The insertion element IS1 is a natural constituent of coliphage P1 DNA

PLASMID

1, 357-365 (1978)

The Insertion

Element IS1 Is a Natural Coliphage Pl DNA

SHIGERU IIDA, Abteilung

Mikrobiologie.

J~RG MEYER,

Biozentrum

Constituent

of

AND WERNER ARBER

der Universitiit,

CH-40.56 Basel, Switzerland

Accepted February 24, 1978 The presence of one copy of the insertion element IS1 in Pl DNA at map unit 20 of the physical genome map is revealed by restriction enzyme cleavage patterns and electron microscopy. This IS1 element is cleaved once by the restriction endonuclease PstI and extends about 500 to 600 base pairs to the left and 200 to 300 base pairs to the right of the unique PstI cleavage site of Pl DNA. Two PlCm derivatives, PlCm246 and PlCm89, carrying a chloramphenicol resistance determinant contain DNA insertions with two terminal directly repeated IS1 elements. Insertion of such El-mediated transposition elements may occur at the IS1 site in the PI aenome or at other sites. The significance of IS1 as a natural constitutent of Pl DNA is discussed.

Temperate coliphage Pl contains circularly permuted linear double-stranded DNA of molecular weight of about 66 x lo6 with a terminal redundancy of 9 to 12% of the genome. In the prophage state, PI replicates as an autonomous plasmid at about one copy per host chromosome (Ikeda and Tomizawa, 1968; Yarmolinsky, 1977). Pl DNA carries an invertible segment of 3 kb (Lee et al., 1974) which is homologous to the G region in coliphage Al.(Chow and Bukhari, 1976) and flanked by long (0.62kb) inverted repetitions (Lee et al., 1974). In the course of restriction enzyme cleavage analysis of DNA from specialized transducing Pl phages carrying a chloramphenico1 resistance determinant derived from the R plasmid NRl (Mise and Arber, 1976; Iida and Arber, 1977), suggestive evidence for the presence of an ISI-like’ element in Pl DNA accumulated. IS elements are known to be normal constitutents of F and R plasmids and the chromosome of Escherichiu cofi (Starlinger and Saedler, 1976). We now present experimental evidence that, in fact, Pl DNA carries an IS1 element ’ Abbreviations used: IS, insertion sequence; bp, base pairs; kb, kilobases. 357

at map unit 20, which contains the unique cleavage site for the restriction endonuclease PstI (Bachi and Arber, 1977). These results have been reported in part by Meyer and Iida (1977). MATERIALS

AND METHODS

The bacterial E. cofi strains used were described by Iida and Arber (1977); bacteriophage PI and its derivatives are listed in Table 1; phage hrl4cZZ::ISl was described by Hirsch et al. (1972). Special care was given to the handling of material containing drug resistance determinants to prevent its escape from the laboratory. The preparation of the DNA and DNA fragments and gel electrophoresis were done according to Bachi and Arber (1977) and Brack et al. (1976). Denaturation of DNA, heteroduplex formation, and mounting for electron microscopy followed the formamide method given by Davis et al. (1971). PM2 DNA and fd DNA were included in the spreadings as internal standards for double- and single-stranded DNA, respectively. Figure 1 shows the part of the cleavage map of Pl (Bachi and Arber, 1977) that 0147-619x/78/0013-0357$02.00/0 Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form rescrvcd.

358

IIDA, MEYER, AND ARBER TABLE 1 PHAGE Pl STRAINS

Strain

Source or reference

Plclts225

J. Scott

PlCm246

Plaque-forming deletion derivative of PlCmSmSu81 (Iida and Arber, 1977).

PlCm89

Type A Cm-transducing Pl derivative showing the same restriction cleavage pattern as PlCm13 described by Iida and Arber (1977).

PlCmO

New designation for PlCM isolated by Kondo and Mitsuhashi (1964).

is relevant for this work. Map unit positions given in this paper always refer to this physical map of the wild-type PI genome. RESULTS AND DISCUSSION I. Hybridization of PI DNA with an IS1 Probe Reveals in the Electron Microscope an IS1 Element at Map Unit 20 of the PI DNA

A number of experimental observations, some of which will be outlined below, had suggested that bacteriophage PI DNA might carry an IS1 element at map unit 20 (which corresponds to the Pstl site on the physical Pl map; Bachi and Arber, 1977). This expectation was confirmed by annealing the strand-separated IS1 probe hr14cZZ::ISl DNA to the restriction cleavage fragment PstI

MAP UNITS

0

10

20

30

40

FIG. 1. The relevant part of the restriction endonuclease cleavage map of the Pl genome (Blchi and Arber, 1977). The genome of the reference PI strain is about 90 kb, corresponding to a molecular weight of 58.6 x 106,and is arbitrarily subdivided into 100map units. The unique PstI cleavage site is chosen as the reference point of the map and placed at map unit 20. The location of the inverted repeats and of the invertible segment in between is also shown.

FIG. 2. Electron micrograph of a heteroduplex molecule between the BglII-2 fragment of Plclts225 DNA and the separated heavy strand of hrlCII::ISl DNA. There is one 800 bp duplex region due to hybridization of the IS1 sequences of Pl and hrlCII::ISl DNA. PI indicates the single-stranded parts of the intact BgIII-2 fragment of PI DNA, and A indicates the broken ends of hrl4 DNA. The bar represents 1000 bp.

Bglll-2 of Plclts225 DNA. The hybrid molecules visualized in the electron microscope (Fig. 2) had a duplex region of 800 bp in length corresponding to the annealed IS1 element. Most of the molecules inspected had one single-stranded end of 1.35 kb and another of about 13.4 kb in common, which was in agreement with the position of IS1 within the Bglll-2 fragment at map unit 20 (Figs. 1 and 6). The other ends were often shorter than expected for hrl4cZZ::ISl, which was evidently because of breaks in the A DNA. IS1 is known to contain one cleavage site for the restriction endonuclease Pstl (Grindley, 1977). As will be shown below, the unique Pstl cleavage site on Plclts225 is contained within the unique IS1 element of this phage. The phage strain Plkc studied by Bachi and Arber (1977) is also cleaved only once by Pstl at map unit 20. Both Plkc and Plclts225 are thus likely to contain only one IS1 in their genomes. However, other phage Pl strains do contain more than one ISI, as was shown recently

INSERTION ELEMENT

by De Bruijn and Bukhari (1977) and by experiments described below.

359

IS1 IN PHAGE Pl DNA A

Eco RI*Pstl Eco RI Pl

2. Some PlCm Derivatives of PI carry ISl-Flanked Insertions at Map Unit 20

Iida and Arber (1977) have obtained a number of PlCmSmSu and PlCm derivatives upon growth of phage Pl in a host strain harboring the R plasmid NRl . Restriction cleavage analysis has shown that the genome of phage strain PlCmSmSu81 carries the intact r-determinant part of NRl inserted at Pl map unit 20 (S. Iida, unpublished data). Let us recall here that IS1 elements occur as direct repeats at the two junctions of the r determinant with the RTF DNA in NRl and in other R plasmids (Hu et al., 1975). PlCm246 is a plaque-forming deletion

_e

-

+130

I FIG. 3. EcoRI digestion and EcoRI:PsrI double digestion patterns of Pl and PlCm246 DNA. Plclts225 and PlCm246clts225 DNA isolated from phage particles were digested and run on a 0.7% agarose gel as described by Biichi and Arber (1977). EcoRI digest of PI (A) and of PlCm246 (C); EcoRI:PsfI double digest of PI (B) and PlCm246 (D). The numbers on the right-hand side of slab (A) identify the EcoRI fragments as defined by Blichi and Arber (1977), and those on the right-hand sides of slabs (B), (C), and (D) indicate newly appearing (+) or disappearing (-) bands.

0 EcoRI*Pstl EcoRI PlCm 246

IS1

Cm IS1

FIG. 4. Interpretation of the data shown in Fig. 3. (A) EcoRI-4 fragment of Pl DNA is cleaved by PsrI within the IS1 element to yield new fragments 8b and 13a. (B) The corresponding region of the PlCm246 genome carries a Cm insertion together with an additional IS1 element. The Cm insertion contains one EcoRI cleavage site. The fragment numbers given in parentheses are those assigned in Fig. 3 and fragment sizes in kilobases are as determined from their electrophoretic mobility in the gel.

derivative of PlCmSmSu81. Its EcoRI and EcoRI:PstI cleavage patterns are compared in Fig. 3 with those of a Pl control. In the EcoRI digestion of PlCm246 DNA, two new bands labeled 8a (4.8 kb) and 8c (3.7 kb) appear, while the triple band EcoRI-4,5,6 (6.3 kb) of Pl is reduced to a double band EcoRI-5, 6 in the PlCm246 digest, as was also confirmed by studying BgfII and Barn HI digestion products (data not shown). Thus, in PlCm246 the genome segment corresponding to fragment EcoRI-4 carries a Cm insertion of about 2.2 kb, which includes one EcoRI cleavage site (Fig. 4). PlCm246 still contains a full equivalent of the Pl genome. In the EcoRI:PstI double digestion of Pl DNA, fragment EcoRI-4 is cleaved by PstI to yield fragments 8b (4.2 kb) and 13a (2.1 kb), which are oriented as shown in Fig. 4A (Bachi and Arber, 1977). Surprisingly, the same two fragments appeared in the EcoRI:PstI double digest of PlCm246 DNA [together with the two new fragments 15a (1.37 kb) and 20a (0.59 kb) (Fig. 3)]. Instead, both fragments 8a and 8c had disappeared from the double digest. These data are interpreted in Fig. 4 to indicate that PlCm246 contains two directly repeated ISI elements flanking the Cm insertion carried at map unit 20.

360

IIDA,

MEYER,

It should be noted here that an EcoRI: PstI double digestion of PlCmO DNA also yields the same fragments 8b and 13a as PlCm246. PlCmO [this is a new designation (see Yarmolinsky, 1977) for PlCM isolated by Kondo and Mitsuhashi (1964)] is therefore another Pl derivative carrying an ISl-mediated Cm insertion at map unit 20.

AND ARBER

of the insertion has the location and the size of the IS1 element at map unit 20 of the Pl genome. This is direct evidence for the presence of two copies of IS1 at either end of the Cm insertion. They are inserted in the same orientation but antipolar to the IS1 at map unit 20 (Fig. 6A). No snapback structure was observed in the singlestranded BglII-2 fragment of the control Pl DNA (Fig. 5C). 3. Not All ISI-Mediated Transpositions A comparison of structure and size of Result in Insertions Linked to the the Cm insertions carried by PlCm246 and Naturally Occurring IS1 Element PlCm89 shows that the insertions in both of PI DNA phages carry the Cm determinants flanked The study of PlCm transposition deriva- by directly repeated IS1 elements. Their tives of Pl revealed that a majority of inser- structure is thus basically similar to that of tions obtained are not located at map unit transposon Tn9 described in phage Acam 20. One example is given here and others (MacHattie and Jackowski, 1977; Cohen, will be presented elsewhere. Restriction 1976). hcam had received Tn9 from PlCmO cleavage analysis suggested that PlCm89 (Gottesman and Rosner, 1975). Differences DNA carried an insertion in the region between these transposons, however, are common to fragments BglII-2, EcoRI-2, noted with respect to the size of the DNA and BamHI-4. The insertion had no BglII carried between the two flanking IS1 elecleavage site, so that the BglII-2 fragment ments. Our comparative measurements of PlCm89 was about 2.4 kb (determined indicate that the Cm transposon carried by gel electrophoresis) larger than the cor- by PlCm89 is slightly (0.1 kb) smaller than responding fragment of the Pl control. Tn9 in PlCmO and measures about 2.6 kb The size and site of the Cm insertion were (including the two IS1 elements). The Cm also determined by heteroduplex mapping transposon in PlCm246 is larger and measusing the BglII-2 fragments of Pl and ures about 3.0 kb, including both flanking PlCm89: 2.58 kb (kO.05 kb:18 molecules) IS1 elements. However, since one of these of DNA were inserted at 0.54 kb (~0.02 kb) IS1 coincides with the naturally occurring from one end of the DNA fragment (Figs. IS1 at map unit 20, the insertion gain in SD and 6A). PlCm246 as compared to Pl DNA is only Single-stranded preparations of fragment 2.2 kb, i.e., 0.8 kb for the second IS1 and BglII-2 of PlCm89 DNA did not form a 1.4 kb (calculated from the electrophoretic snap-back structure between the two ends mobility of the restriction fragments in the of the Cm insertion. Intramolecular rean- gel) for the Cm marker and material adjacent nealing, however, did occur between 800 to it. These data clearly show that Cm transbp-long sequences present at either end posons derived from the r determinant of of the Cm insertion and a region 1.35 kb the R factor NRl do not have a unique size. from the other end of the fragment (Figs. We assume that they originate in ISl-medi5A and 5B). The resulting looped molecules ated deletion formation. IS1 might also be belonged to one of two classes which dif- responsible for tandem duplications leading fered in the size of the loop and in the length to the structure -ISl-Cm-ISl-Cm-ISlas of one single-stranded end but which had observed in PlCmOcZr 1002or PlCm92cl ts225 the other single-stranded end and the doublestranded part in common (Fig. 6B, Table 2). p Some PlCmO strains carry one and others two The region duplexing the terminal parts copies of Tn9.

INSERTION ELEMENT

IS1 IN PHAGE Pl DNA

361

FIG. 5. Structures observed in denatured and reannealed BglII-2 fragments of Pl and PlCm89 DNA. (A) and (B) Single-stranded fragments of PlCm89 DNA with IS1 sequences reannealed (A) representing a class I and (B) representing a class II molecule as defined in Fig. 6B. The length measurements are given in Table 2. (C) Single-stranded fragments of Pl DNA do not form a snap-back structure. The circular molecule is fd DNA. (D) Heteroduplex formed between fragments of Pl and PlCm89 DNA, showing an insertion loop of 2.6 kb at 0.54 kb from one end (arrowhead). The bars represent 1000 bp.

and for higher oligomeric repetitive struc- molecules had two loops with a doubletures occasionally formed in PlCm phages stranded stem, one having the character(Meyer and Iida, in preparation). istics of either of the two possible loops observed with BglII-2 fragments; the other was formed by the characteristic inverted 4. Electron Microscope Mapping of the repetitive sequences (duplex) and the 3 kb IS1 Elements of PlCm89 Relative to the invertible DNA segment (single-stranded) Inverted Repeats on Pl DNA (Lee et al., 1974) spanning from map unit 30 To map the location of the IS1 element to 34 (Bachi and Arber, 1977). These strucin Pl DNA by a method independent of tures allowed us to determine the distance restriction fragment analysis, we examined from the end of the naturally occurring single-stranded phage PlCm89 DNA after IS1 element to the start of the leftside inintrastrand reannealing (Fig. 7A). Most verted repeat: This distance is about 9 kb

362

IIDA, MEYER, AND ARBER A PlCmB9 Pl

IS1 &$j

IS1

\

I

0.5

B PstI

Class I

“IYkII

Class II

,H-----)

FIG. 6. Schematic representation of the structure of the BglII-2 fragments of PI and PlCm89 DNA. (A) The position of the IS1 element in Pl DNA and the location and structure of the Cm insertion of PlCm89 were deduced from molecules as depicted in Figs. 2 and 5. The orientations of the IS1 elements are given relative to each other. The numbers give the length in kilobases. (B) Two classes of looped molecules were observed after denaturation and intrastrand reannealing of the IS1 elements in PlCm89 DNA fragments: Class 1 molecules have a small loop and a long and an intermediate end (Fig. 5A); class II molecules have a large loop and an intermediate and a short end (Fig. 5B; Table 2). The cleavage site of the restriction endonuclease PstI visualized in the molecule shown in Fig. 8 is indicated in this scheme.

(8.95 ? 0.31 kb, 20 molecules), which corresponds to 10 map units, and is in agreement with the data from restriction cleavage analysis. 5. Visualization of the PstI Cleavage Site within ISI As already mentioned above, ISI contains one PstI cleavage site. The location

of this site was visualized when phage PlCm89 DNA was cleaved with restriction endonuclease PstI, denatured, and prepared for electron microscopy. Intrastrand reannealing occurred between the remaining part of the IS1 at map unit 20 on Pl DNA and that of the right IS1 of the Cm insertion, and it resulted in single-stranded looped molecules with a double-stranded stem of 0.51 + 0.06 kb (25 molecules measured; Fig. 8). The size of the loops corresponded well to the class I of small loops obtained with the single-stranded BglII-2 fragments (Fig. 6B). In this analysis we also focused our attention to PstI cleavage fragments containing the 3-kb invertible DNA segment. About half of these molecules had a single-stranded end of 9.13 2 0.43 kb (15 molecules), which is about 180 bp longer than the distance between the stem of the loop formed by the inverted repeats and the start of the IS1 element. The apparent difference in the position of the PstI cleavage site within IS 1 (180 vs 290 bp from the right-hand end) is insignificant in view of the precision of EM measurements, especially of singlestranded DNA. 6. Biological Significance of the IS1 Present in the PI Genome Restriction cleavage and heteroduplex analysis revealed that Plclts225 carries one IS1 element at physical map unit 20. Phage Plclts225 was isolated from Plkc by hydrox-

TABLE 2 LENGTH

MEASUREMENTS

OF LOOPED

MOLECULES

IN DENATURED

BglII-2

FRAGMENTS

OF

PlCm89 DNA”

ClasG

Number of molecules measured

Single-stranded loop (kb)

Single-stranded ends (kb)*

Double-stranded part (kb)

1

35

12.85 + 0.27

a = 1.31 2 0.03 b = 2.24 ? 0.04

0.82 r 0.02

II

36

14.49 -c 0.51

a = 1.37 i 0.03 c = 0.54 k 0.02

0.79 ? 0.03

B As depicted in Figs. 5A and 5B. * As defined in Fig. 6B. c Corresponding to reannealed ISl.

INSERTION ELEMENT

IS1 IN PHAGE PI DNA

363

9kb FIG. 7. Relative localization of IS1 elements and the inverted repeats of PI DNA. (A) Single-stranded DNA from phage PlCm89. Reannealing of the IS1 elements resulted in this case in the formation of a class II loop (a) as defined in Fig. 6. The location of the IS1 element could be mapped with respect to the loop (b) formed by the invertible segment and the flanking inverted repeats. The lengths of the molecular ends were irregular. Because of single-stranded breaks, the total length of ,the DNA was often shorter than the length of the virion DNA. The bar represents 1000 bp. (B) Relative position of the stem loops in phage DNA allowing determination of the distance between IS1 and the inverted repeats. Only the relevant regions are shown.

ylamine mutagenesis (Scott, 1968). Our Plkc data). Furthermore, PUP7 and PlCmO/P7 is cleaved by PsrI only once. Furthermore, heteroduplex analysis demonstrated a nonPlCmO, isolated as recombinant between homology region around map unit 20 of the Plkc and an R plasmid now called pSM14 PI genome (Yun and Vapnek, 1977). Alto(Kondo and Mitsuhashi, 1964; Cohen, gether these facts seem to indicate that the 1976), has no Z?stI-cleavable site other than presence of a complete IS1 at map position those at map unit 20, where Tn9 is inserted. 20 is not essential for phage propagation Therefore, we can conclude that wild-type and lysogenization, including its autonoPlkc has one IS1 element as a natural con- mous replication as a plasmid. stituent. In some PlCm phages such as PlCm246 The Pl-related phage W, formerly called and also PlCmO, the Cm element is car+Amp (Smith, 1972), and a Pl/~s&,~+ hy- ried at the IS1 locus and it is flanked by brid phage (Arber and Wauters-Willems, two directly repeated IS1 elements. How1970) do not have a PstI cleavage site at ever, six sites other than the IS1 locus on map unit 20 (Iida and Arber, unpublished the Pl genome are by now identified to have

364

IIDA,

MEYER,

AND ARBER

deserve further investigation, in particular since IS l-mediated transposition and deletion formation is known to occur under the recA- condition (Reif and Saedler, 1975; Gottesman and Rosner, 1975; Iida and Arber, 1977). ACKNOWLEDGMENTS

FIG. 8. Preparation of single-stranded Purl cleavage products of PlCm89 DNA under intrastrand reannealing conditions. lntrastrand reannealing occurred between the cleaved parts of IS1 and resulted in the formation of looped molecules which had the size of class I molecules (Figs. 5A and 6B) and a doublestranded stem representing 500 bp of IS1 (arrowhead). The bar represents 1000 bp.

received IS l-mediated Cm transposons without loss of essential functions. These sites are spread on the left half of the Pl genetic map (Iida, Meyer, and Arber, in preparation; Yarmolinsky, 1977). Thus, IS1 at map unit 20 can be an insertion site for IS 1 transposons but other sites can serve for this purpose as well. In analogy to transposition outside of map unit 20, the insertion of an ISl-flanked Cm transposon at map unit 20 might be expected to result in a structure containing three IS1 elements. For reasons that remain to be elucidated, such a structure has not been observed. Recombination of Pl is mainly dependent on the host recombination system. In the recA- condition, however, rare recombination has been observed, and it occurs then more preferentially in the left half of the Pl genetic map (Hertman and Scott, 1973). There is some low probability that Pl plasmid can integrate into the host chromosome even under the recA- condition as demonstrated by integrative suppression (Chesney and Scott, 1977). The involvement of IS1 in the recA independent recombination of Pl and in the integration of Pl into the chromosome of recA- cells should thus

We thank T. Bickle, D. Pfeifer, and P. Starlinger for DNA preparations and B. Bachi, V. Pirrotta, C. Tschudi, and R. Yuan for restriction endonucleases. We are grateful to B. Bachi, L. A. MacHattie, H. Saedler, J. R. Scott, P. Starlinger, and M. B. Yarmolinsky for communication of their results prior to publication, and we appreciate the stimulating discussion with P. Starlinger. This work was supported by the Swiss National Science Foundation Grant No. 3.466.75. Note added in proof: We have now studied the presence of IS1 elements in Pl DNA by DNA-DNA hybridization experiments using a 32P-labeledAc1857cam 1 carrying two ISI. Only fragments containing map unit 20 in the EcoRl, Bglll and BamHl digestions of Plclts225 DNA were hybridizable to the probe. This result confirms that the Pl DNA used in this work carries only one ISl. No hybridization was detected between the probe and the region of phage P7 corresponding to the map unit 20 of PI.

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INSERTION

ELEMENT

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STARLINGER, P., AND SAEDLER, H. (1976). IS elements in microorganisms. Curr. Top. Microbial. Immunol. 75, 111-152. YARMOLINSKY, M. B. (1977). Genetic and physical structure of bacteriophage Pl DNA. In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 721-732. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. YUN, T., AND VAPNEK, D. (1977). Electron microscopic analysis of bacteriophages Pl, PlCm, and P7. Determination of genome sizes, sequence homology, and location of antibiotic resistance determinants. Virology 77, 376-385.