Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

PLASMID

10, I1 1-I 18 (1983)

Analysis of IS21-Mediated Mobilization of Plasmid pACYC184 by R68.45 in Escherichia G. RIESS,’ B. MASEPOHL,

co/i

AND A. FUEHLER

Lehrstuhl fir Genetik, Universitiit Bielefeld, Postfach 8640, D-4800 Bielefeld 1, Federal Republic of German) Received November I 1, 1982 Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS2 I, at a frequency of IO-‘lo-’ per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegratesof pACYC I84 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS2 1 in direct orientation. After resolution of the cointegrate in recA+recipients,the mobilizing plasmid R68.45 lost one copy of IS2I becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS2 I transposesto the mobilized plasmid. Insertion sites and orientations of IS2 I in 33 pACYC 184::IS2 I insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYCl84 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.

The P-type plasmid R68.45, isolated by Haas and Holloway ( 1976), is well-known for its chromosome mobilizing ability (Cma)* in various gram-negative bacteria (reviewed by Holloway, 1979). R68.45 is a derivative of plasmid R68, which, on the basisof restriction enzyme analysis and heterocluplex experiments, is identical to plasmids RP 1, RP4, and RK2 (Burkhardt et al., 1979; Currier and Morgan, 1981). Plasmid R68.45 carried a tandem duplication of a DNA region already present on the parent plasmid R68. This DNA region, located close to the kanamycin-resistance gene, is 2120 bp in length and characterized by a sequence of five restriction sites (Riess et al., 1980). Additional restriction sites for different restriction endonucleases have been mapped by Willetts et al. (1981) and Currier and Morgan (198 1). Willetts et al. (198 I) reported that this DNA region behaves ’ Present address:University of California, Division of Natural Sciences, Thimann Laboratories, Santa Crux. California, 95064. * Abbreviations used Cma, chromosome mobilizing ability; Ap, ampicillin; Km, kanamycin; Tc, tetracycline; Cm, chloramphenicol; Sm. streptomycin; Rf, rifampicin; Nx, nahdixic acid; bp, base pairs; kb, kilobases.

like an insertion element designated IS2 1. It has been suggestedthat the molecular basis for chromosome mobilization by R68.45 might involve the formation of a cointegrate intermediate during transposition of IS2 1 (Riess, 1981;Willetts et al., 1981). Cointegrate formation between a molecule carrying a transposable element and a target molecule is a common feature in the mechanism of transposition (reviewed by Kleckner, 1981). In this paper we report on the mobilization of Escherichia coli vector plasmids like pACYC184 and pBR325 by R68.45 as a model system for analyzing the mechanism of chromosome mobilization in more detail. We have investigated the end-products of the mobilization processand report the structures of the mobilized plasmids, the cointegrate molecule and the P-type plasmid after resolution of the cointegrate. MATERIALS

AND

METHODS

Strains and media. Bacterial strains and plasmids used in this paper are described in Table 1. Antibiotics added to Penassayagar were used at the following concentrations:

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0147-619X/83 $3.00 Co~~righl

0 1983 by Academic

Press. Inc

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RIESS,MASEPOHL,AND PUEHLER TABLEI BACTERIALSTRAINSAND PLASMIDS~ Strain or plasmid

Genotype or phenotype

Source or reference

Escherichia coli CSHSI CSH52 CSH56 CSH65 S302.IO

ara, A(lac. pro), rpsL. [hi rec.4derivative of CSHS I ara. A(lac, pro), gy.4, fhi leu, lac, gyr.4, rpsL. rhi rifderivative of CSH52

Miller ( 1972) Miller ( 1972) Miller ( 1972) Miller (1972) R. Simon, Univ. Bielefeld

R68 R68.45 pACYC184 pBR325

AP,~ Km, Tc, Tra+ Ap, Km, Tc, Tra’, Cma’ Tc, Cm Ap, Tc, Cm

Haas and Holloway ( 1976) Haas and Holloway ( 1976) Chang and Cohen ( 1978) Bolivar (1978)

’ Genetic symbols are those used by Bachmann and Low (1980). Plasmid phenotype symbols are those proposed by Novick et al. (1976). The symbol Cma’ was introduced by B. W. Holloway for plasmids that mobilize the bacterial chromosome (Haas and Holloway, 1978). b See footnote 2 for abbreviations.

ampicillin 100 &ml, chloramphenicol 50 pg/ ml, kanamycin 50 &ml, nahdixic acid 100 &ml, rifampicin 20 pg/ml, streptomycin 200 j&ml, tetracycline 5 &ml. Plasmid isolation by cesium chloride-ethidium bromide gradient centrtfugation and agarose gel electrophoresis. These methods were described by Puehler et al. (1979) and Riess et al. ( 1980). Matings. Matings were carried out by mixing 0.5 ml of a logarithmic-phase donor and recipient strain in a sterile Eppendorf tube, spinning down the cells, and resuspending the pellet in 0.1 ml of saline buffer. The mating suspension was spread on a membrane filter (Sartorius SM 11306) placed on Penassayagar. Matings were carried out overnight at 37°C. Mating mixtures were harvested by vortexing filters in saline buffer. Appropriate dilutions were plated on selective agar. Transformation. For plasmid transformation E. coli strains were grown in Luria broth and made competent by the CaC12shock procedure (Cohen et al., 1972). Cleared lysate preparation of multicopy plasmid DNA for restriction analysis. Fresh overnight cultures (5 ml) were pelleted and the pellets resuspended in 100 ~1 of sucrose (20%). In ice, cells were lysed by adding 25 ~1 of lysozyme (10 mg/ml), 25 ~1EDTA (0.2 M, pH8), and 50 ~1of Triton X- 100 ( 10%).After spinning down the cell debris, proteins in the

supematant were extracted with an equal volume of phenol and the plasmid DNA was precipitated by adding 3X volumes of ethanol (-20°C) and 0. IX volume of sodium acetate (3 M) and cooling to -70°C for 5 min. After centrifugation, plasmid DNA pellets were resuspendedin the appropriate restriction buffer containing RNase ( 1 unit/ml) and then used for restriction analysis. Restriction endonucleasedigestion of DNA. Restriction endonucleases EcoRl, and PstI were isolated by W. Arnold, University of Bielefeld, FRG. KpnI, SalI, and Hind111 were purchased from Boehringer-Mannheim, and HpaI from New England Biolabs, Beverly, Massachusetts.All enzymes were used under the conditions recommended by the suppliers. DNA fragments were separatedby agarosegel electrophoresis using a Tris-acetate buffer (40 mM Tris, 10 ttIM sodium acetate, 1 mM EDTA, pH 7.8). When necessary, slab gels were calibrated using DNA fragments of known sizes present in an EcoRI/HindIII double digest of Xc1857 S7 DNA isolated from the E. coli strain CSH45 (Miller, 1972). RESULTS

Mobilization of E. coli Plasmids by R68.45 and R68 We reported previously that the IS21 tandem structure of plasmid R68.45 is responsible

PLASMID

MOBILIZATION

for the mobilization of chromosomal genesin E. coli (Riess et al., 1980). This has now been found to apply to the mobilization of E. coli plasmids pACYC184 and pBR325. For mobilization experiments donor strains containing both the P-type plasmid R68 or R68.45 and the plasmid pACYC 184 or pBR325 were constructed by transforming cells haboring the P-type plasmid with vector plasmid DNA. Transformants were tested routinely for plasmid encoded antibiotic resistancemarkers before they were used as donors in the matings described in Table 2. R68 and R68.45 were transferred to recipient cells in filter matings at a frequency of approximately one exconjugant per donor cell; only R68.45 was able to mobilize the plasmids pACYC184 and pBR325, at frequencies of 10-3-10-5 per donor cell.

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BY R68.45

mycin (Km) resistance, whereas the recA donor strain gave 100% cotransfer of these antibiotic markers. These results can be explained by assuming that in recA+ exconjugants, cointegrates between R68.45 and pACYC 184 are to some extent resolved, whereasin recA exconjugants such a resolution cannot occur. This assumption was confirmed by plasmid analysis of exconjugants (see below). Mobilization of pACYC184 by R68.45 Occurs via Cointegrate Formation

Exconjugants of the matings between recA+ and recA donors with recA recipients all contained plasmids with a higher molecular weight than plasmid R68.45. No plasmids of the size of pACYC 184 were found. This result is consistent with the assumption that these plasmids are cointegratesbetween R68.45 and The Influence of the recA Function in pACYC184 and that the cointegrate cannot Donor and Recipient Strains on the be resolved in recA cells. For the matings inMobilization Process volving recA+ recipients, the plasmid patterns for exconjugant clones were more compliMatings to demonstrate the mobilization of plasmid pACYC 184by R68.45 were carried cated. Besides exconjugants harboring coinout using recA+ and recA donor and recipient tegrate plasmids, exconjugants containing two strains. We found the same mobilization fre- plasmids were also identified. In the latter case quencies, when the donor was recA+ and the the larger plasmid had a molecular weight recipients were either recA+ or recA (5 X 10e4). similar to that of the P-type plasmid: the The mobilization frequency was slightly en- smaller plasmid was either identical to plasmid hanced, when the donor was recA (1 X 10-3). pACYCl84 or significantly enlarged by inrecA+ and recA exconjugant clones were used sertion of an IS21 element, as shown in the in further matings to test cotransfer of P-type next section. The results obtained with recA+ and vector plasmid. The secondary recA+ do- exconjugants show clearly that the cointegrate nor strain showed up to 80% cotransfer of between R68.45 and pACYC184 can be rechloramphenicol (Cm) resistance and kana- solved in these cells. Some 30-60% of the TABLE 2 SELF-TRANSMISSIBILITY OF R68.45 AND R68 AND MOBILIZATION OF E. coli PLASMIDS pACYC184

Donor strain CSH5I(R68,45)(pACYC184) CSH51(R68)(pACYCl84) CSH5I(pACYC184) CSH5 I(R68.45XpBR325) CSHS I(R68)(pBR325) CSH5 l(pBR325)

Recipient strain CSH65 CSH65 CSH65 CSH65 CSH65 CSH65

R-plasmid transfer frequency/donor cell and selected marker 9.5 x IO-’ 9.3 x IO-’ 8.7 X IO-’ 9.2 X IO-’ -

Km’ Km’ Km’ Km’

AND pBR325

Mobilization frequency/donor cell and selected marker 5X 1X 12 X 9 X 12 X 12 X

10m4 lOm8 lOm8 10e4 IO-* IO-*

Cm’ Cm’ Cm’ Cm’ Cm’ Cm’

114

RIESS, MASEPOHL,

transferred pACYC184 plasmids had the increased molecular weight. This percentage varied from mating to mating for no obvious reason. The existence of pACYC 184 plasmids with unchanged molecular weight could be due to the fact that plasmid dimers can be mobilized and then yield pACYC 184 with no change in molecular weight following resolution in the recA+ exconjugant. In fact about 50% of all of the cointegrates between R68.45 and pACYC 184 that were analyzed consisted of the P-type plasmid and two copies of pACYC 184 carrying the IS2 1 insertion in one copy (data not shown). Typical plasmid profiles from recA+ and recA exconjugant clones are shown in Fig. 1.

AND

PUEHLER

The restriction maps of R68.45 and of one special R68.45-pACYC184 cointegrate are compared in Fig. 2. They differ in the HindIIISalI fragment of R68.45 which contains the IS2 1 tandem (Fig. 2a,b). It was concluded that plasmid pACYC 184 is located in the cointegrate between two IS21 copies in the same orientation. The Structure of the P-Type Plasmid after Resolution of the Cointegrate

The resolved P-type plasmids were isolated from the products of secondary matings; exconjugants were selected for acquisition of Ptype plasmid-encodedantibiotic resistanceand screened for loss of the pACYC 184-encoded Cm resistance. PstI digests of three indepenThe Structure of Cointegrates between dently isolated plasmids, when compared to R68.45 and pACYC184 R68.45, showed that after mobilization and resolution of the cointegrate the P-type plasRestriction analysis of six independently mid had become a normal R68 plasmid with isolated cointegrates between R68.45 and only one IS21 element. The restriction map pACYC 184 from recA exconjugants revealed of the HindIII-Sa/I fragment of the P-type that each junction contains just single copies plasmid after resolution is shown in Fig. 2c. of IS2 1 in the sameorientation. The IS2 1 tandem originally found in R68.45 was no longer The Structure qf the Mobilized part of the cointegrate plasmid. These conpACYC184 Plasmids clusions were drawn from results obtained afThe pACYCl84 plasmids showing an inter digestion of cointegrate plasmid DNA with different restriction enzymes. creased molecular weight after mobilization c d

Q b

t

chromosome

chromosome mobilized pACYC18L

pACYC184

pACYCl&

FIG. 1. Plasmid content of recA and recA’ exconjugants, after mobilization of pACYC184 by plasmid R68.45. (a) Control: donor strain CSHS1 (R68.45)(pACYC184). (b) recA exconjugant S302.10 carrying a cointegrate between R68.45 and pACYC184. (c) Control: donor strain CSHSI (R68.45)(pACYC184). (d) recA+ exconjugant CSH65 carrying the P-type plasmid and the mobilized plasmid pACYC 184.

PLASMID MOBILIZATION

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mO8CCllnOax, H PP PP I--J P

8

>’

bl

1OCU KU 1200 H PP H 111 4

’ IS21

cl

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Txoem H PP I--J

b

1700

2700 E I

em P

S

P --J b

: pACYC1BL

115

(d) Insertions of IS21 in plasmid pACYC 184 were preferentially found between 0.3 and 0.8 kb and between 1.7 and 3.0 kb. The latter region encompassesthe Tc-resistance gene.

s

IS 21

1521

BY R68.45

IS21

S

1521

FIG. 2. Partial restriction maps of R68.45, a cointegrate of R68.45 and pACYC184, and of the P-type plasmid after mobilization. (a) The restriction map of the HindIllSufi fragment of plasmid R68.45 containing an IS2 I tandem is shown. (b) This HindIII-S&I fragment is changed dramatically in the cointegrate of R68.45 and pACYC184. (c) After resolution of the cointegrate the same HindHISu/l fragment only contains one IS21 copy. (E = EcoRI, P = PsfI, H = HindIII, S = Sun, Hp = &x11). Fragment sizes are given in bp.

by R68.45 were separated from the P-type plasmid by transforming E. co/i CSH52 with cleared lysate plasmid DNA preparations. Transformants were retested for pACYC 184 and P-type plasmid-encoded antibiotic resistance markers. In no case was cotransformation of the P-type and the E. cofi plasmid detected. Twelve out of 33 mobilized pACYC 184 plasmids with increased molecular weight (pBM lOOl-pBM1033) conferred sensitivity to tetracycline (Tc). Restriction analysis of cleared lysate plasmid DNA with EcoRI, HindUI, &I, and HpaI in different combinations revealed that: (a) The increased molecular weight of mobilized pACYC184 plasmids was due to insertion of one copy of IS2 I. (b) All insertions of IS21 occurred in the same orientation with respect to the single EcoRI restriction site on pACYC 184, namely EcoRI . . . PstI-PstI-1lpaI. (c) Tetracycline-sensitive pACYC 184 plasmids had an IS21 inserted in the Tc-resistance gene.

These results are summarized in Fig. 3. It should be noted that Cm resistance was used for selecting mobilized plasmids so that IS2 1 insertions in the Cm resistance would not be detected. DISCUSSION

The aim of this work was to study the role of insertion element IS2 1 in the mobilization of plasmid DNA by the P-type plasmid R68.45 and the following conclusions can be drawn: (i) The IS21 tandem structure of R68.45 described by Riess et al. ( 1980), Willetts et al. (198 I), and Currier and Morgan (198 1) is responsible for the mobilization of the E. coli plasmids pACYC 184 and pBR325. (ii) Mobilization of plasmid pACYC 184 occurs via cointegrate formation with the Ptype plasmid R68.45 due to IS2 1 transposition in the donor cell. (iii) After conjugational transfer of the cointegrate plasmid to a recipient a functional recrl gene product is necessary for the resolution of the cointegrate. Thus transposition of IS2 1, leading to cointegrate formation between R68.45 and the target plasmid, is a necessaryprecondition for its mobilization. Furthermore, the cointegrate plasmid is absolutely stable in recA recipient cells, indicating that IS2 1 does not provide its own resolution system, unlike members of the Tn3 family (reviewed by Kleckner, 1981). Additionally, resolution of cointegrates is relatively slow in rec,4+cells. This is important because it means that a cointegrate can be transferred by conjugation with a relatively high probability. These observations also explain the slightly higher mobilization efficiency of pACYC 184 from recA donors. Cointegrates between R68.45 and pACYC184, once formed, cannot be resolved in the recA background and thus reach a higher proportion in a population of R68.45 and pACYC184 carrying donor cells.

116

RIESS, MASEPOHL, AND PUEHLER

FIG. 3. Insertion sites and orientations of insertion element IS2 I found in plasmid pACYC184. Thirtythree insertions in pACYC I84 have been mapped following mobilization by R68.45. Each arrow represents one IS21 insertion. All insertions occurred in the same orientation with respect to the EcoRI restriction site, namely: EcoRI Purl-&I-&al.

A more detailed insight into the IS2 1 transposition processwas obtained after structural analysis of cointegrates of R68.45 and pACYC 184, and of the plasmid end products formed upon the resolution. The results are summarized in Fig. 4. First, it is important to note that in the cointegrate the pACYCl84 DNA is bordered by one copy of IS2 1 at each junction, in direct orientation, so that the original IS21 tandem of R68.45 is no longer present. The samestructure has been observed for R68.45 prime plasmids carrying fragments of the E. coli chromosome (unpublished data) and for a cointegrate between RP4 and parts of the Agrobacferium Ti plasmid (DePicker et al., 1980). Thus it is very likely that the cointegrate plasmid shown in Fig. 4 represents a general model for R68.45 prime plasmids, when pACYC184 is replaced by other DNA of chromosomal or plasmid origin. Resolution of the cointegrate via the cellular recombi-

nation system results in two plasmid end products. One of them is the R68 plasmid, since the IS21 tandem structure is absent in the cointegrate. Of special interest is the structure of the mobilized pACYCl84 plasmid. Here we showed that IS2 1 is inserted at many different sites into pACYC 184, but always in one orientation. A similar phenomenon was reported by Barth ef al. (1978) for the transposition of transposon Tn7 to plasmid RP4. In contrast, Willetts et al. (198 I) have demonstrated insertions of IS2 1 into the pBR325derived vector plasmid pED815 in both orientations. We have confirmed this finding by analyzing eight independently isolated IS2 1 insertions into plasmid pBR325. The IS2 1 insertions were located at different positions on the pBR325 plasmid with a 4:4 distribution with respect to orientation (unpublished results). Intrinsic properties of target DNA may be responsible for this difference.

0

FIG. 4. The different steps in the mobilization of the E. coli plasmid pACYC184 by the P-type plasmid R68.45. (a) Plasmids in the donor cell are shown. The initial step in mobilization is the transposition of IS21 to the target molecule pACYCl84. (b) The cointegrate between R68.45 and pACYCI84 is a precondition for the mobilization of pACYC184 to a recipient cell and at the same time represents the endproducts of the mobilization process using rrcA recipient cells. In the cointegrate pACYC184 is bordered by one copy of IS2 I at each junction. (c) The plasmid endproducts of the mobilization process in recA+ recipient cells are shown. After resolution of the R68.45 cointegrate the P-type plasmid only contains one copy of IS2 I and has become indistinguishable from R68. IS2 I transposes to many different siles of the pACYCl84 genome, but is inserted only in one orientation.

\,

TC

lszl

%m’ pACYC 184 :: IS21

118

RIESS, MASEPOHL,

Taking into account current models for the transposition process (reviewed by Kleckner, 1981) we would have expected to find in the cointegrate, pACYCl84 plasmid DNA flanked by a copy of IS21 at one end and the original tandem IS21 at the other end, or, if tandem IS2 1 transposesasa unit, pACYC I84 plasmid DNA flanked by tandem IS21 copies. Furthermore, the loss of one copy of IS2 1 by the P-type plasmid following resolution is in contrast to other observations, where transposon donors were unchanged during transposition. In summary our results suggestthat the insertion element IS2 1 is similar to members of class I transposable elements as classified by Kleckner (198 1). Insertion of IS2 I into the target plasmid seems to be of low sequence specificity, and may cause gene inactivation, and transposition of IS2 1 is recA-independent. However, resolution of IS2 1 mediated transposition intermediates is r&-dependent. This observation agrees with results reviewed by Kleckner ( 1981). Cointegrate structures generated by class I transposable elements-e.g., IS 1-are stablein recA and someeven in rec,4’ hosts. The experiments described in this paper do not indicate whether IS21 can also transpose via direct transposition (Galas and Chandler, 198l), since they are based on mobilization, where cointegration is a precondition. ACKNOWLEDGMENTS We thank W. KIipp, N. S. Willetts, V. Krishnapillai for stimulating discussionsand S. Malmivaara for typing the manuscript. This work was supported by a grant from Deutsche Forschungsgemeinschaft(Pu28/8). REFERENCES BACHMAN,B. J., AND Low, K. B. (1980). Linkage map of Escherichia co/i K-12. edition 6. Microbial. Rev. 44, l-56. BARTH, P. T., GRINTER, N. J., AND BRADLEY, D. E. (1978). Conjugal transfer system of plasmid RP4: Analysis by transposon Tn7 insertion. J. Bacreriol. 133,4352.

BOLIVAR, F. (1978). Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EC&I sites for selection of

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PUEHLER

EcoRI generated recombinant molecules. Gene 4, I2 I 136.

BURKARDT,H.-J., RIESS,G.. AND PUEHLER,A. (1979). Relationship of group PI plasmids revealed by heteroduplex experiments: RP I, RP4, R68 and RK2 are identical. J. Gen. Microbial. 114, 341-348. CHANG,A. C. Y.. ANDCOHEN,S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15 cryptic miniplasmid. J. Bncreriol. 134, 1141-I 156. COHEN,S. N.. CHANG, A. C. Y.. AND Hsu, L. (1972). Nonchromosomal antibiotic resistancein bacteria: Genetic transformation of Escherichiu coli by R-factor DNA. Proc. h’utl. .kad. Sci. CT.4 69, 21 IO-21 14. CURRIER,T. C.. AND MORGAN,M. K. ( I98 I). Restriction endonucleaseanalysesof the incompatibility group PI plasmids RK2. RP I, RP4. R68 and R68.45. Curr. Microbiol. 5, 323-327.

DEPICKER.A.. DEBLOCK,M., INZE. D.. VANMONTAGU, M., .&NDSCHELL,J. (1980). IS-like element IS8 in RP4 plasmid and its involvement in cointegration. Gene IO, 329-338.

GALAS, D. J.. AND CHANDLER,M. (1981). On the molecular mechanism of transposition. Proc. Nat. Acad. Sci. LSA 78, 4858-4862.

HAAS,D.. AND HOLLOWAY,B. W. (1976). R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol. Gen. Genet. 144, 243-25 I.

HAAS,D., AND HOLLOWAY,B. W. (1978). Chromosome mobilization by R plasmid R68.45: A tool in Pseudomonas genetics. Mol. Gen. Genet. 158, 229-237. HOLLOWAY,B. W. ( 1979).Plasmidsthat mobilize bacterial chromosomes. Plasmid 2, I-19. KLECKNER, N. (1981). Transposable elements in procaryotes. rlnnu. Rev. Genet. 15, 341-404. MILLER, J. H. (1972). “Experiments in molecular genetics,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. NOVICK, R. P., CLOWES, R. C., COHEN, S. N., CURTISIII, R.. DATTA, N., AND FALKOW,S. (1976). Uniform nomenclature for bacterial plasmids: a proposal. Bacterial. Rev. 40, I68- 189. PLIEHLER,

A., BURKARDT,

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W. (1979).

Cloning of the entire region for nitrogen fixation from Klebsiella pneumoniae on a multicopy plasmid vehicle in Escherichia coli. Mol. Gen. Gena. 176, 17-24. RIESS, G. (I 98 1). Die Tandemduplikation der InscrtionssequenzIS2I im Plasmid R68.45 iti verantwortlich fuer die Mobilisierung des Escherichiu coli Chromosoms. Thesis, Univ. of Bielefeld, FRG. RIE.SS,G., HOLLOWAY,B. W., AND PUEHLER,A. ( 1980). R68.45, a plasmid with chromosome mobilizing ability (Cma) carries a tandem duplication. Genet. Rex 36, 99-109. WILLETTS, N. S., CROWTHER,C., AND HOLLOWAY, B. W. (1981). The insertion sequence IS21 of R68.45 and the molecular basis for mobilization of the bacterial chromosome. Plusmid 6, 30-52.