Karin Hammer-Jespersen

Regulation of the deo operon in Escherichia coli

SummaryThe synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.

Multiple regulation of nucleoside catabolizing enzymes: Regulation of thedeo operon by thecytR anddeoR gene products

SummaryThe protein and repressor nature of two regulatory gene products inE. coli has been demonstrated, employing mutants with either amber or thermosensitive mutations. The regulatory genes are thecytR and thedeoR genes, both of which contribute to the regulation of the synthesis of nucleoside catabolizing enzymes.Enzyme levels in strains with concurrent mutations in both regulatory genes are considerably higher than the sum of the levels in strains with acytR or adeo R mutation alone, indicating a certain co-operativity between the two repressor proteins.The glucose repression of enzyme levels observed in the double regulatory mutant is similar to that found in acytR mutant, and much more pronounced than the glucose effect in adeo R mutant.A model of the promoter-operator region in thedeo operon is proposed.

Mutants of Escherichia coli unable to metabolize cytidine: Isolation and characterization

SummaryStrains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.

Overlapping transcriptional units in the deo operon of Escherichia coli K-12: Evidence from phage Mu-1 insertion mutants

Four closely-linked genes of Escherichia coli, comprising the deoxyribonucleoside (deo) operon, viz. dra, tpp, drm, and pup†, which code for enzymes involved in the catabolism of nucleosides and deoxynucleosides, are known to form two transcriptional units, one initiated from a promoter to the left of dra and one initiated between tpp and drm. Thus a potentially polar mutation induced by phage Mu-1 in tpp has no effect on the level of drm gene product. We show here, however, that if the level of transcription of all four genes is increased by the insertion of a regulatory mutation (deoR− or cytR−) then a Mu-induced polar mutation in tpp does in fact drastically reduce the level of drm gene product. The interpretation of this result is that two species of mRNA are produced; one is tetracistronic, being transcribed from a promoter located to the left of dra, and one is dicistronic, being transcribed from a promoter lying between tpp and drm. The two distal genes are therefore transcribed into two separate mRNA species. In the wild-type (deoR+ cytR+) most of the drm and pup gene products are synthesized from the short message and thus there is no polar effect of a Mu-1-induced tpp mutation on drm. The long message is controlled by the deoR and cytR gene products, whereas the remaining inducibility of drm in deoR− cytR− strains with a polar tpp mutation suggests that the short message is controlled by another, unknown, regulatory system.

Multiple regulation of nucleoside catabolizing enzymes: Effects of a polar dra mutation on the deo enzymes

SummaryStrains with an amber, polar mutation in the dra1 gene have been isolated. The mutation was introduced into a set of isogenic strains, wild type or with concurrent regulatory mutations, and further characterized by suppression and heat inactivation experiments.The effect of the polar dra mutation on the three remaining genes of the deo operon, the tpp, drm and pup genes, was determined by estimating the enzyme levels in the various dra-mutants. The effect was found to be non-coordinate, indicating the formation in the cells of two types of transcripts: A tetracistronic unit, containing the message from all four genes, and a dicistronic unit, covering the two distal genes only.

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: Effects of 3:5′ cyclic AMP and CRP protein

SummaryThe regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.

Evidence for the existence of three promoters for the deo operon of Escherichia coli K12 in vitro.

Abstract Expression of the deo operon of Escherichia coli K12 has been studied in vitro using the Zubay (1973) system for DNA primed transcription and translation. The kinetics for messenger RNA synthesis of all four structural deo genes have been followed by use of rifampicin and streptolydigin. The results show that all three deo promoters initiate mRNA synthesis in vitro ; expression of the internal deo promoter was demonstrated using a phage lambda DNA template constructed so as to be devoid of the other two promoters. The relative amounts of mRNA made from the three deo promoters in vitro correspond to previous findings in vivo . At one of the deo promoters initiation of transcription starts immediately whereas a lag in initiation is seen for the two other deo promoters and also for the lac promoter. This demonstrates that the times required for RNA polymerase to reach a constant rate of initiation from the three deo promoters in vitro are different. The time lag in initiation of transcription, characteristic for a given deo promoter, is found not to be inversely proportional to the promoter strength.

Fusion of the lac genes to the promotor for the cytidine deaminase gene of Escherichia coli K-12.

SummaryPhage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage λ (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming λ phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.