Circular Deoxyribonucleic Acid Shigella dysenteriae Y6R · 2012. 6. 22. · Shigella dysenteriae...
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JOURNAL OF BACTERIOLOGY, Nov. 1969, p. 803-808 Vol. 100, No. 2 Copyright 0 1969 American Society for Microbiology Printed in U.S.A. Circular Deoxyribonucleic Acid from Shigella dysenteriae Y6R MARK G. RUSH,1 C. N. GORDON, AND ROBERT C. WARNER2 Department of Biochemistry, New York University School of Medicine, ivew York, New York 10016 Received for publication 18 August 1969 Circular deoxyribonucleic acid was isolated from Shigella dysenteriae Y6R and was found to consist of six species having molecular weights of 106, 1.3 X 106, 2.6 x 106, 3.8 x 106, 20 x 106, and 24 x 106 daltons. These size classes were par- tially resolved by sucrose density gradient centrifugation. The minicircles (106 and 1.3 x 106) were found to have a buoyant density in CsCl of 1.710 g/ml. The 3.8 x 106 dalton class had a density of 1.707 g/ml. The two largest species had a density of 1.702 g/ml. Two other strains, S. sonnei II and S. dysenteriae 60, also contained circular deoxyribonucleic acid. Covalently closed circular duplex deoxyribonu- cleic acid (DNA) is more resistant to irreversible denaturation than native DNA in any other topological configuration (15; M. Rush and R. C. Warner, submitted for publication). It is therefore possible to treat a cellular lysate with alkali and then neutralize it, with the result that, of all of the DNA molecules present, only those having closed strands will remain intact. The separation of native, circular, duplex DNA from alkali- denatured, single-stranded DNA is accomplished by nitrocellulose chromatography, since single- stranded material binds to nitrocellulose whereas native DNA does not. We report here the isola- tion by this technique of six different species of circular DNA present in Shigella dysenteriae Y6R. MATERIALS AND METHODS S. dysenteriae Y6R was obtained from I. Tessman; S. dysenteriae 60 and S. sonnei II, from W. L. Barks- dale. Ethidium bromide was obtained from Boots Pure Drug Co. Ltd.; lysozyme, from Worthington Biochemical Corp., Freehold, N.J.; and 32P-ortho- phosphoric acid, from E. R. Squibb & Sons, New York, N.Y. The methods employed for preparative and ana- lytical ultracentrifugation (13, 14) and for electron microscopy (12, 13) were described previously. Preparation of circular DNA. S. dysenteriae Y6R was grown at 37 C to stationary phase in 1 liter of modified 3XD medium (3), washed with four 100-ml portions of 0.15 M ethylenediaminetetraacetate, pH 8.0, and frozen in 100 ml of this buffer. Cells were lysed by thawing in the presence of 10 pg of lysozyme per ml, and circular DNA was isolated as previously 1 Present address: Department of Biology, California Institute of Technology, Pasadena, Calif. 91109. 2 Present address: Department of Molecular and Cell Biology University of California, Irvine, Calif. 92664. described for the penicillinase plasmid of Staphy- lococcus aureus (12). An average of 100 pug of pure form I (covalently closed, circular duplex DNA) was obtained per liter of culture. 32P-labeled circular DNA was prepared by adding 20 mc of carrier-free ortho- phosphoric acid to cells in X medium (8) 1 hr before harvesting. A specific activity of 4,000 counts per min per pg of DNA was obtained. RESULTS Basic properties of S. dysenteriae Y6R circular DNA. As indicated in the ultracentrifuge scans A I h r B FIG. 1. Analytical ultracentrifugationi of isolated circular DNA. (A) Ultraviolet scan of bands present after 41 min of zonal centrifugation through 3 M CsCl, 0.15 M NaCl, 0.015 M Na citrate at 40,000 revl min and 20 C. (B) Ultraviolet scan of bands present after 36 hr of centrifugation in CsCl at 48,000 revl min and 25 C. The lighter sharp band (1) has a buoyant density of 1.702; the heavier broad band (h) has an average density of 1.711. The very heavy band (r) is denatured DNA of Micrococcus luteus, a marker with a density of 1.742. A total of about I pg of circular DNA was present in each experiment. An arrow indi- cates the meniscus. By estimating the fraction of total DNA representing minicircular molecules (1.3 X 106 and I X 106 daltons) from the scan in A, and by assuming a 50% preparative yield, it can be calculated that each cell contains about 10 molecules of this size. 803
Transcript of Circular Deoxyribonucleic Acid Shigella dysenteriae Y6R · 2012. 6. 22. · Shigella dysenteriae...
JOURNAL OF BACTERIOLOGY, Nov. 1969, p. 803-808 Vol. 100, No. 2Copyright 0 1969 American Society for Microbiology Printed in U.S.A.
Circular Deoxyribonucleic Acid fromShigella dysenteriae Y6R
MARK G. RUSH,1 C. N. GORDON, AND ROBERT C. WARNER2
Department of Biochemistry, New York University School of Medicine, ivew York, New York 10016
Received for publication 18 August 1969
Circular deoxyribonucleic acid was isolated from Shigella dysenteriae Y6R andwas found to consist of six species having molecular weights of 106, 1.3 X 106,2.6 x 106, 3.8 x 106, 20 x 106, and 24 x 106 daltons. These size classes were par-tially resolved by sucrose density gradient centrifugation. The minicircles (106and 1.3 x 106) were found to have a buoyant density in CsCl of 1.710 g/ml. The3.8 x 106 dalton class had a density of 1.707 g/ml. The two largest species had adensity of 1.702 g/ml. Two other strains, S. sonnei II and S. dysenteriae 60, alsocontained circular deoxyribonucleic acid.
Covalently closed circular duplex deoxyribonu-cleic acid (DNA) is more resistant to irreversibledenaturation than native DNA in any othertopological configuration (15; M. Rush and R. C.Warner, submitted for publication). It is thereforepossible to treat a cellular lysate with alkaliand then neutralize it, with the result that, of allof the DNA molecules present, only those havingclosed strands will remain intact. The separationof native, circular, duplex DNA from alkali-denatured, single-stranded DNA is accomplishedby nitrocellulose chromatography, since single-stranded material binds to nitrocellulose whereasnative DNA does not. We report here the isola-tion by this technique of six different species ofcircular DNA present in Shigella dysenteriaeY6R.
MATERIALS AND METHODSS. dysenteriae Y6R was obtained from I. Tessman;
S. dysenteriae 60 and S. sonnei II, from W. L. Barks-dale. Ethidium bromide was obtained from BootsPure Drug Co. Ltd.; lysozyme, from WorthingtonBiochemical Corp., Freehold, N.J.; and 32P-ortho-phosphoric acid, from E. R. Squibb & Sons, NewYork, N.Y.The methods employed for preparative and ana-
lytical ultracentrifugation (13, 14) and for electronmicroscopy (12, 13) were described previously.
Preparation of circular DNA. S. dysenteriae Y6Rwas grown at 37 C to stationary phase in 1 liter ofmodified 3XD medium (3), washed with four 100-mlportions of 0.15 M ethylenediaminetetraacetate, pH8.0, and frozen in 100 ml of this buffer. Cells werelysed by thawing in the presence of 10 pg of lysozymeper ml, and circular DNA was isolated as previously
1 Present address: Department of Biology, California Instituteof Technology, Pasadena, Calif. 91109.
2 Present address: Department of Molecular and Cell BiologyUniversity of California, Irvine, Calif. 92664.
described for the penicillinase plasmid of Staphy-lococcus aureus (12). An average of 100 pug of pureform I (covalently closed, circular duplex DNA) wasobtained per liter of culture. 32P-labeled circular DNAwas prepared by adding 20 mc of carrier-free ortho-phosphoric acid to cells in X medium (8) 1 hr beforeharvesting. A specific activity of 4,000 counts per minper pg of DNA was obtained.
RESULTSBasic properties of S. dysenteriae Y6R circular
DNA. As indicated in the ultracentrifuge scans
A
I h rB
FIG. 1. Analytical ultracentrifugationi of isolatedcircular DNA. (A) Ultraviolet scan of bands presentafter 41 min of zonal centrifugation through 3 MCsCl, 0.15 M NaCl, 0.015 M Na citrate at 40,000 revlmin and 20 C. (B) Ultraviolet scan of bands presentafter 36 hr of centrifugation in CsCl at 48,000 revlmin and 25 C. The lighter sharp band (1) has a buoyantdensity of 1.702; the heavier broad band (h) has anaverage density of 1.711. The very heavy band (r) isdenatured DNA of Micrococcus luteus, a marker witha density of 1.742. A total of about I pg of circularDNA was present in each experiment. An arrow indi-cates the meniscus. By estimating the fraction of totalDNA representing minicircular molecules (1.3 X106 and I X 106 daltons) from the scan in A, and byassuming a 50% preparative yield, it can be calculatedthat each cell contains about 10 molecules of this size.
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804 RUSHY GORDON, AND WARNER J. BACTERIOL.
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CIRCULAR DNA FRO
TABLE 1. Mean contour lengths and estimatedmolecular weights of the six circular DNA species
present in S. dysenteriae Y6RM
No. of EstimatedSpecies molecules Mean length 1 SD (OM) molecular
measured wt (daltons)
1 134 0.487 :1: 0.014 0.97 X 1062 35 0.652 ± 0.017 1.3 X 1063 30 1.30 4 0.03 2.6 X 1064 30 1.91 4 0.03 3.8 X 1065 16 10.2 0.10 20 X 1066 23 12.2 0.12 24 X 106
a Molecular weights were calculated by assum-ing a value of 2 X 106 daltons/,m for the sodiumsalt of DNA.
Fig. 1B, depends upon base composition andnot size, it was necessary to resolve the mixturebefore assigning a buoyant density to each sizeclass.
Isolation of individual molecular species of S.dysenteriae Y6R circular DNA. After a prelim-inary ethidium bromide-CsCl gradient clarifica-tion (Fig. 3) to remove the small amount (1 to2%) of contaminating linear host cell DNApresent in the preparation, 32P-form I DNA waspartially resolved into its component fractionsby means of successive sucrose gradient cen-trifugations (Fig. 4). Thus, fractions 1 to 6 of thegradient represented in Fig. 4A contained amixture of large rings (20 x 106 and 24 X 106daltons), and fractions 19 to 28 of the samegradient contained an unresolved mixture ofthe remaining components. As can be seen inFig. 4B, these components were partially resolvedafter centrifugation through a new gradient. Bymeans of this procedure, pure samples of mini-circular DNA (fractions 20 to 23 in Fig. 4B)and of 3.8 X 106 daltons DNA (fractions 8 to12 in Fig. 4B) were obtained. The 2.6 X 106dalton species was only partially resolved inthis gradient.
Properties of individual molecular species ofS. dysenteriae Y6R circular DNA. An electronmicrograph of a preparation of minicircularmolecules is shown in Fig. 5. This preparationcontained two distinct classes of DNA, as isevident from the contour length histogrampresented in Fig. 6, and formed a single broadpeak in buoyant CsCl (48,000 rev/min) with a
density of 1.710. Resolved 3.8 X 106 daltonmolecules had a density of 1.707, and a mixtureof the large rings (20 x 106 and 24 x 106 daltons)formed a single sharp band with a density of1.702.
Isolation of circular DNA from two otherShigella strains. To learn whether the presenceof any or all of these six circular DNA species
OM S. D YSENTERIAE 805
is typical of the genus Shigella, we attempted toisolate covalently closed molecules from twoother strains, S. sonnei II and S. dysenteriae 60.Both strains did contain such forms. Althoughthey were apparently not the same size as thosepresent in Y6R, the measurements were notsufficiently precise to establish clearly that theyare different. S. sonnei II contained three specieswith molecular weights of 1.4 x 106, 3.4 x 106,and 4.8 X 106 daltons, whereas S. dysenteriae60 contained only one species with a molecularweight of 2 X 106 daltons.
DISCUSSIONBoth form I and form II of circular duplex
DNA have been isolated from a wide spectrumof bacterial sources. In some cases, these mole-cules have been identified as episomes (2, 4) or as
plasmids (6, 10), allowing for detailed correla-tions among their genetic, biochemical, andphysical characteristics. In others, the functionalsignificance of the isolated molecules has re-
mained obscure (1, 5, 11); the circular DNA
w
20-
z
w
10
(n
z
0
01
I 10 20 30
FRACTION NUMBERFiG. 3. Sedimentation equilibrium of purified 32P_
labeled circular DNA in CsCl containing ethidiumbromide. The solution contained 700 ,ug of ethidiumbromide, 75 ,ug ofDNA (1.3 X 103 counts per min per,g), and 0.07 mmoles of tris(hydroxymethyl)aminomethane-hydrochloride pH 7.5, in 7.0 ml of CsCI,density 1.59 by refractometry. Fractions were col-lected by means ofa microsiphon after 44 hr ofcentrifu-gation in a type 65 angle head rotor at 50,000 rev/milland 20 C, and were assayed by scintillation counting.The density increases from right to left. Although some
of the species in the circular DNA preparation differslightly in buoyant density (Fig. IB), only a singleheavy band (I) was formed during preparative ethidiumbromide-CsCl gradient centrifugation. Fractions 6 to12 were combined, dialyzed against 0.015 M NaCl,0.0015 M Na citrate, and concentrated by flash evapora-tion to a volume of 100 ,uliters.
VOL. 100, 1969
RUSH, GORDON, AND WARNER
0
FRACTION NUMBERFJG. 4. Sucrose gradient sedimentation of NP-labeled circular DNA. Linear gradients (13 ml) prepared from
5 to 23% (w/v) sucrose in 0.15 X NaCI, 0.015 X citrate were centrifuged at 40,000 rev/min in a Spinco SW 41rotor. (A) DNA (60 mg) from the heavy ethidium peak represented in Fig. 3 after 5 hr of centrifugation at 4 C.Fractions 19 to 28 were pooled, dialyzed, and concentrated to a volume of 200 pliters. (B) Combined fractions 19to 28 from A, and another gradient similar to A, after 8.5 hr of centrifugation at S C through another gradient.The ordinates are the total counts per minute in each fraction.
0 n00 F:Ni, : .
FIG. 5. Electron micrograph of resolved minicircular DNA, showing members of the two size classes withmean contour lengths of0.49 and 0.65>m.
806 J. BACTERIOL.
CIRCULAR DNA FROM S. DYSENTERIAE
w
-J
030.w
0
U.0 20-
Lu 0.652
0.386 0.446 0.506 0.566 0.626 0.686
CONTOUR LENGTH
FIG. 6. Contour length distribution of minicircularDNA. Ofthe 169 molecules randomly chosen, 134 were
of the smaller class (mean length: 0.487 0.014 pm),and 35 were of the larger class (mean length: 0.652 i
0.017 pm).
isolated from S. dysenteriae Y6R, and describedin this paper, falls into the latter class. Never-theless, it is tempting to speculate on the possiblefunctions of such species. In light of presentknowledge regarding the types of genetic charac-teristics determined by extrachromosomal ele-ments (6), resistance to antimicrobial agentssuch as antibiotics, heavy metals, and othermetabolic poisons would appear to be the bestcandidates. If these were the characteristicsdetermined, one might even suspect that, becauseof its size, the 20 x 106 dalton species is a sexfactor, the 3.8 X 106 dalton species is a non-transferable plasmid, and the 24 X 106 daltonmolecule is a recombinant of the two. It is alsopossible that one or more of the species is due toa phage, present either as an autonomous pro-
phage or as an integrated one in which occasionalinduced cells yield progeny phage DNA. How-ever, preliminary investigations pertaining tosuch properties in S. dysenteriae Y6R were
unrevealing. Circular DNA was isolated fromtwo other strains of Shigella in an attempt tosee whether any of these six DNA molecules ischaracteristic of Shigella. However, as previouslynoted, the circular DNA molecules isolated fromthese strains appear to be unrelated to those fromY6R.
Because of their unusually small size, the 106and the 1.3 X 106 dalton species (minicircular
DNA) present in Y6R are of special interest.Such molecules can code for only a limited num-ber of proteins or specific ribonucleic acids, andit is not apparent why a cell possessing morethan 10 copies of such a molecule would havean evolutionary advantage over one in whichthis information is present in only one copy.Nevertheless, minicircular DNA has been iden-tified both in bacterial (1, 5) and in animal (16)cells.The possibility of preparing pure samples of
the various size classes of circular DNA fromY6R and other Shigella strains should allowdetailed biochemical investigations of the nature,origin, and replication of these molecules.
ACKNOWLEDGMENTS
This research was supported by Public Health Service grantGM 06967 from the National Institute of General MedicalSciences, by a grant for postdoctoral research from Merck andCo. (M.G.R.), by Public Health Service career award K3 GM14899 (R.C.W.), and by a predoctoral fellowship from PublicHealth Service training grant 5 Ti GM 1234 (C.N.G.)
The expert assistance of Dymetria Rush is gratefully acknowl-edged.
ADDENDUM IN PROOFCircular DNA has also been isolated from Shigella
by H. S. Jansz, J. Zandberg, J. H. vander Pol, andE. F. J. van Bruggen (Eur. J. Biochem. 9:156, 1969).
LITERATURE CITED
1. Cozzarelli, N. R., R. B. Kelly, and A. Kornberg. 1968. Aminute circular DNA from Escherichia coli 15. Proc. Nat.Acad. Sci. U.S.A. 60:992-999.
2. Freifelder, D. 1968. Studies on Escherichia coli sex factors. IV.Molecular weights of the DNA of several F' elements. J.Mol. Biol. 35:95-102.
3. Guthrie, G. D., and R. L. Sinsheimer. 1960. Infection of pro-toplasts of Escherichia coll by subviral particles of bacterio-phage OX 174. J. Mol. Biol. 2:297-305.
4. Hickson' F. T., T. H. Roth, and D. R. Helinski. 1967. Circular,DNA forms of a bacterial sex factor. Proc. Nat. Acad. Sci.U.S.A. 58:1731-1738.
5. Lee, C. S., and N. Davidson. 1968. Covalently closed mini-circular DNA in Micrococcus lysodelkticus. Biochem. Bio-phys. Res. Commun. 31:757-762.
6. Novick, R. P. 1969. Extrachromosomal inheritance in bac-teria. Bacteriol. Rev. 33:210-263.
7. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance toinorganic salts in Staphylococcus aureus. J. Bacteriol. 95:1335-1342.
8. Pouwels, P. H., H. S. Jansz, J. van Rotterdam, and J. A.Cohen. 1966. Structure of the replicative form of bacterio-phage XX 174. Physico-chemical studies. Biochim. Biophys.Acta 119:289-300.
9. Riou, G., and C. Paoletti. 1967. Preparation and properties ofnuclear and satellite deoxyribonucleic acid of Trypanosomacruzi. J. Mol. Biol. 28:377-382.
10. Roth, T. F., and D. R. Helinski. 1967. Evidence for circularDNA forms of a bacterial plasmid. Proc. Nat. Acad. Sci.U.S.A. 58:650-657.
11. Rownd, R. R., R. Nakaya, and A. Nakamura. 1966. Molecu-lar nature of the drug resistance factors of the enterobac-teriaceae. J. Mol. Biol. 17:376-393.
807VOL. 100, 1969
RUSH, GORDON, AND WARNER
12. Rush, M. G., C. N. Gordon, R. P. Novick and R. C. Warner.1969. Penicillinase plasmid DNA from Staphylococcusaureus. Proc. Nat. Acad. Sci. 63, in press.
13. Rush, M. G., A. K. Kleinschmidt, W. Hellmann, and R. C.Warner. 1967. Multiple-length rings in preparations ofOX 174 replicative form. Proc. Nat. Acad. Sci. U.S.A. 58:1676-1683.
14. Rush, M. G., and R. C. Warner. 1967. Multiple-length rings
of OX 174 replicative form. II. Infectivity. Proc. Nat. Acad.Sci. U.S.A. 58:2372-2376.
15. Vinograd, J., and J. Lebowitz. 1966. Physical and topologicalproperties of circular DNA. J. Gen. Physiol. 49:103-125.
16. Vinograd, J., R. Radloff, and W. Bauer. 1967. A dye-buoyantdensity method for the detection and isolation of closedcircular duplex DNA: the closed circular DNA in HeLacells. Proc. Nat. Acad. Sci. U.S.A. 57:1514.
808 J. BACTERIOL.
