Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion

Abstract

The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of ,3-galactosidase was regulated by the tyrR+ gene product. Transducing phage carrying the aroF-lac fusion were isolated, and a A aroF-lac lysogen was used to select for aroFo mutants. A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo.In Escherichia coli K-12 the aroF-tyrA operon codes for two enzymes involved in the biosynthesis. of tyrosine: the tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7phosphate (DAHP) synthetase Tyr (aroF) and the bifunctional enzyme, chorismate mutase T-prephenate dehydrogenase (tyrA) (19). Regulatory mutants with increased expression of this operon have been isolated by using selections for resistance to the tyrosine analog 4-aminophenylalanine. Mutations in these strains have been mapped either in the repressor gene tyrR (22), which is now known to regulate the expression of a number of genes involved in the biosynthesis and transport of the aromatic amino acids (4, 12, 23), or in aroFo (previously designated aroK) (16). aroFo+ is the putative operator sequence at which the tyrR+ gene product binds in the presence of tyrosine to repress transcription of the operon. This paper describes the fusion of the structural genes of the lac operon to the promoter-operator region of aroF, using the method of Casadaban (5). X aroF-lac transducing phage which carry the fusions were isolated, and a A aroFlac lysogen was used to select for strains with aroFo mutations. In addition, we describe the construction of an appropriate plasmid vector onto which such operator mutations can be readily transferred by recombination in vivo as a preliminary step to detailed molecular analyses. MATERIALS AND METHODSOrganisms. Bacterial strains, all derivatives of E. coli K-12, are described in Table 1. Xpl(209) was obtained from M. Casadaban (5) and X(TnJO)tyrR+ (6), K cI Aint9 h80, A cI, and Mu cts62 were from our laboratory stocks.Media. The minimal medium (MM) used was half-strength 56 described by Monod et al. (18) and supplemented with glucose (0.2%), or lactose (0.2%) where appropriate, plus thiamine and required amino acids. Nutrient media used were nutrient broth (Oxoid) and Luria broth (17). Lactose indicator media have been described by Miller (17). 5-Bromo-3-chloro-2-indolyl-p-D-galactoside (XG) was used at a concentration of 40 pug/ml and tetracycline was used at 20 ,ug/ml in nutrient medium, and ampicillin at 50 ,ug/ml and trimethoprim at 5 ,ug/ml were used in MM. For enzyme assays, cells were grown in MM alone or MM plus 10-3 M tyrosine, 10-3 M tryptophan, and 10-3 M phenylalanine.Transductions. Transductions with bacteriophage P1 kc and K have been described previously (6).Propagation of A phage. UV-induced lysates and plate lysates were prepared by the methods of Miller (17).Selection of A lysogens. X lysogens were selected as described by Casadaban (5).Cross-feeding tests. Derivatives of JP587 unable to accumulate dehydroshi...

Construction of a tyrP-lac operon fusion strain and its use in the isolation and analysis of mutants derepressed for tyrP expression

“…The putative tyrP operator mutations were transferred onto pMU518 by recombination in vivo by a modification of the strategy used by Cobbett et al (16). The strains containing these mutations (see below) were made RecA+ by transferring the F-prime F637 into them.…”

Isolation of an aroF-lac plasmid by recombination in vivo

“…The genome of such transducing phage would be analogous in structure to Xpl(209) (7) but with the aroF regulatory region interposed between the trp-lac DNA and the 'Mu cts DNA. Because X aroF-lac transducing phage are stable in the lysogenic state, they can be isolated as prophages by transducing an appropriate tyrR /lac recipient strain and selecting for Lac' transductants (3). Recombination between a X 'Mu cts aroF-lac prophage and a plasmid carrying the 'Mu cts trp-lac region of Xpl(209) was expected to transfer the aroF-lac fusion onto the plasmid (Fig.…”