Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac

J Gowrishankar, J Pittard
1982 Journal of Bacteriology  
Bacteriophage Mu dl (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage
more » ... on enzyme cleavage map of A ppheA-lac for the enzymes HindlIl and PstI is presented. Bacteriophage Mu dl (lac Apr) of Casadaban and Cohen (6) has been widely used to obtain lac operon fusions to aid in the study of the regulation of expression of a variety of operons in Escherichia coli. In this report, we describe a general method for the isolation, from a Mu dl lysogen, of specialized transducing lambda derivatives carrying the promoter-lac fusion; we applied this method to the preparation of X ppheA-lac phage from a strain in which Mu dl (lac Apr) had been used to fuse the lac genes to the promoter ofpheA, the structural gene for the enzyme chorismate mutase P-prephenate dehydratase (EC 5.4.99.5/4.2.1.51) (13). As discussed in this and in the accompanying paper (14), A ppheA-lac offered several advantages over the original pheA::Mu dl (lac Apr) fusion in elucidation of the mechanisms of transcriptional control of the pheA operon. MATERIALS AND METHODS Bacterial sains and phage. The bacterial strains used were all derivatives of Escherichia coli K-12 and are listed in Table 1 . Xpl(209) was obtained from M. Casadaban (5). A vir, A cI h80 Aint, X cI b2, Mu-1, and Mu cts from our laboratory stocks. A ppheA-lac was constructed for this study (see Fig. 2 ). Chemicals. The chemicals used were obtained commercially and not further purified. 5-Bromo-4chloro-3-indolyl-,3-D-galactoside (X-gal) and o-nitrophenyl-p-D-galactoside were obtained from Sigma Chemical Co., St. Louis, Mo. Barium prephenate, prepared by the method of Cotton and Gibson (9), was a gift from B. E. Davidson. Growth media. Unless otherwise specified, the minimal medium used was half-strength medium 56, as described by Monod et al. (23), supplemented with 0.2% glucose or other carbon sources as indicated, thiamine (10 pLg/ml), and appropriate growth factors. The nutrient media used were Luria broth and nutrient agar (Oxoid Ltd., London). MacConkey agar was also from Oxoid. When the minimal medium was supplemented with repressing concentrations of the aromatic amino acids and vitamins (end products), these were added in the following concentrations: L-phenylalanine, 10-3 M; L-tyrosine, 10-3 M; L-tryptophan, 5 x 10-4 M; shikimic acid, 10' M; p-aminobenzoic acid, 106 M; p-hydroxybenzoic acid, 4 x 10-6 M; and 2,3dihydroxybenzoic acid, 5 x 10-1 M. Oxoid 3 agar was routinely used in the preparation of plates except for minimal plates with carbon sources other than glucose, for which the purer Oxoid 1 agar was used. Tetracycline was used at a final concentration of 5 .g/ml in minimal medium and 15 ig/ml in nutrient medium; ampicillin, at a final concentration of 25 pg/ ml; rifampin, at 100 1Lg/ml; and X-gal, at 40 ag/ml. P1 kc transuction and conjugatin. Transduction and conjugation of P1 kc were carried out by the methods previously described (24, 25). Preparation of Mu dl (lc Apr) lysate (from MAL103) and Its use in mut is. The method for preparing the Mu lysate described by Casadaban and Cohen (6) was followed. Preparation of A lysates. A lysates were prepared by UV induction of lysogens or by lytic propagation in soft agar overlays on nutrient plates, as described by Miller (22). Selection for A lysogens. A lysogens were obtained by spotting the phage lysate on a lawn of the sensitive bacterial strain; colonies from the center of the cleared areas were purified and tested for lysogeny by demonstration of their resistance to A cI b2 and sensitivity to A vir phage. In an alternate method based on that described by Debarbouille and Schwartz (11), sensitive cells grown in Luria broth supplemented with 0.4% maltose were harvested in midexponential phase and mixed with A phage at a multiplicity of infection of 0.1 to 0.5; after adsorption for 30 min at 37°C and further expression for 30 min, an excess of A cI h80 Aint or X cI b2 was 1122 on May 8, 2020 by guest
doi:10.1128/jb.150.3.1122-1129.1982 fatcat:2zhem3eqcnggbdlfgkolustz44