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A stable derivative of pBR322 conferring increased tetracycline resistance and increased sensitivity to fusaric acid
PLASMID
7, 290-293 (1982)
A Stable Derivative of pBR322 Conferring Resistance and Increased Sensitivity
increased Tetracycline to Fusaric Acid
G. L. HERRIN, JR., D. R. RUSSELL,AND G. N. BENNETT Department of Biochemistry, Rice University, Houston, Texas 77001 Received December 4, 1981 A hybrid trp-tet promoter was formed on pBR322 by insertion of a segment containing part of the trp promoter at the CIaI site. The product plasmid, pDR42, conferred resistance to higher concentrations of tetracycline than pBR322. Cells bearing pDR42 were sensitive to lower concentrations of fusaric acid than were those bearing pBR322. Since the difference in growth on fusaric acid between the E. coli RR1 alone and the strain with pDR42 is greater than is the case with pBR322, an improved selection of tetracycline-sensitive (Tc’) colonies out of a background of pDR42 specified tetracycline-resistant (Tc’) colonies was observed.
A modification of a medium developed by Bochner et al. (I) for direct selection of Tc”’ bacteria was reported recently by Maloy and Nunn (2). The selection depends on the sensitivity of cells to lipophilic chelating agents such as fusaric acid. The proposed method of action of fusaric acid involves the effect of the tetracycline resistance protein (M, = 34,000) on metal ions in the membrane. The binding of metal cations by tetracycline in the membrane may aid in the transport of tetracycline across the membrane. The tetracycline resistance protein is also believed to bind these cations, thus lowering their effective concentration and preventing entrance of tetracycline. By binding additional metal ions fusaric acid may then cause a lethal reduction in the metal ion concentration in the membrane when the tetracycline resistance protein is present (I). If this proposed mechanism is correct one would expect plasmid bearing strains producing higher levels of Tc’ to be more sensitive to fusaric acid. In addition the slow growth rate of strains expressing a higher Tc’ should allow a better selection of Tc” mutants on fusaric acid. ’ Abbreviations used: Tc’, tetracycline resistance; Tc”, tetracycline sensitive; Ap’, ampicillin resistance; Km’, kanamycin resistance.
0147-619X/82/030290-04%02.00/0 Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
290
In describing the use of this selection system, Bochner et al. (I ) observed a high background of Tc’ E. coli when using a medium which included glucose. Maloy and Nunn (2) circumvented this problem by eliminating glucose from the medium, which increased the positive selection efficiency for Tc” cells to greater than 80% by slowing the growth rate. The incubation time for this medium was 24-48 h and little background problem was observed in most cases. However, neither of the above groups noted that alteration of fusaric acid concentration significantly affected the selection efficiency. In our work using pBR322 derivatives in E. coli strain RRl, we have found that the fusaric acid sensitivity varied inversely with the level of Tc’ conveyed by the plasmid. Plasmids with increased Tc’ have been described; however, they were relatively large and unstable (3). Perhaps such powerful expression from this region is destabilizing to the plasmid. We constructed a pBR322 derivative (pDR42) that exhibited increased Tc’ (Fig. 1). This plasmid contains a hybrid promoter derived from a portion of the E. coli trp operon promoter and the pBR322 Tc’ promoter. The construction involved the insertion of a segment of the trp promoter into the ClaI site of pBR322. The trp promoter
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SHORT COMMUNICATIONS
TC hg/ll
FIG. 1. Tetracycline resistance of pBR322 and pDR42 in E. coli RRl. pDR42,O; pBR322, A. All plating was done in triplicate by centrifuging 5 ml of an overnight L-broth culture and resuspending the pellet in 5 ml M9 minimal media (4). Serial dilutions were made and 0.1 ml of the appropriate dilution was plated. Plates were incubated at 37°C for 20-48 h. The percentage control axis refers to the ratio of the average number of colonies on a plate containing tetracycline to the average number of colonies on a plate containing 30 mg/liter ampicillin. The tetracycline resistance of E. co/i RR1 and E. coli RR1 bearing pRGl0 or pACYCl77 (see Fig. 4) was tested and found to be less than 2 mg/liter.
DNA was isolated by cleaving pDRl2 (5) with HpaII, electrophoresis on a 5% polyacrylamide gel, and fragment elution (6). The back half of the trp promoter was obtained by cleaving the 350-bp fragment with TuqI and isolating the 7 1-bp HpaII-TaqI fragment as described above. The 71-bp fragment was then inserted at the ClaI site of pBR322 using T4 DNA ligase (5). The DNA was introduced into RR1 by transformation and colonies were screened for increased Tc’ by replica plating. Plasmid DNA, designated pDR42, was isolated from tl~a
II t
2120
Alu I :
2116
one of the selected colonies. Detailed restriction mapping has shown that pDR42 contains a trp-tet promoter fusion such that the upstream (back half) portion of the trp promoter from the TaqI site is fused to the downstream (front half) portion of the Tc’ promoter at the ClaI site. The sequence of pDR42 is known since it differs from pBR322 only by the 71-bp insert into the CluI site (see Fig. 2). This insertion regenerates a CluI site where the TuqI and CluI ends were joined but destroys both HpuII and CluI sites where the HpuII end of the 7 1-bp fragment was joined to the vector. The restriction pattern produced from pDR42 upon cleavage with several restriction enzymes for pDR42 is as expected for the proper insertion of the 7 1-bp fragment into the CluI site of pBR322 (as sequenced by Sutcliffe (7)). pDR42 appears to be as stable and is present in approximately the same copy number as pBR322 in E. coli strain RRl. To further characterize the nature of the increased Tc’ of pDR42, the trp-tet and tet promoters were isolated on restriction fragments and individually inserted into pKO-1 (8), a plasmid designed to allow comparison of relative promoter strengths. The experiment showed the trp-tet promoter expressed higher in vivo activity than the tet promoter (data not shown) in agreement with the Tc’ levels shown in Fig. 1. In addition to studying tetracycline sensitivity, the fusaric acid sensitivity of RR1 cells bearing plasmids and RR1 cells alone was examined. The comparison of pDR42 to pBR322 (Fig. 3) shows that pDR42 is Hha I
Pvu II
Hmc II
Taq I
I I
2076
2067
FIG. 2. Restriction map of 7 1-bp fragment inserted into the CfaI site of pBR322 to construct pDR42. The region from HpoII to PvuII (2120 to 2067 using Sutcliffe’s nomenclature (7)) is derived from pBR322. The region in the shaded block from PvuII to TugI is an IS-bp region of DNA derived from pDRl2 (5) that correspondsto the back half of the E. coli trp promoter (the sequenceof the rrp promoter DNA from PvtrII at -39 to TaqI at -21 is 5’-CTGTTGACAATTAATCAT-3’, the CTG of the PvuII site, and the T of the TuqI sites are underlined). The insertion of this fragment into the ClaI site regenerates a C/n1 site and produces a trp-ret promoter fusion.
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SHORT COMMUNICATIONS
20.
I
12
14
16
FIG. 3. Fusaric acid resistance of pBR322 and pDR42 in E. coli RRl. All conditions were the same as described for Fig. 1 with the exception that fusaric acid ampicillin plates were used in the selection. For each 2 mg/liter increase in fusaric acid concentration ap proximately 2-3 additional h growth was necessary for colony formation. pDR42, 0, pBR322, A. The percentage control axis refers to the ratio of the average number of colonies on a plate containing the indicated quantity of fusaric acid plus 30 mg/liter ampicillin to the average number of colonies on an ampicillin plate. The plate composition was: agar 20 g/liter; nutrient broth 8 g/liter; NaCl 5 g/liter; cysteine 30 mg/liter. Fusaric acid resistance of E. coli RR1 and RR1 containing pACYC177 or pRGl0 was also checked and found to be greater than 20 mg/liter although growth was slow at this concentration.
more sensitive to fusanic acid suggesting that adjustment of this concentration could aid in the selection process. The rapid drop in survival after passing a critical fusaric acid concentration should be noted from Fig. 3, since this can be important in choosing the optimal fusaric acid concentration. E. coli RR1 was tested for tolerance to fusaric acid and found to be viable up to a concentration of 20 mg/liter although at this high concentration a growth period of 2 days was necessary. To check the relative ability of Tc” colonies to be selected from a background of a Tc’ strain carrying pBR322 or pDR42 the following mixtures were made: pACYC 177 (an Ap’, Tc”, Km’ plasmid) + pDR42; pACYC177 + pBR322; pRGl0 (a pBR322 derivative in which the Tc’ gene has been inactivated by insertion of a lac operator fragment into the BamHI site) + pDR42; pRGl0 + pBR322. The results of these experiments are shown in Fig. 4. It should be
noted from Fig. 4 that pDR42 allows increased efficiency of selection with either pACYC 177 or pRGl0 using a fusaric acid concentration of only 10 mg/ml. Selection for Tc” cells was also accomplished using pBR322 although colonies were surrounded by pBR322 background. The selection had an efficiency of greater than 90% for 1 TcScell in lo4 Tc’ cells (Fig. 4) when using pDR42 which has a strong promoter controlling the Tc’ gene. In addition to this high efficiency, shorter growth times were made possible by modification of the fusaric acid concentration. The ease of fusaric acid selection against Tc’ and the
‘“On
TcR
cells/ml
FIG. 4. Efficiency of selection of Tc’ bacteria in mixtures containing Tc’ bacteria. Replica plating by stippling was performed to verify that colonies on fusaric acid medium were Tc”. A lawn of small colonies was produced on plates of pACYC177 + pBR322 in cases where high amounts of RRI cells bearing pBR322 were used. Larger Tc” pACYC177 colonies were present. The percentage of those colonies from original fusaric acidampicillin selection plates which were exclusively Tc’ upon replica plating is shown as a function of the number of Tc’ cells present in the mixture plated on the original fusaric acid selection plate. The fusaric acid concentration was 10 mg/liter for pDR42 mixtures and 14 mg/liter for pBR222 mixtures. The number of Tc” cells/ml in the final mixture was approximately 5 X 10’. The screening test for pACYC177 was growth on a kanamycin (40 mg/liter)-ampicillin plate. For pRG IO, a positive test was a blue colony on the 5-bromo-rlchloro-3-indolyl-@-galactoside-ampicillin plates. The colonies were screened for Tc’ on 20 mg/liter tetracycline. pDR42 + pACYCl77, 0, pDR42 + pRGl0, 0; pBR322 + pACYC177,b pBR322 + pRG10, A.
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possibility for producing increased transcription of DNA inserted into the unique restriction sites within the Tc’ gene may make pDR42 useful as a cloning vector when these characteristics are sought. The increased tetracycline resistance, increased fusaric acid sensitivity, and increased transcription of the Tc’ gene by pDR42 as compared to pBR322 support the proposed mechanism of fusaric acid selection. The high expression of the Tc’ protein in pDR42 may account for the increased fusaric acid sensitivity of cells bearing this plasmid. ACKNOWLEDGMENTS We thank R. Gayle for pRGl0 and A. Chang for pACYCl77. The work was supported by NIH Grant GM26437 and D.R.R. was supported in part by NIH Training Grant GM 07833.
REFERENCES 1. BOCHNER,B. R., HUANG, H., SCHIEVEN, G. L., AND AMES, B. N., J. Bacterial. 145, 926-933
(1981). MALOY, S. R., AND NUNN, W. D., J. Bacterial. 145,1110-1112(1981). TACON, W., CAREY, N., AND EMTAGE, S., Mol. Gen. Genet. 177, 427-438 (1980).
MILLER, J. H., “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1972. RUSSELL,D. R., AND BENNETT, G. N., Gene 17, 9-18 (1982). SUMNER,W., II, AND BENNETT,G. N., Nucl. Acids Res. 9, 21052119 (1981). SUTCLIFFE, J. G., Cold Spring Harbor Symp. Quant. Biol. 43, 77-90 (1978). MCKENNEY, K., SCHIMATAKE, H., COURT, D., SCHMEISSNER,U., AND BRADY, C., In “Gene Amplification and Analysis” (M. Rosenberg, J. G. Chirikjian, and T. Papas, Eds.), 2, pp. 383415. Elsevier/North-Holland, New York, 1981.
7, 290-293 (1982)
A Stable Derivative of pBR322 Conferring Resistance and Increased Sensitivity
increased Tetracycline to Fusaric Acid
G. L. HERRIN, JR., D. R. RUSSELL,AND G. N. BENNETT Department of Biochemistry, Rice University, Houston, Texas 77001 Received December 4, 1981 A hybrid trp-tet promoter was formed on pBR322 by insertion of a segment containing part of the trp promoter at the CIaI site. The product plasmid, pDR42, conferred resistance to higher concentrations of tetracycline than pBR322. Cells bearing pDR42 were sensitive to lower concentrations of fusaric acid than were those bearing pBR322. Since the difference in growth on fusaric acid between the E. coli RR1 alone and the strain with pDR42 is greater than is the case with pBR322, an improved selection of tetracycline-sensitive (Tc’) colonies out of a background of pDR42 specified tetracycline-resistant (Tc’) colonies was observed.
A modification of a medium developed by Bochner et al. (I) for direct selection of Tc”’ bacteria was reported recently by Maloy and Nunn (2). The selection depends on the sensitivity of cells to lipophilic chelating agents such as fusaric acid. The proposed method of action of fusaric acid involves the effect of the tetracycline resistance protein (M, = 34,000) on metal ions in the membrane. The binding of metal cations by tetracycline in the membrane may aid in the transport of tetracycline across the membrane. The tetracycline resistance protein is also believed to bind these cations, thus lowering their effective concentration and preventing entrance of tetracycline. By binding additional metal ions fusaric acid may then cause a lethal reduction in the metal ion concentration in the membrane when the tetracycline resistance protein is present (I). If this proposed mechanism is correct one would expect plasmid bearing strains producing higher levels of Tc’ to be more sensitive to fusaric acid. In addition the slow growth rate of strains expressing a higher Tc’ should allow a better selection of Tc” mutants on fusaric acid. ’ Abbreviations used: Tc’, tetracycline resistance; Tc”, tetracycline sensitive; Ap’, ampicillin resistance; Km’, kanamycin resistance.
0147-619X/82/030290-04%02.00/0 Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
290
In describing the use of this selection system, Bochner et al. (I ) observed a high background of Tc’ E. coli when using a medium which included glucose. Maloy and Nunn (2) circumvented this problem by eliminating glucose from the medium, which increased the positive selection efficiency for Tc” cells to greater than 80% by slowing the growth rate. The incubation time for this medium was 24-48 h and little background problem was observed in most cases. However, neither of the above groups noted that alteration of fusaric acid concentration significantly affected the selection efficiency. In our work using pBR322 derivatives in E. coli strain RRl, we have found that the fusaric acid sensitivity varied inversely with the level of Tc’ conveyed by the plasmid. Plasmids with increased Tc’ have been described; however, they were relatively large and unstable (3). Perhaps such powerful expression from this region is destabilizing to the plasmid. We constructed a pBR322 derivative (pDR42) that exhibited increased Tc’ (Fig. 1). This plasmid contains a hybrid promoter derived from a portion of the E. coli trp operon promoter and the pBR322 Tc’ promoter. The construction involved the insertion of a segment of the trp promoter into the ClaI site of pBR322. The trp promoter
291
SHORT COMMUNICATIONS
TC hg/ll
FIG. 1. Tetracycline resistance of pBR322 and pDR42 in E. coli RRl. pDR42,O; pBR322, A. All plating was done in triplicate by centrifuging 5 ml of an overnight L-broth culture and resuspending the pellet in 5 ml M9 minimal media (4). Serial dilutions were made and 0.1 ml of the appropriate dilution was plated. Plates were incubated at 37°C for 20-48 h. The percentage control axis refers to the ratio of the average number of colonies on a plate containing tetracycline to the average number of colonies on a plate containing 30 mg/liter ampicillin. The tetracycline resistance of E. co/i RR1 and E. coli RR1 bearing pRGl0 or pACYCl77 (see Fig. 4) was tested and found to be less than 2 mg/liter.
DNA was isolated by cleaving pDRl2 (5) with HpaII, electrophoresis on a 5% polyacrylamide gel, and fragment elution (6). The back half of the trp promoter was obtained by cleaving the 350-bp fragment with TuqI and isolating the 7 1-bp HpaII-TaqI fragment as described above. The 71-bp fragment was then inserted at the ClaI site of pBR322 using T4 DNA ligase (5). The DNA was introduced into RR1 by transformation and colonies were screened for increased Tc’ by replica plating. Plasmid DNA, designated pDR42, was isolated from tl~a
II t
2120
Alu I :
2116
one of the selected colonies. Detailed restriction mapping has shown that pDR42 contains a trp-tet promoter fusion such that the upstream (back half) portion of the trp promoter from the TaqI site is fused to the downstream (front half) portion of the Tc’ promoter at the ClaI site. The sequence of pDR42 is known since it differs from pBR322 only by the 71-bp insert into the CluI site (see Fig. 2). This insertion regenerates a CluI site where the TuqI and CluI ends were joined but destroys both HpuII and CluI sites where the HpuII end of the 7 1-bp fragment was joined to the vector. The restriction pattern produced from pDR42 upon cleavage with several restriction enzymes for pDR42 is as expected for the proper insertion of the 7 1-bp fragment into the CluI site of pBR322 (as sequenced by Sutcliffe (7)). pDR42 appears to be as stable and is present in approximately the same copy number as pBR322 in E. coli strain RRl. To further characterize the nature of the increased Tc’ of pDR42, the trp-tet and tet promoters were isolated on restriction fragments and individually inserted into pKO-1 (8), a plasmid designed to allow comparison of relative promoter strengths. The experiment showed the trp-tet promoter expressed higher in vivo activity than the tet promoter (data not shown) in agreement with the Tc’ levels shown in Fig. 1. In addition to studying tetracycline sensitivity, the fusaric acid sensitivity of RR1 cells bearing plasmids and RR1 cells alone was examined. The comparison of pDR42 to pBR322 (Fig. 3) shows that pDR42 is Hha I
Pvu II
Hmc II
Taq I
I I
2076
2067
FIG. 2. Restriction map of 7 1-bp fragment inserted into the CfaI site of pBR322 to construct pDR42. The region from HpoII to PvuII (2120 to 2067 using Sutcliffe’s nomenclature (7)) is derived from pBR322. The region in the shaded block from PvuII to TugI is an IS-bp region of DNA derived from pDRl2 (5) that correspondsto the back half of the E. coli trp promoter (the sequenceof the rrp promoter DNA from PvtrII at -39 to TaqI at -21 is 5’-CTGTTGACAATTAATCAT-3’, the CTG of the PvuII site, and the T of the TuqI sites are underlined). The insertion of this fragment into the ClaI site regenerates a C/n1 site and produces a trp-ret promoter fusion.
292
SHORT COMMUNICATIONS
20.
I
12
14
16
FIG. 3. Fusaric acid resistance of pBR322 and pDR42 in E. coli RRl. All conditions were the same as described for Fig. 1 with the exception that fusaric acid ampicillin plates were used in the selection. For each 2 mg/liter increase in fusaric acid concentration ap proximately 2-3 additional h growth was necessary for colony formation. pDR42, 0, pBR322, A. The percentage control axis refers to the ratio of the average number of colonies on a plate containing the indicated quantity of fusaric acid plus 30 mg/liter ampicillin to the average number of colonies on an ampicillin plate. The plate composition was: agar 20 g/liter; nutrient broth 8 g/liter; NaCl 5 g/liter; cysteine 30 mg/liter. Fusaric acid resistance of E. coli RR1 and RR1 containing pACYC177 or pRGl0 was also checked and found to be greater than 20 mg/liter although growth was slow at this concentration.
more sensitive to fusanic acid suggesting that adjustment of this concentration could aid in the selection process. The rapid drop in survival after passing a critical fusaric acid concentration should be noted from Fig. 3, since this can be important in choosing the optimal fusaric acid concentration. E. coli RR1 was tested for tolerance to fusaric acid and found to be viable up to a concentration of 20 mg/liter although at this high concentration a growth period of 2 days was necessary. To check the relative ability of Tc” colonies to be selected from a background of a Tc’ strain carrying pBR322 or pDR42 the following mixtures were made: pACYC 177 (an Ap’, Tc”, Km’ plasmid) + pDR42; pACYC177 + pBR322; pRGl0 (a pBR322 derivative in which the Tc’ gene has been inactivated by insertion of a lac operator fragment into the BamHI site) + pDR42; pRGl0 + pBR322. The results of these experiments are shown in Fig. 4. It should be
noted from Fig. 4 that pDR42 allows increased efficiency of selection with either pACYC 177 or pRGl0 using a fusaric acid concentration of only 10 mg/ml. Selection for Tc” cells was also accomplished using pBR322 although colonies were surrounded by pBR322 background. The selection had an efficiency of greater than 90% for 1 TcScell in lo4 Tc’ cells (Fig. 4) when using pDR42 which has a strong promoter controlling the Tc’ gene. In addition to this high efficiency, shorter growth times were made possible by modification of the fusaric acid concentration. The ease of fusaric acid selection against Tc’ and the
‘“On
TcR
cells/ml
FIG. 4. Efficiency of selection of Tc’ bacteria in mixtures containing Tc’ bacteria. Replica plating by stippling was performed to verify that colonies on fusaric acid medium were Tc”. A lawn of small colonies was produced on plates of pACYC177 + pBR322 in cases where high amounts of RRI cells bearing pBR322 were used. Larger Tc” pACYC177 colonies were present. The percentage of those colonies from original fusaric acidampicillin selection plates which were exclusively Tc’ upon replica plating is shown as a function of the number of Tc’ cells present in the mixture plated on the original fusaric acid selection plate. The fusaric acid concentration was 10 mg/liter for pDR42 mixtures and 14 mg/liter for pBR222 mixtures. The number of Tc” cells/ml in the final mixture was approximately 5 X 10’. The screening test for pACYC177 was growth on a kanamycin (40 mg/liter)-ampicillin plate. For pRG IO, a positive test was a blue colony on the 5-bromo-rlchloro-3-indolyl-@-galactoside-ampicillin plates. The colonies were screened for Tc’ on 20 mg/liter tetracycline. pDR42 + pACYCl77, 0, pDR42 + pRGl0, 0; pBR322 + pACYC177,b pBR322 + pRG10, A.
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SHORT COMMUNICATIONS
possibility for producing increased transcription of DNA inserted into the unique restriction sites within the Tc’ gene may make pDR42 useful as a cloning vector when these characteristics are sought. The increased tetracycline resistance, increased fusaric acid sensitivity, and increased transcription of the Tc’ gene by pDR42 as compared to pBR322 support the proposed mechanism of fusaric acid selection. The high expression of the Tc’ protein in pDR42 may account for the increased fusaric acid sensitivity of cells bearing this plasmid. ACKNOWLEDGMENTS We thank R. Gayle for pRGl0 and A. Chang for pACYCl77. The work was supported by NIH Grant GM26437 and D.R.R. was supported in part by NIH Training Grant GM 07833.
REFERENCES 1. BOCHNER,B. R., HUANG, H., SCHIEVEN, G. L., AND AMES, B. N., J. Bacterial. 145, 926-933
(1981). MALOY, S. R., AND NUNN, W. D., J. Bacterial. 145,1110-1112(1981). TACON, W., CAREY, N., AND EMTAGE, S., Mol. Gen. Genet. 177, 427-438 (1980).
MILLER, J. H., “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1972. RUSSELL,D. R., AND BENNETT, G. N., Gene 17, 9-18 (1982). SUMNER,W., II, AND BENNETT,G. N., Nucl. Acids Res. 9, 21052119 (1981). SUTCLIFFE, J. G., Cold Spring Harbor Symp. Quant. Biol. 43, 77-90 (1978). MCKENNEY, K., SCHIMATAKE, H., COURT, D., SCHMEISSNER,U., AND BRADY, C., In “Gene Amplification and Analysis” (M. Rosenberg, J. G. Chirikjian, and T. Papas, Eds.), 2, pp. 383415. Elsevier/North-Holland, New York, 1981.