Stimulatory effect of low ATP pools on transport of purine nucleosides in cells of Escherichia coli.

Cells of Escherichia coli contain two nucleoside-transport systems. Energy-starved cells of a strain containing only one of these systems and, in addition, carrying a mutation in the Ca2+- and Mg2+-dependent ATPase (ATP phosphohydrolase 3.6.1.3) are still able to transport nucleosides. The rate is only slightly lower than the rate measured in unstarved cells. Freshly harvested uncA cells transport purine nucleosides at a higher rate than cells from the isogenic strain containing a functional ATPase. If cells from the latter strain are treated with arsenate, transport rates increase to the same levels as found in uncA cells. The presence of an uncA mutation has no effect on the transport rates for cytidine, deoxycytidine, and uridine, nor has arsenate treatment. These findings indicate that ATP is not required as energy donor for nucleoside transport. The enhanced transport rate for purine nucleosides after treatment with arsenate seems to suggest a regulatory relationship between the transport of these nucleosides and the cellular levels of ATP or a closely related metabolite.

Nucleoside transport systems in Escherichia coli K12: specificity and regulation.

Two nucleoside transport systems have been verified and separated by mating and recombination experiments. The recipient strain was a mutant which is negative for transport of all nucleosides. The two systems differ in specificity and in regulation. One system transports pyrimidine and adenine nucleosides. It is regulated by the cytR gene. The other system transports all nucleosides and is regulated by the cytR as well as by the deoR genes. Enzyme assays performed on whole cells of strains, able or unable to transport nucleosides, indicate that the nucleoside catabolizing enzymes are located inside the permeability barrier of the cell.

Analysis of the regulatory region of the Escherichia coli nupG gene, encoding a nucleoside-transport protein.

The S1-mapping procedure, when applied to an 800-bp fragment upstream of the nupG structural gene of Escherichia coli revealed one transcription-initiation site. The corresponding promoter is negatively regulated by the cytR and the deoR repressors. The expression of the gene is activated by the complex between cAMP and its receptor protein. In strains lacking cAMP the promoter expression is reduced and it is no longer regulated by the cytR repressor, whereas the deoR regulation is retained.

Studies on the sequence and structure of the Escherichia coli K-12 nupG gene, encoding a nucleoside-transport system.

The nupG gene, encoding one of the two active nucleoside-transport systems in Escherichia coli K-12, has been cloned on the multicopy plasmid pBR322 and derivatives thereof. The recombinant plasmids complemented a chromosomal nupG mutation. A genetic map was determined by digestion with restriction endonucleases and the nucleotide sequence of a 3-kb stretch of DNA has been determined on fragments cloned into M13 phages. An open reading frame of 1254 bp, encoding a protein with a calculated molecular mass of 45.333 kDa, was deduced to be the coding region of nupG. Minicell-forming strains carrying plasmids containing this gene were shown to produce a hydrophobic, membrane-bound polypeptide with an apparent molecular mass of approximately 43 kDa.

Nucleoside transport in cells and membrane vesicles from Escherichia coli K12.

Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells. The strains used carried mutations in the nucleoside catabolizing enzymes. This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment. Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine. Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles. These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport. Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts. This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside.

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

Strains 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.

Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

The 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.