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A multiple plasmid-containing Escherichia coli strain: Convenient source of size reference plasmid molecules
PLASMID
1, 417-420 (1978)
SHORT COMMUNICATION A Multiple
Plasmid-Containing Escherichia co/i Strain: Convenient Source of Size Reference Plasmid Molecule9
FRANCIS L. MACRINA,* DENNIS J. KOPECKO,~ KEVIN R. JONES,* DEBORAH J. AYERS,+ AND SARA M. MCCOWENS *Departments of Microbiology and SBiology, Virginia Commonwealth University, Richmond, Virginia 23298; and TDepartment of Bacterial Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. 20012
Accepted April 13, 1978 A strain of Escherichia co/i is described that contains eight plasmid species ranging in size from 1.36 x 106to 35.8 x 106daltons. This strain can be employed as a single source of covalently closed circular deoxyribonucleic acid molecules of different sizes for use as references in agarose gel electrophoretic analysis.
The utility of agarose gel electrophoresis in the study of bacterial extrachromosomal deoxyribonucleic acid (DNA) molecules (plasmids) has been well established during the past few years. Plasmid size may be estimated conveniently by this method using either purified plasmid DNA or plasmid-containing cell lysates that have been cleared of most chromosomal DNA by highspeed centrifugation (I). Such size estimations are made possible by the inclusion of plasmid molecules of known molecular size in the same electrophoretic run. Meyers et al. (I ) have shown that there is a linear relationship between the logarithm of the relative migration of covalently closed circular (CCC) DNA molecules and the logarithm of the plasmid molecular size. Availability of circular reference molecules of known size is usually limited by the necessity of utilizing several 1 Requests for reprints should be addressed to F. L. Macrina, Box 847, MCV Station, Richmond, Virginia 23298. The strain described in this communication may be obtained from F.L.M. or from Dr. E. M. Lederberg, Piasmid Reference Center, Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305. 417
bacterial strains, each containing a single molecular species, in order to obtain a full complement of molecular sixes. We recently analyzed a strain of Escherichia coli, designated V517 in our laboratory, that contains multiple plasmid species of eight distinct sizes. We report here the characterization of these plasmids and suggest that this strain will be a useful single source of plasmid reference molecules. E. coli V517 was forwarded to P. V. Phibbs (Department of Microbiology, Virginia Commonwealth University) by J. J. Farmer (Center for Disease Control, Atlanta) as a citrate-utilizing clinical isolate. With the exception of this latter phenotype, it displays a typical E. coli biotype by the Analytical Profile Index scheme (API System, Analytab Products, Plainview, N. Y. 11803) as used for identification of Enterobacteriaceae. Antibiotic susceptibility testing by the standard agar dilution method (2) revealed this strain to be resistant [based on obtainable peak serum levels of antibiotic (3)] to cephalothin, kanamycin, and amikacin. It was sensitive to ampicillin, tetracycline, chloramphenicol, carbenicillin, gentamicin, tri0147-619X/78/0013-0417$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
418
SHORT COMMUNICATION
methoprim/sulfmethoxazole, and nitrofurantoin. P-Lactamase activity was not detected by the chromogenic cephalosporin assay (4). Plasmid DNA was routinely isolated from 500 to 1000 ml of mid-logarithmic cells (grown in Antibiotic Medium No. 3, Difco Labs, Detroit, Mich.) by dye-buoyant density centrifugation of cleared cell lysates according to the method of Clewell and Helinski (5 ). Optimal cell lysis of V.517 was obtained by allowing the lysozyme spheroplasts to remain in the presence of Brij-58 at 4°C for several hours (or overnight, if more convenient). Such Brij-58treated suspensions were placed in a 60°C water bath for 3 min prior to centrifugation to remove chromosomal DNA [see ref. (5)] Standard reference DNA for gel analysis was as follows: Rldrdl9, 62 x lo6 daltons; R6K, 26 x lo6 daltons; pSC101, 6.02 x IO6 daltons; and pMB8, 1.8 x lo6 daltons. pSC185 [(6); molecular size, 9.2 x IO6 daltons] was used as an internal contour length reference in electron microscopic analysis. Agarose slab gel electrophoresis of DNA was performed according to the methods of Meyers et al. (I ). Migration distances of the DNA components were measured directly from photographs of the gels with dividers and an engineer’s ruler (l/60-in. scale). The distance measured was always that from the bottom of the well to the leading edge of the band. Standard curves and size estimates of the plasmids of V517 were generated by linear regression analysis. CsClethidium bromide analysis of crude cell lysates (7), preparation of DNA samples for electron microscopy, and measurements of open circular molecules photographed in the electron microscope (8) were as previously published. Agarose gel analysis of cleared, concentrated cell lysates (I) of strain V517 revealed multiple plasmids. CCC plasmid DNA, purified by dye-buoyant density centrifugation, was analyzed by both agarose gel electrophoresis and electron micros-
copy. These analyses revealed the presence of eight different size classes of circular DNA in V517. The size determinations of the eight plasmid species are presented in Table 1, and relatively good agreement is seen for values obtained by the two methods. Standard deviation for the size values obtained from gel determinations ranged from 4 to ll%, while the range was from 2 to 5.8% for the values obtained by contour length measurements. We feel that subtle variations in band curvature and band size account for the significantly higher standard deviation of gel measurements relative to the values obtained by electron microscopy. We thus accept values obtained by the latter method as standards. Plasmid isolation and migration properties (from several independent plasmid preparations) have been found to be highly reproducible. Occasionally the 3.7 x 106-dalton (CCC) component tends to separate into two very closely migrating but discrete bands. If this reflects a true size difference, it is undetectable by contour length measurements (see Table 1). Alternatively, this phenomenon may reflect some heterogeneity TABLE I SIZE ESTIMATESOF PLASMIDSIN E.
COLI
V517
Size (x 106daltons) 2 SD Electron microscopy* Plasmid designation
Agarose gel analysis”
tic
Size
pVA517A pVA5 17B pVA5 17C pVA5 17D pVA517E pVA5 17F pVA5 17G pVA5 17H
32.04 2 5.19 t 3.48 e 3.03 2 2.24 2 1.69 2 1.51 t 1.24 -t
5 18 54 29 19 34 76 61
35.84 t 4.82 k 3.67 -t 3.39 c 2.63 + 2.03 k 1.79 2 1.36 2
3.60 0.27 0.20 0.18 0.09 0.10 0.09 0.09
1.0 0.11 0.08 0.09 0.07 0.06 0.07 0.08
(LBased on the results of four independently performed analyses using Rldrdl9, R6K, pSClO1, and pMB8 as CCC reference markers. * Size estimates made from open circular contour lengths using pSC185 as an internal reference. c n = number of molecules measured.
419
SHORT COMMUNICATION
tions upon prolonged storage at 4°C or following repeated freezing and thawing. With the exception of pVA517A, all of the open circular molecules generated by y-irradiation may be seen in Fig. 1A as well as their corresponding CCC forms. The open circular form of pVA517A does not enter this gel under these electrophoresis conditions. The relationship of the relative migration of the open circular molecules (pVA5 17B pVA517H) versus their molecular size was found to be linear by the above-cited logarithmic plots (only a plot of data obtained with CCC molecules is shown; Fig. 1B). In general, the open circular molecules of pVA517B-pVA517H migrated some 10 to 15% slower than the corresponding CCC molecules. At present we have not been able to ascribe phenotypic functions to any of the plasmids of E. co/i V517. Two spontaneously occurring citrate-nonutilizing variants of V517 were found to contain the exact same plasmid complement that the parental strain contained. This work thus provides no evidence supporting the plasmid linkage of citrate utilization in E. cofi. The clinical levels of resistance to the TABLE 2 above mentioned antibiotics were in all cases <20 pg/ml, which is lower than those RELATIVE AMOUNTSOFPLASMID DNA IN E. COLI V517 usually observed with plasmid-linked R-determinants. It seems doubtful, therefore, No. of that any of these resistances are plasmidAmount (%) plasmid copies borne. Plasmid of total per genome In summary, E. coli V5 17 provides a condesignation plasmid DNA” equivalent venient single source of a range of CCC pVAS17A 7.04 - 1 plasmid molecules useful as references in pVA5 17B 12.56 -6 agarose gel analysis. The molecular sizes pVA517C 20.60 -13 of these plasmids span the range of most pVA5 17D 18.30 -12 pVA5 17E 10.30 -9 naturally occurring plasmids isolated from pVA5 17F 12.00 -13 a number of species including the staphpVA517G 9.70 -12 ylococci and streptococci and, further, pVA517H 9.50 -16 encompass the size range of most plas(2Plasmid amounts were estimated from tracings of mids constructed by recombinant DNA photographic negatives of agarose gels made with a methods.
in the superhelix density of this plasmid species. Since these two species usually cannot be distinguished from one another, however, we have treated this component as a single band in our measurements. On the basis of three independent experiments, the amount of total CCC DNA in V517 is 9.1 + 0.6% of the chromosomal DNA. Using this figure in conjunction with the molecular weight (from contour length measurements) and the relative amounts (from densitometry of photographic negatives) of the different species, we have calculated the approximate number of copies of each per chromosomal equivalent (Table 2). Both the closed circular and open circular forms of the plasmids of V517 have been analyzed by agarose gel electrophoresis. Bands corresponding to open circular molecules were identified by electrophoresis of plasmids from V517 that had been y-irradiated to convert the majority of the total molecules to the open circular form as verified in the electron microscope. Small amounts of open circular molecules have been found to accumulate in CCC prepara-
Coming 350 densitometer. Areas under peaks corresponding to plasmid components were automatically computed by this instrument. These numbers represent the average of three independently performed experiments.
ACKNOWLEDGMENTS We thank H. J. Welshimer and H. P. Dalton for help with taxonomic evaluation. Miss Carolyn J.
420
SHORT COMMWNICATION
B) 4.8
2.4
2.0 1.8 LOG. REL. MOBIL.
1.4
FIG. 1. Agarose gel analysis of plasrnids found in E. co/i V517. A. Sixty microliters (absorbance at 260 nm = 1.6) of closed circular DNA was applied to the lane labeled “CCC”, while 30 ~1 of the same material which had been y-irradiated (see text) was applied to the lane labeled “OC”. Electrophoresis was carried out for 2.5 has at 30 mA (-90 V). Agarose concentration was 0.8%, the gel thickness was 0.3 cm, and the path of migration was 10 cm. The gel was stained for 20 min in water containing 1 &ml of ethidium bromide, destained in water for 1 hr, and photographed using Polaroid Type 55 P/N film and a Wratten No. 9 filter. CCC plasmid sizes (X lo6 daltons) are noted to the left while OC sizes are noted to the right. B. Typical migration vs size logarithmic plot of data for CCC molecules.
Pritchett is gratefully acknowledged for assistance in manuscript preparation. This work was supported by USPHS Grant DE04224 and NSF Grant PCM77-00858.
REFERENCES 1. MEYERS, J. A., SANCHEZ, D., ELWELL, L. P., AND FALKOW, S., J. Bacterial. 127, 1529-
4. 5. 6.
1537 (1976).
2. WASHINGTON, J. A., AND BARRY, A. L., In “Manual of Clinical Microbiology,” 2nd ed., pp. 410-417. American Society for Microbiology, Washington, D. C., 1974. 3. SHERRIS, J. C., In “Manual of Clinical Microbiology,” 2nd ed., pp. 439-442. American
7.
Society for Microbiology, Washington, D. C., 1974. O’CALLAGHAN, C. H., MORRIS,A., KIRBY, S. M., AND SHINGLER, A. H., Antimicrob. Ag. Chemother. 1, 283-288 (1972). CLEWELL, D. B., AND HELINSKI, D. R., Biochemistry 9, 4428-4439 (1970). KOPECKO,D. J., BREVET, J., AND COHEN, S. N., J. Mol. Biol. 107, 333-360 (1976). MACRINA, F. L., WEATHERLY, G. G., AND CURTISS, R., J. Bacteria/. 120, 1387-1400 (1974).
8. MACRINA, F. L., REIDER, J. L., VIRGILI, S. S., AND KOPECKO, D. J., Znfect. Zmmun. 17, 215-226 (1977).
1, 417-420 (1978)
SHORT COMMUNICATION A Multiple
Plasmid-Containing Escherichia co/i Strain: Convenient Source of Size Reference Plasmid Molecule9
FRANCIS L. MACRINA,* DENNIS J. KOPECKO,~ KEVIN R. JONES,* DEBORAH J. AYERS,+ AND SARA M. MCCOWENS *Departments of Microbiology and SBiology, Virginia Commonwealth University, Richmond, Virginia 23298; and TDepartment of Bacterial Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. 20012
Accepted April 13, 1978 A strain of Escherichia co/i is described that contains eight plasmid species ranging in size from 1.36 x 106to 35.8 x 106daltons. This strain can be employed as a single source of covalently closed circular deoxyribonucleic acid molecules of different sizes for use as references in agarose gel electrophoretic analysis.
The utility of agarose gel electrophoresis in the study of bacterial extrachromosomal deoxyribonucleic acid (DNA) molecules (plasmids) has been well established during the past few years. Plasmid size may be estimated conveniently by this method using either purified plasmid DNA or plasmid-containing cell lysates that have been cleared of most chromosomal DNA by highspeed centrifugation (I). Such size estimations are made possible by the inclusion of plasmid molecules of known molecular size in the same electrophoretic run. Meyers et al. (I ) have shown that there is a linear relationship between the logarithm of the relative migration of covalently closed circular (CCC) DNA molecules and the logarithm of the plasmid molecular size. Availability of circular reference molecules of known size is usually limited by the necessity of utilizing several 1 Requests for reprints should be addressed to F. L. Macrina, Box 847, MCV Station, Richmond, Virginia 23298. The strain described in this communication may be obtained from F.L.M. or from Dr. E. M. Lederberg, Piasmid Reference Center, Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305. 417
bacterial strains, each containing a single molecular species, in order to obtain a full complement of molecular sixes. We recently analyzed a strain of Escherichia coli, designated V517 in our laboratory, that contains multiple plasmid species of eight distinct sizes. We report here the characterization of these plasmids and suggest that this strain will be a useful single source of plasmid reference molecules. E. coli V517 was forwarded to P. V. Phibbs (Department of Microbiology, Virginia Commonwealth University) by J. J. Farmer (Center for Disease Control, Atlanta) as a citrate-utilizing clinical isolate. With the exception of this latter phenotype, it displays a typical E. coli biotype by the Analytical Profile Index scheme (API System, Analytab Products, Plainview, N. Y. 11803) as used for identification of Enterobacteriaceae. Antibiotic susceptibility testing by the standard agar dilution method (2) revealed this strain to be resistant [based on obtainable peak serum levels of antibiotic (3)] to cephalothin, kanamycin, and amikacin. It was sensitive to ampicillin, tetracycline, chloramphenicol, carbenicillin, gentamicin, tri0147-619X/78/0013-0417$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
418
SHORT COMMUNICATION
methoprim/sulfmethoxazole, and nitrofurantoin. P-Lactamase activity was not detected by the chromogenic cephalosporin assay (4). Plasmid DNA was routinely isolated from 500 to 1000 ml of mid-logarithmic cells (grown in Antibiotic Medium No. 3, Difco Labs, Detroit, Mich.) by dye-buoyant density centrifugation of cleared cell lysates according to the method of Clewell and Helinski (5 ). Optimal cell lysis of V.517 was obtained by allowing the lysozyme spheroplasts to remain in the presence of Brij-58 at 4°C for several hours (or overnight, if more convenient). Such Brij-58treated suspensions were placed in a 60°C water bath for 3 min prior to centrifugation to remove chromosomal DNA [see ref. (5)] Standard reference DNA for gel analysis was as follows: Rldrdl9, 62 x lo6 daltons; R6K, 26 x lo6 daltons; pSC101, 6.02 x IO6 daltons; and pMB8, 1.8 x lo6 daltons. pSC185 [(6); molecular size, 9.2 x IO6 daltons] was used as an internal contour length reference in electron microscopic analysis. Agarose slab gel electrophoresis of DNA was performed according to the methods of Meyers et al. (I ). Migration distances of the DNA components were measured directly from photographs of the gels with dividers and an engineer’s ruler (l/60-in. scale). The distance measured was always that from the bottom of the well to the leading edge of the band. Standard curves and size estimates of the plasmids of V517 were generated by linear regression analysis. CsClethidium bromide analysis of crude cell lysates (7), preparation of DNA samples for electron microscopy, and measurements of open circular molecules photographed in the electron microscope (8) were as previously published. Agarose gel analysis of cleared, concentrated cell lysates (I) of strain V517 revealed multiple plasmids. CCC plasmid DNA, purified by dye-buoyant density centrifugation, was analyzed by both agarose gel electrophoresis and electron micros-
copy. These analyses revealed the presence of eight different size classes of circular DNA in V517. The size determinations of the eight plasmid species are presented in Table 1, and relatively good agreement is seen for values obtained by the two methods. Standard deviation for the size values obtained from gel determinations ranged from 4 to ll%, while the range was from 2 to 5.8% for the values obtained by contour length measurements. We feel that subtle variations in band curvature and band size account for the significantly higher standard deviation of gel measurements relative to the values obtained by electron microscopy. We thus accept values obtained by the latter method as standards. Plasmid isolation and migration properties (from several independent plasmid preparations) have been found to be highly reproducible. Occasionally the 3.7 x 106-dalton (CCC) component tends to separate into two very closely migrating but discrete bands. If this reflects a true size difference, it is undetectable by contour length measurements (see Table 1). Alternatively, this phenomenon may reflect some heterogeneity TABLE I SIZE ESTIMATESOF PLASMIDSIN E.
COLI
V517
Size (x 106daltons) 2 SD Electron microscopy* Plasmid designation
Agarose gel analysis”
tic
Size
pVA517A pVA5 17B pVA5 17C pVA5 17D pVA517E pVA5 17F pVA5 17G pVA5 17H
32.04 2 5.19 t 3.48 e 3.03 2 2.24 2 1.69 2 1.51 t 1.24 -t
5 18 54 29 19 34 76 61
35.84 t 4.82 k 3.67 -t 3.39 c 2.63 + 2.03 k 1.79 2 1.36 2
3.60 0.27 0.20 0.18 0.09 0.10 0.09 0.09
1.0 0.11 0.08 0.09 0.07 0.06 0.07 0.08
(LBased on the results of four independently performed analyses using Rldrdl9, R6K, pSClO1, and pMB8 as CCC reference markers. * Size estimates made from open circular contour lengths using pSC185 as an internal reference. c n = number of molecules measured.
419
SHORT COMMUNICATION
tions upon prolonged storage at 4°C or following repeated freezing and thawing. With the exception of pVA517A, all of the open circular molecules generated by y-irradiation may be seen in Fig. 1A as well as their corresponding CCC forms. The open circular form of pVA517A does not enter this gel under these electrophoresis conditions. The relationship of the relative migration of the open circular molecules (pVA5 17B pVA517H) versus their molecular size was found to be linear by the above-cited logarithmic plots (only a plot of data obtained with CCC molecules is shown; Fig. 1B). In general, the open circular molecules of pVA517B-pVA517H migrated some 10 to 15% slower than the corresponding CCC molecules. At present we have not been able to ascribe phenotypic functions to any of the plasmids of E. co/i V517. Two spontaneously occurring citrate-nonutilizing variants of V517 were found to contain the exact same plasmid complement that the parental strain contained. This work thus provides no evidence supporting the plasmid linkage of citrate utilization in E. cofi. The clinical levels of resistance to the TABLE 2 above mentioned antibiotics were in all cases <20 pg/ml, which is lower than those RELATIVE AMOUNTSOFPLASMID DNA IN E. COLI V517 usually observed with plasmid-linked R-determinants. It seems doubtful, therefore, No. of that any of these resistances are plasmidAmount (%) plasmid copies borne. Plasmid of total per genome In summary, E. coli V5 17 provides a condesignation plasmid DNA” equivalent venient single source of a range of CCC pVAS17A 7.04 - 1 plasmid molecules useful as references in pVA5 17B 12.56 -6 agarose gel analysis. The molecular sizes pVA517C 20.60 -13 of these plasmids span the range of most pVA5 17D 18.30 -12 pVA5 17E 10.30 -9 naturally occurring plasmids isolated from pVA5 17F 12.00 -13 a number of species including the staphpVA517G 9.70 -12 ylococci and streptococci and, further, pVA517H 9.50 -16 encompass the size range of most plas(2Plasmid amounts were estimated from tracings of mids constructed by recombinant DNA photographic negatives of agarose gels made with a methods.
in the superhelix density of this plasmid species. Since these two species usually cannot be distinguished from one another, however, we have treated this component as a single band in our measurements. On the basis of three independent experiments, the amount of total CCC DNA in V517 is 9.1 + 0.6% of the chromosomal DNA. Using this figure in conjunction with the molecular weight (from contour length measurements) and the relative amounts (from densitometry of photographic negatives) of the different species, we have calculated the approximate number of copies of each per chromosomal equivalent (Table 2). Both the closed circular and open circular forms of the plasmids of V517 have been analyzed by agarose gel electrophoresis. Bands corresponding to open circular molecules were identified by electrophoresis of plasmids from V517 that had been y-irradiated to convert the majority of the total molecules to the open circular form as verified in the electron microscope. Small amounts of open circular molecules have been found to accumulate in CCC prepara-
Coming 350 densitometer. Areas under peaks corresponding to plasmid components were automatically computed by this instrument. These numbers represent the average of three independently performed experiments.
ACKNOWLEDGMENTS We thank H. J. Welshimer and H. P. Dalton for help with taxonomic evaluation. Miss Carolyn J.
420
SHORT COMMWNICATION
B) 4.8
2.4
2.0 1.8 LOG. REL. MOBIL.
1.4
FIG. 1. Agarose gel analysis of plasrnids found in E. co/i V517. A. Sixty microliters (absorbance at 260 nm = 1.6) of closed circular DNA was applied to the lane labeled “CCC”, while 30 ~1 of the same material which had been y-irradiated (see text) was applied to the lane labeled “OC”. Electrophoresis was carried out for 2.5 has at 30 mA (-90 V). Agarose concentration was 0.8%, the gel thickness was 0.3 cm, and the path of migration was 10 cm. The gel was stained for 20 min in water containing 1 &ml of ethidium bromide, destained in water for 1 hr, and photographed using Polaroid Type 55 P/N film and a Wratten No. 9 filter. CCC plasmid sizes (X lo6 daltons) are noted to the left while OC sizes are noted to the right. B. Typical migration vs size logarithmic plot of data for CCC molecules.
Pritchett is gratefully acknowledged for assistance in manuscript preparation. This work was supported by USPHS Grant DE04224 and NSF Grant PCM77-00858.
REFERENCES 1. MEYERS, J. A., SANCHEZ, D., ELWELL, L. P., AND FALKOW, S., J. Bacterial. 127, 1529-
4. 5. 6.
1537 (1976).
2. WASHINGTON, J. A., AND BARRY, A. L., In “Manual of Clinical Microbiology,” 2nd ed., pp. 410-417. American Society for Microbiology, Washington, D. C., 1974. 3. SHERRIS, J. C., In “Manual of Clinical Microbiology,” 2nd ed., pp. 439-442. American
7.
Society for Microbiology, Washington, D. C., 1974. O’CALLAGHAN, C. H., MORRIS,A., KIRBY, S. M., AND SHINGLER, A. H., Antimicrob. Ag. Chemother. 1, 283-288 (1972). CLEWELL, D. B., AND HELINSKI, D. R., Biochemistry 9, 4428-4439 (1970). KOPECKO,D. J., BREVET, J., AND COHEN, S. N., J. Mol. Biol. 107, 333-360 (1976). MACRINA, F. L., WEATHERLY, G. G., AND CURTISS, R., J. Bacteria/. 120, 1387-1400 (1974).
8. MACRINA, F. L., REIDER, J. L., VIRGILI, S. S., AND KOPECKO, D. J., Znfect. Zmmun. 17, 215-226 (1977).