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Insertion Element (IS1 Insertion Sequence/Chloramphenicol Resistance Transposon Tn9/Integrative Recombination) L

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Insertion Element (IS1 Insertion Sequence/Chloramphenicol Resistance Transposon Tn9/Integrative Recombination) L Proc. Nati. Acad. Sci. USA Vol. 75, No. 3, pp. 1490-1494, March 1978 Genetics Chromosomal integration of phage X by means of a DNA insertion element (IS1 insertion sequence/chloramphenicol resistance transposon Tn9/integrative recombination) L. A. MACHATTIE AND J. A. SHAPIRO Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Communicated by Albert Dorfman, January 10, 1978 ABSTRACT Phage Xcamll2, which contains the chlor- carries a deletion of the gal-attB-bio region of the Escherichia amphenicol resistance transposon Tn9 and has a deletion of attP coil chromosome. MGBO is a gal+bio+ transductant of and the int gene, will lysogenize Escherichia coli K-12. Pro- phage integration occurs at different chromosomal sites, in- MADO. MS6 is a galE indicator for detection of XgalE + T- cluding lacYand maiB, but not at attB. All Xcamll2 prophages transducing particles and S1653 is a gal deletion strain for de- are excised from the chromosome after induction but with tection of Xgal + particles (3). Strain 200PS is a thi lacY strain various efficiencies for different locations. Heteroduplex from the Pasteur collection. Strain QL carries a complete lac analysis of XplacZ transducing phages isolated from a lacY:: deletion, strain X9003 carries the nonpolar M15 lacZ deletion, Xcamll2 prophage reveals an insertion sequence 1 (IS1) element and either will serve as indicator for XplacZ phage in a blue at theloint of viral and chromosomal DNA. Two lines of evi- dencekdicate that Xcamll2 encodes an excision activity that plaque assay (8). recognizes the ISI element: (i) prophage derepression increases Media. Our basic minimal medium and complete TYE the frequency of excision from IacYto yield lac+ revertants, and medium have been described (9). For selecting chlormpheni- (i) Xcamll2 infection increases reversion of a gaIT::ISl col-resistant (Cmr) clones, we supplemented media with 50Mug mutation about 50-fold. Our results indicate that the ISI termini of chloramphenicol per ml. Our tetrazolium indicator medium of Tn9 can replace attP as a site for X insertion in the bacterial has been described (7). chromosome and that excision events are catalyzed by an ISI- Genetic Methods. Basic techniques are described elsewhere encoded protein under X repressor and N gene control. (3, 7). Tn9-containing phage are distinguished from cat phage We have argued that DNA insertion elements and specific re- by a turbid plaque assay (5); we have improved this by use of combinases provide a mechanism for joining unrelated chro- TYE agar, 30 ml per plate, containing 4,ug of chloramphenicol mosome segments (1, 2). The smallest insertion elements are per ml. Survival of Xcamll2 lysogens at 420 was determined the insertion sequences (IS elements) that causepolar mutations by plating appropriate dilutions of TYE-glucose (0.4%) cultures (3, 4). To study the role of IS elements in integration and exci- grown at 32' on TYE-glucose agar at 320 and 420. Unless sion events, we have developed a model system using bacter- specified otherwise, all cultures were grown at 32°. iophage X and the transposable Tn9 element, which encodes Electron Microscopy. We used a Siemens 101 microscope resistance to chloramphenicol. Tn9 contains the structural gene to photograph heteroduplex DNA preparations which were for chloramphenicol acetyltransferase (cat+) bracketed by made and measured as described (11). For separate calibration direct repeats of the IS1 element (5). Xcamll2 is a Tn9-con- of duplex and single-stranded length measurements, we used taining phage that has lost the attP region and integrase gene as internal length standards the duplex length from the right so that it has no viral integration system (Fig. 1). In this paper, X cohesive end to the imm2l substitution (assumed to represent we report the isolation of stable Xcamll2 lysogens, show that 0.206 of the X+ DNA molecule) and the single-stranded immX the IS1 element is the joint between host and viral DNA seg- portion of the immunity nonhomology bubble (assumed to ments in one of these lysogens, and present genetic evidence represent 0.083 X+). that the Xcamll2 genome directs the synthesis of an IS1 exci- sion activity. RESULTS Transduction by Xcamll2. We observed that Xcamll2 will MATERIALS AND METHODS transduce E. coil K-12 to a stable chloramphenicol-resistant Bacteriophages. XcamlO5 and Xcamll2 (5) are deletion (Cmr) phenotype despite the absence of a viral integration mutants of the original Xcam phage, which had acquired Tn9 system. Comparison of transduction by Xcamll2 and its from PlCm (6). XMGB22 is a derivative of int+attP+ sibling XcamlO5 (Fig. 1) reveals that loss of the Ximm434ctslb5l5b519, which acquired Tn9 from a chromo- attP-int segment depresses transduction at a low multiplicity somal site and simultaneously lost integrase activity. XCI857S7 by only 14-fold in an attB + strain (MGBO) and not at all in an free of Xgal was obtained by induction of a lysogen carrying attB-deleted strain (MADO) (Table 1). Although the conditions the S165 gal deletion (7). We have previously described are different, these effects are much less severe than the effect XdgalS188 (3), Xcamll2imm2l and Xcamll2imm2l Acat-IS1 of an int point mutation on X insertion into the bacterial chro- (5). mosome. The Wnt6 mutation decreases lysogenization of an Bacteria. MADO is an F-thi rha trkA trkD strain which attB + strain by more than 10,000-fold and of an attB-deleted strain by 500-fold (12). This difference is not explained by Tn9 The costs of publication of this article were defrayed in part by the transposition from the X genome to the bacterial chromosome payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: Cmr, chloramphenicol resistance phenotype; Cms, this fact. chloramphenicol sensitivity phenotype; IS, insertion sequence. 1490 Downloaded by guest on October 1, 2021 Genetics: MacHattie and Shapiro Proc. Natl. Acad. Sci. USA 75 (1978) 1491 434 Table 1. Transduction by XcamlO5 and Xcamll2 Tn9~~ imm A |@JN SAR Mean relative 0.1 0.2 0.3 0.4 0.5 ..6 0.7. .08 0.9 1.0 attP- transduction /05_ 21tP112 imm Phage Host int attB frequency 104 _ 108 XcamlO5 MGBO + + 100 XcamlO5 MADO + - 1.2 i 0.4 FIG. 1. The genome ofAcam and its derivatives. The coordinates Xcamll2 MGBO - + 7.0 ± 1.4 of the various A markers are their fractional distances from the left Xcamll2 MADO - - 1.3 ± 0.4 end of the X+ DNA molecule. The Tn9 element is inserted at position 0.498 and measures 0.053 unit (5). Endpoints of deletions adjacent All transductions were done at multiplicities of infection less than to Tn9 are: 105,0.466; 104,0.561; 108,0.525; and 112,0.608. The attP 1 plaque-forming unit per 50 host cells. The last column gives the site is at position 0.574 and the nt gene extends from 0.576 to 0.60 mean of transduction frequency (Cmr transductants formed per in- (10). fected cell) relative to XcamlO5 on MGBO (integrase-promoted transduction) in the same experiment repeated three times. In these experiments (one on growing cells, two on starved cells), the absolute because tests on repurified independent Cmr transductants frequencies of integrase-promoted transduction varied from 0.3 to showed that more than half of them are stable lysogens (5/7 5%. MGBO transductants and 9/12 MADO transductants). Thus, Xcamll2 appears to have an alternative mechanism for chro- boxes that bound the A sequences and the cat + gene indicate mosome integration. None of the Cmr transductants carries a ISI elements or a shorter sequence that recombines more or less detectable auxotrophic mutation (267 clones examined), but efficiently with the end of an ISI element in a specific excision selection on tetrazolium indicator media has permitted us to event. We postulate the presence of these elements based on isolate lac- and mabl Cmr Acamll2 lysogens of MGBO. Re- the structure of Xcamll2 and to explain the following results: version of the lac and mal mutations is accompanied by loss of (i) efficient prophage excision to yield two sharp density species Cmr and of phage production. Hence, the specific mutations of cat + and cat phage progeny (Table 2), (ii) the occurrence result from Xcamll2 insertion. (Subsequent experiments with of both Cmr and Cms cured survivors at comparable frequen- a different strain have yielded Xcamll2 insertions in glpR.) cies (Table 2), and (iii) the fact that most cured survivors revert Properties of Xcamll2 Lysogens. We have examined to lac +. Xcamll2 lysogens of strain MGBO in a number of different Structure of XplacZPhages Derived from MGB18. In order ways. Table 2 summarizes our results. All of the lysogens pro- to examine the connection between host and Xamll2 se- duce chloramphenicol-transducing phage when induced, and quences directly, we isolated blue-plaque-forming XlacZ phages all are stably lysogenic by two criteria: (i) the efficiency of among induced progeny of X9003 lac+ tranductants plated killing by prophage induction at 42° (>99%) and (ii) phage on lawns of X9003 or QL on plates containing a chromogenic production by isolated subclones (>99%). Among the first eight substrate for fl-galactosidase (8). We thus isolated four inde- lysogens isolated, only strains MGB5 and MGB7 appear to be pendent XplacZ phages (XXJS18, XXJS19, ASJX20, and XXJS21). double lysogens and segregate clones with reduced phage yields We also obtained a transducing particle for the lacY point (between 4 and 12% of all clones tested). The single-lysogen mutation in strain 200PS. This plaque-forming phage (XXJS25) segregants (such as MGB155 and MGB156) still produce bursts also carries the lacZ gene; so the lacY mutation must lie between greater than one per induced cell.
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