purF-lac fusion and direction of purF transcription in Escherichia coli

Bacteriophage Mu d1 (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 map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.

Section: Resultsmentioning

confidence: 90%

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

Gowrishankar¹,

Pittard²

1982

“…It seems to us that this simple interpretation is not valid. Thus it is known that addition of adenine to cultures of E. coli causes repression of the pur genes (17,25), including purF (25), but it is also known that in E. coli (16), as well as in S. typhimurium (Table 2), the presence of adenine elevates the ATP pool and somewhat reduces the GTP pool. Therefore, although GTP may be required for a putative pur repressor to bind to purF DNA, it is hardly this binding of GTP that causes repression of purF expression in vivo.…”

Section: Resultsmentioning

confidence: 99%

Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression

Houlberg¹,

Jensen²

1983

Data are presented which indicate that the repression of pur gene expression seen after the addition of preformed purines to cultures of Salmonella typhimurium is the consequence of the presence or the formation of the purine bases, hypoxanthine and guanine. This conclusion is based on the following observations. First, it was impossible to find a correlation between the size of any individual purine nucleotide pool and the level of the first four enzymes in the de novo biosynthetic pathway. Second, adenine plus guanosine served as a perfect source of purine nucleotides, but their presence caused no repression of pur gene expression if the cells lacked purine nucleoside phosphorylase activity. This enzyme is needed to convert adenine and guanosine to hypoxanthine and guanine, but not for their conversion to nucleotides. Third, addition of guanine to a strain lacking guanine phosphoribosyltransferase (gpt) resulted in a repression of the level of the purine de novo biosynthetic enzymes, a reduction of the growth rate, and a fall in the pools of ATP and GTP. Addition of hypoxanthine to a strain lacking hypoxanthine phosphoribosyltransferase (hpt) had a similar, although weaker, effect. If the cells lacked both hypoxanthine and guanine phosphoribosyltransferases (hpt gpt), their basal level of the purine de novo biosynthetic enzymes was repressed in minimal medium. Such cells grow slower than wild-type cells and excrete purines, probably due to the inability to salvage endogenously formed hypoxanthine and guanine.

“…By using the method of Casadaban, the Xpl(209) prophage adjacent to an operon fusion can be induced and may form k transducing phage which carry the fusion. This has been achieved by others who identified such phage by their ability to produce blue plaques on medium containing XG (5,20,21). JP3107, J. BACTERIOL.…”

Section: Resultsmentioning

confidence: 98%

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

Cobbett¹,

Morrison²,

Pittard³

1984

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...