0021-9193/78/0133-0430$02.00/0 JOURNAL OF BACTERIOLOGY, Jan. 1978, p. 430-432 Copyright (© 1978 American Society for Microbiology

Vol. 133, No. 1 Printed in U.S.A.

Physical Properties of Plasmid Morl74, Which Determines Bacteriocin Production in Proteus morganii 174 J. A. WILLIAMS* AND K. KRIZSANOVICH-WILLIAMSt Microbial Genetics Unit, South African Medical Research Council, Department of Microbiology, University of Pretoria, South Africa Received for publication 1 August 1977

Plasmid Morl74 has a molecular weight of 3.6 x 106 and a buoyant density of 1.6994 g/cm3. The covalently closed circular form has a sedimentation coefficient of 22S. There are 30 to 40 plasmid copies per genome equivalent, but growth in chloramphenicol results in amplification of the copy number to 600. In Proteus morganii 174, Morl74 coexists with a cryptic plasmid of molecular weight 15.8 x 10' and a buoyant density of 1.7170 g/cm3. Proteus morganii 174 produces a bacteriocin active against a number of P. morganii strains (11). This bacteriocin, morganocin 174, interferes with the energy metabolism of sensitive cells. Characteristics of this interference include inhibition of macromolecular synthesis and intracellular accumulation of glutamine and proline, efflux of K+, and rapid depletion of intracellular ATP (12). Although the genetic determinant of morganocin 174, plasmid Morl74, is nonconjugative, conjugal transfer may be mediated by a mobilizing plasmid, kanamycin re-

"cleared" lysate was centrifuged to equilibrium in cesium chloride (CsCl)-ethidium bromide (EtBr). The satellite band obtained was subjected to a second equilibrium banding in CsClEtBr. After removal of EtBr with isopropanol (8), the DNA was banded in CsCl (2) in a Spinco model E analytical ultracentrifuge equipped with a photoelectric scanner. Two DNA bands with buoyant densities of 1.6994 and 1.7170 g/cm3 and molecular weights of 2.4 x 106 and 16.9 x 106 (2), respectively, were observed (Table 1). When plasmid DNA isolated as described

TABLE 1. Physical properties of plasmid DNA isolated from P. morganii 174 and P. morganii 165 morr(Morl 74) Contour Mol wtb Mol Wtd Buoyant denMol wt' P. morganu strai Plasmid (ltm) (x106) SccC sity (x106) (x(gm) Morl74 1.85 3.6 1.6994 2.408 8.1 Cryptic 15.8 1.7170 16.89 165 morr(Morl74) Morl74 1.85 3.6 22 3.7 a Average of seven molecules each; ColEl DNA (contour length, 2.15 Um [1]) was mounted on the same grids as a standard. b Calculated from contour length, assuming 1.96 x 10' daltons per ,Lm (1). Sedimentation constant of CCC form. d Determined from the equation Sccc = 0.034 M041 (1). e Computed from the width of the band at equilibrium in a CsCl gradient in the analytical ultracentrifuge 174

c

(2). above was sedimented through a 15 to 50% neutral sucrose gradient, two discrete bands were again resolved (Fig. 1), which on analysis in the analytical ultracentrifuge corresponded to the two buoyant density bands initially observed. DNA from the two peaks from the sucrose gradient were mounted for electron microscopy by the aqueous method (6) and shadowed with PtPd at an angle of 8°. Circular DNA molecules t Present address: Department of Microbiology, University were observed. DNA from the slower-sedimentof Iowa, Iowa City, IA 52242. ing band, corresponding to p = 1.6994 g/cm3,

sistance factor R772 (11). Transfer of Morl74 to a susceptible indicator strain renders it both morganocinogenic and immune to homologous bacteriocin (12). The purpose of this investigation was to determine physical characteristics of Morl74. Plasmid DNA was initially isolated from P. morganii 174. Cells were lysed by a lysozymesodium dodecyl sulfate procedure (8), and the

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43 1 (OC) DNA sedimented at 16S (Fig. 2). This agrees with the Sccc/Soc ratio of 1.33 to 1.35, as expected for a plasmid of 2 x 106 to 6 x 106 daltons (5). The molecular weight of Morl74 calculated from the Sccc value (1), 3.7 x 106, agrees with the molecular weight obtained from the contour length rather than that obtained from the equilibrium centrifugation. Colicinogenic plasmids have been divided into two groups according to their molecular weight and number of copies per genome equivalent (7). Group I plasmids have molecular weights in the region of 5 x 106 and exist as multiple copies in the cell; group II plasmids have molecular weights of 62 x 106 to 94 x 10W and are found as one to two copies per genome equivalent. To determine the copy number of Morl74, NOTES

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FIG. 1. Centrifugation of plasmid DNA from P. morganii 174 through 15 to 50% neutral sucrose gradient. Plasmid DNA from I liter of P. morganii 174 grown in broth was isolated and banded twice in CsCI-EtBr gradients (8). EtBr was extracted with CsCI-saturated isopropanol, and CsCI was removed by dialysis against 20 mM tris(hydroxymethyl)aminomethane, 10 mM ethylenediaminetetraacetic acid, and 60 mM KCI, pH 7.3. A 1-ml sample was sedimented through a 15 to 50% neutral sucrose gradient in the above buffer in a Spinco SW27 rotor at 22,500 rpm for 15 h at 2°C. Fractions (800 p1) were colected from the bottom of the tube through a 22-gauge needle and monitored at 252 nm through an LKB Uvicord II. Sedimentation is shown from right to left.

had a contour length of 1.85 am, equivalent to a molecular weight of 3.6 x 106. The other DNA species had a contour length of 8.1 ,um, which corresponds to a molecular weight of 15.8 x 10' (Table 1). As either of these DNA species could have been the Morl74 plasrid, a anamycin-sensitive defective segregant of a P. morganui 165 morr(Morl74 R772) transconjugant (11) was selected for further study, since DNA isolated from P. morganii 165 did not yield a satellite band on centrifugation in a CsCl-EtBr gradient. Plasmid DNA from P. morganii 165 morr(Morl74) was examined by electron microscopy. A single plasmid species with a contour length of 1.85 pm was observed, identifying the morganocinogenic plasmid Morl74 as the smaller of the two plasmids isolated from P. morganii 174. No function has yet been found associated with the larger plasmid. Differentially labeled Morl74 [3H]DNA and ColEl ['4C]DNA were isolated from P. morganii 165 mor'(Morl74) and Escherichia coli W3110(ColEl) by sedimentation of cleared lysates through 15 to 50% neutral sucrose gradients (4). Portions from the respective plasmid peaks were cosedimented through a 5 to 20% neutral sucrose gradient (4) to calculate Morl74 DNA S values relative to those of CoIEl DNA (5). The covalently closed circular (CCC) DNA sedimented at 22S, whereas the open circular

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FIG. 2. Sedimentation of Morl74 [3HJDNA and ColEl [4CJDNA through a 5 to 20% neutral sucrose gradient. P. morganii 165 morr(Mor1 74) and E. coli W3110(ColE1) were grown for 3 h at 37°C in nutrient broth supplemented with 250 pg of adenosine and 2 pCi of [HJthymidine per ml and in Penassay broth supplemented with 100 pg of deoxyadenosine and 1 pACi of ['4Clthymidine per ml, respectively. Cells from 50 ml of cultures were lysed with lysozyme-sodium dodecyl sulfate (8), and the lysates were sedimented through 15 to 50% neutral sucrose gradients as in Fig. 1. Portions (100 ,ul) from the respective plasmid peaks were cosedimented through a 5 to 20% neutral sucrose gradient in 30 mM tris(hydroxymethyl)aminomethane and 5 mM NaCI, pH 8.0, in a Spinco SW65 rotor at 50,00X rpm for 135 min at 2°C. Twelvedrop fractions were collected through a 22-gauge needle directly into 5 ml of scintiUation fluid. Sedimentation is shown from right to left. Symbols: 0, Morl 74 [3HJDNA; *, ColE) [14C]DNA.

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NOTES

P. morganii 165 morr(Morl74) was grown in the presence of [3H]thymidine, and lysates were banded in CsCl-EtBr. The plasmid copy number was determined from the relative amounts of radioactivity in plasmid and chromosomal DNA bands, assuming a chromosome mass of 2 x 109 daltons for P. morganii. Cells in both exponential (Fig. 3A) and stationary (not shown) growth phases were found to contain 30 to 40 plasmid copies per genome equivalent. Growth of cells in the presence of chloramphenicol resulted in amplification of the plasmid to 600 copies per genome equivalent (Fig. 3B). Amplification of the plasmid copy number after growth in chloramphenicol has also been demonstrated for bacB

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FIG. 3. Determination of Mor 74 copy number. A 1% inoculum of P. morganii 165 morr(Morl74) in 4 ml of nutrient broth containing 1X) pCi of [3H]thymidine was incubated at 37°C; after 2 h, 2 ml was withdrawn and lysed (8). The remaining 2 ml was supplemented with a further 100,uCi of [3H]thymidine and 200 pg of chloramphenicol and incubated for 6 h before lysis. The total lysates were centrifuged to equilibrium in CsCl-EtBr in a Spinco SW41 rotor at 32,000 rpm for 66 h at 15°C. Fractions (4 drops) were collected from the bottom of the gradient through a 22-gauge needle directly into 5 ml of scintillation fluid. (A) Cells in exponential phase of growth; (B) cells treated with chloramphenicol.

teriocinogenic plasmids ColEl (3) and CloDF13 (9).

The physical data on Morl74 agrees with the nonconjugative nature of this plasmid. A plasmid of molecular weight 3.6 x 106 could only code for approximately six proteins of average molecular weight 30,000, and this would be insufficient to meet the requirements for transfer (10). The small size of Morl74, as well as its multiple copy number, identifies it with the group I colicinogenic plasmids. We thank J. C. van der Walt for her technical assistance.

LITERATURE CITED 1. Bazaral, M., and D. Helinski. 1968. Characterization of multiple circular DNA forms of colicinogenic factor El from Proteus mirabilis. Biochemistry 7:3513-3520. 2. Chervenka, C. H. 1969. A manual of methods for the analytical ultracentrifuge. Spinco Division of Beckman Instruments, Inc., Palo Alto, Calif. 3. Clewell, D. B. 1972. Nature of Col E, plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676. 4. Clewell, D. B., and D. R. Helinski. 1969. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 61:1159-1166. 5. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405. 6. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. Methods Enzymol. 21D:413-428. 7. Hardy, K. G. 1975. Colicinogeny and related phenomena. Bacteriol. Rev. 39:464-515. 8. Sidikaro, J., and M. Nomura. 1975. In vitro synthesis of the E3 immunity protein directed by ColE3 plasmid deoxyribonucleic acid. J. Biol. Chem. 250:1123-1131. 9. Veltkamp, E., W. Barendsen, and H. J. Nijkamp. 1974. Influence of protein and ribonucleic acid synthesis on the replication of the bacteriocinogenic factor Clo DF13 in Escherichia coli celLs and minicells. J. Bacteriol. 118:165-174. 10. Willetts, N. S. 1967. The elimination of the Flac+ from Escherichia coli by mutagenic agents. Biochem. Biophys. Res. Commun. 27:112-117. 11. Williams, J. A. 1977. Mobilization of morganocin 174 plasmid and kinetics of morganocin production in Proteus and Escherichia coli hosts. Antimicrob. Agents Chemother. 11:514-520. 12. Williams, J. A., and K. Krizmanovich-Williams. 1977. Mode of action of morganocin 174. Antimicrob. Agents Chemother. 12:395-400.