Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12.

Uracil phosphoribosyltransferase from Escherichia coli K12 was purified to homogeneity as determined by polyacrylamide gel electrophoresis. For this purpose a pyrimidine-requiring strain harboring the upp gene on a ColE1 plasmid was used, which showed 15-times higher uracil phosphoribosyltransferase activity in a crude extract. When this strain was grown under conditions of uracil starvation, an additional 10-times elevation of the enzyme activity was obtained. The molecular weight of uracil phosphoribosyltransferase was determined to be 75000; the enzyme consists of three subunits with a molecular weight of 23500. Uracil phosphoribosyltransferase is specific for uracil and some uracil analogues. The apparent Km values for uracil and PRib-PP were 7 microM and 300 microM, respectively. As an effector of enzyme activity, GTP lowered the Km for PRib-PP to 90 microM and increased the Vmax value 2-fold, but had no effect on the Km for uracil. The effect of GTP was found to be pH-dependent. The enzymatic characterization of uracil phosphoribosyltransferase and the observed regulation of its synthesis emphasizes the role of the enzyme in pyrimidine salvage.

Transport of pyrimidine nucleosides in cells of Escherichia coli K 12.

1. The transport of pyrimidine mucleosides into cells of Escherichis coli has been investigated in mutant strains which cannot metabolize these nucleosides. Such cells transport and concentrate purimidine mucleosides several hindredfold. 2. The transport is inhibited by energy poisons and by sulfhydryl reagents. 3. Pyrimidine mucleosides compete mutually for transport. Adenosine is also a strong competitor while guanosine and inosine are weak competitors. 4. The rate of pyrimidine mucleoside transport is shown to be under control of the cytR and deoR gene products, which are also known to regulate the synthesis of nucleoside-catabolizing enzymes. The transport system is repressed by growth on glucose, as is the synthesis of the enzymes.

Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli.

Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-alpha-1-diphosphate (PPRibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing K(m) for PPRibP by about fivefold without affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PPRibP displayed constant velocities. This indicated that PPRibP and GTP influenced the structure of the enzyme. Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PPRibP. Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP.

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.

Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene.

Mutants defective in utilization of uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding uracil phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated Mr of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids.

Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12.

The upp gene coding for uracil phosphoribosyltransferase was subcloned on a 5-kb EcoRI restriction fragment along with the purMN operon. By a combination of complementation, deletion and minicell analyses, the upp gene was located adjacent to and divergently transcribed from the purMN operon. All three gene products could be identified in minicell extracts. The cloned upp gene shows an elevated expression upon uracil starvation. The nucleotide sequence and transcription start of the gene were determined. The sequence yields an open reading frame of 624 nucleotides encoding a protein of 22.5 kDa which is in agreement with the previously determined subunit Mr of the purified enzyme. A putative 5-phosphoribosyl-alpha-1-diphosphate (PRPP) binding site has been identified which is similar to the PRPP binding site of the yeast uracil phosphoribosyltransferase.

The gene encoding the periplasmic cyclophilin homologue, PPIase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP-CRP complex and negatively regulated by the CytR repressor.

The rot gene in Escherichia coli encodes PPIase A, a periplasmic peptidyl-prolyl cis-trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA-binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP-CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP-CRP to two sites separated by 53 bp. Moreover, one of the cAMP-CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP-CRP/DNA nucleoprotein complex consisting of the two cAMP-CRP molecules and CytR bound in between. The two regulators bind co-operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP-CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo- and deoxyribonucleoside catabolism or transport.

Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area.

The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6. To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred. With one exception, these mutant Tsx proteins could still serve as a colicin K receptor. DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication. In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease. The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function. HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane.

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.