Bacteriophage X carrying the Escherichia coli chromosomal region of the replication origin ( transducing phages / dnaA - ilv region / minichromosome )

A transducing phage Xasn was isolated. The late gene region of its genome was found to have been substituted by an Escherichia coli chromosomal segment containing the genes bgIR, bgIC, gimS, uncA, and asn. Restriction endonuclease cleavage mapping and electron microscopic analysis of the Xasn DNA revealed that the size of the bacterial segment is approximately 1.75 X 107 daltons, corresponding to about 26.4 kilobases. The circular DNA of Xasn was digested with restriction endonuclease EcoRI, diluted, and sealed with DNA ligase. When the reaction mixture was used to transform a recipient E. coli strain, a small plasmid of about 1 X 107 daltons (named pMCR115) was obtained. Restriction endonuclease cleavage mapping of pMCR115 and other evidence suggested that it contained the replication origin (oriC) of the E. coli chromosome. The chromosome of Escherichia coli is a single molecule of circular DNA of approximately 2.5 X 109 daltons (1). A cycle of replication is initiated from a unique origin (1-), which has been proposed to reside somewhere near the dnaA and iiv genes on the chromosome (4-7). The origin was named oriC by Hiraga (7). Now we have undertaken to determine its exact location. One of the approaches we have chosen for this purpose was to insert the genome of bacteriophage X within the dnaA-ilv region, and to isolate a series of X strains with the capability of transducing various markers in the region. If any one of the transducing X strains contained oriC in the phage genome, digestion of the genome with a restriction endonuclease would release an E. colh DNA fragment which would form, upon ligation and subsequent transformation into a suitable cell, a small plasmid capable of autonomous replication. In this way, we expected to clone oriC-using the X genome as a vehicle. This in vivo cloning should ensure identification of ornC in its native integrity. Thus we obtained a series of X strains with the capability of transducing consecutively a set of markers in the dnaA-tlv region of the E. coil chromosome. Among them, one phage strain called Xasn was found to contain a bacterial DNA segment from which we were able to construct a small autonomously replicating plasmid named pMCR115. This paper describes the isolation and characterization of these phage strains and the plasmid. Especially, data will be presented to demonstrate that the plasmid pMCR115 carries the asn gene of E. coil, but lacks the replication origin of phage X originally present in Xasn DNA. Based on these data and other observations, we conclude that a nucleotide sequence which may be interpreted as oriC is located very close to the asn gene. This nucleotide sequence was found to reside, according to cleavage mapping and electron microscopic analysis, in the region some 24,000 base pairs away from the bgiB gene toward the ilv gene. MATERIALS AND METHODS Bacterial and Phage Strains, Media, and Transduction. Bacterial strains used were derivatives of E. coli K-12 as listed in Table 1. A tna-transducing phage strain Xtna imm21 (11) was a generous gift from W. J. Brammar. Other phage strains were our laboratory stocks. L-broth (12) was used for transduction with Plvdr (13), and also to grow cells lysogenic for XcI857S7. Medium E (14), with 2 ,tg of thiamine per ml, was used as a synthetic minimal medium, which was supplemented as required; e.g., with 0.4% glucose for selecting asparagine prQtotroph (Asn+) clones, or with 1% glycerol, 25 ,tg of indole per ml, and 100 jtg of 5-methyltryptophan per ml for selecting clones with active tryptophanase (Tna+) (11). In both cases the medium was further supplemented with 0.2% Casamino acids (Difco), and in the following cases with 0.02% Casamino acids. For selecting clones with active membrane-bound Mg2+,Ca2+-ATPase (Unc+), the medium was supplemented with 0.5% sodium succinate (15). For selecting clones that used salicin (Sal+) or arbutin (Arb+), it was supplemented with 0.5% salicin or with 0.5% arbutin, respectively (16). Enzymes. Restriction endonucleases EcoRI, HindIII, and Bam I and T4 DNA ligase were purchased from the Miles Laboratory. T Okazaki and M. Takanami generously supplied us Bam I and HindIII, respectively. The method described by Robinson and Landy (17) was used for endonuclease digestions and ligase reactions. Preparation of DNA and Transformation. The method of Meyers et al. (18) was used for isolation of plasmid DNA. Phage DNA was prepared from particles that were purified by CsCl equilibrium density gradient centrifugation. The phage suspension was treated with 1/100 vol of 0.25 M Na2EDTA (pH 7.9), 1/50 vol of 1 M sodium dodecyl sulfate, and 1 vol of phenol; after low-speed centrifugation the aqueous phase was recovered. It was dialyzed overnight against 10mM Tris-HCl, pH 7.9/1 mM Na2EDTA/10 mM NaCl. DNA transformation was carried out as described by Cohen et al. (19). Agarose Gel Electrophoresis. A slab-gel preparation contained 0.7% or 1.2% agarose II (Wakenyaku Co.) in a buffer solution composed of 10.8 g of Tris base, 0.93 g of Na2EDTA, and 5.5 g of boric acid per liter. Electrophoresis was carried out at 40 V for 12 hr; the specimen was stained with 7 ,tg of ethidium bromide per ml in Tris borate buffer, and was illuminated by a UV lamp (Maruzen Electric Co., FL20 X 1). Electron Microscopy. The method for heteroduplex analysis of X DNA by electron microscopy was a modified version (20) of the formamide technique (21). A Parlodion flake (Mallinkrodt Co.) was generously given by T. Ando and T. Arai. Abbreviation: MDal, megadalton. 5099 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. E. coli K-12 strains used Strain Relevant markers Source (ref.) "W3110"* trpB9578, tna-2, str C. Yanofsky KY7243 trpB9578, tna-2, str (Xtna imm21::tna) This work KH693 dnaA46, bglB, bglR, tna T. Horiuchit KY7302 trpB9578, tna-2, str, bglR (Xtna imm21::tna) This work KH712 bglR, Agal-attx-bio T. Horiuchit KY7126 bglR, Agal-attx-bio (XcI857S7::bglB) This work SH97 dna-167, trpE, tyr This work KY7200 trpE, tyr (XcI857S7::bglB) This work KL16-99 Hfr/recAl B. J. Bachmann (8) KY2350 glmS, gal, lac T. Satot KY2377 uncA401, gal, lac, ton T. Satot ER F+/asn-31, thi B. J. Bachmann (9) KH716 F+/asn-31, thi, rif T. Horiuchit KY7512 F-/asn, thi, rif, recA This work For gene symbols, see Bachmann et al. (10). The mark:: indicates that the phage genome is inserted in the bacterial gene or the vicinity. A designates deletion. * A derivative of W3110 carrying markers as indicated. t Unpublished results.