of R100 have been isolated recently, and it is thought that the mercury hypersensitivity of these strains reflects a mercury uptake function in the absence of the usual mercury-detoxifying enzyme (S. Silver, T. Foster, and R. Rownd, personal communication). It is therefore possible that the region on pCU3 defining mercuric sensitivity may be structurally and functionally related to that part of the mer region of R100 that determines mercuric uptake.
-2\
z w
0
x w
a. 0
-4
2
3
CONCENTRATION OF HgCI2
4
5
(x103M)
FIG. 1. Survival of strain CR34B (0) and strain CR34G (0) on Penassay agar containing different concentrations of HgC42. Dilutions of Penassay broth cultures in the late exponential phase ofgrowth were spread on Penassay agar plates containing varying concentrations of HgCl2 and incubated for 2 days at 37°C. The differential sensitivity of the strains to HgCl2 and CoCl2 could also be readily recognized by replicating colonies directly onto nutrient agar plates containing HgCl2 or CoCl2 in the range of 1Cr-
to
1043
lt5 M.
open circular molecules are observed and measured, the proportion of the three measured classes may not accurately reflect the in vivo proportion of these molecules. For example, the smallest molecules may be nicked less frequently so that fewer of them assume the open circular form; on the other hand, the largest molecules may tend to be nicked more than once and become linear. With this important reservation stated, we are able to report that in preparations of plasmid DNA from CR34G(R64-11), the proportion of the three size classes of molecules was 4:5:5. This suggests that fusion or recombination between R64-11 and pCU3 probably occurs frequently within the cells. In CR34G,
We thank F. Bonhoeffer, S. Bresler, H. Goldfine, and P. Hanawalt for the CR34 strains used. V.N.I. is grateful to the Medical Research Council of Canada for support of this research.
LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Bazaral, M., and D. Helinski. 1968. Circular DNA forms of colicinogenic factors El, E2 and E3 from E. coli. J.
Mol. Biol. 36:185-194. 3. Dempsey, W. B., and N. S. Willets. 1976. Plasmid cointegrates of prophage lambda and R factor R100. J. Bacteriol. 126:166-176. 4. Gross, J. D. 1972. DNA replication in bacteria. Curr. Top. Microbiol. Immunol. 57:39-74. 5. Hirota, Y. 1960. The effect of acridine dyes on mating type factors in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 46:57-61. 6. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J. Bacteriol. 95:1335-1342. 7. Palchaudhuri, S., E. Bell, and M. R. J. Salton. 1975.
Electron microscopy of plasmid deoxyribonucleic acid from Neiseria gonorrhoeae. Infect. Immun. 11:1141-1146. 8. Wang, P. Y., and V. N. Iyer. 1977. Suppression and enhancement of temperature sensitivity of dnaB mutations of Escherichia coli K-12 by conjugative plasmids. Plasmid 1:19-33. 9. Wechsler, J., and J. D. Gross. 1971. Escherichia coli mutants temperature sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284.
