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1.
Mol Microbiol ; 4(3): 381-92, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2192230

ABSTRACT

The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities. The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.


Subject(s)
Acyltransferases/genetics , Carbon-Nitrogen Ligases , DNA, Bacterial/analysis , Escherichia coli/genetics , Hydroxymethyl and Formyl Transferases , Nucleotide Deaminases/genetics , Acyltransferases/biosynthesis , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/enzymology , Humans , Ligases/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Nucleotide Deaminases/biosynthesis , Phosphoribosylaminoimidazolecarboxamide Formyltransferase , Restriction Mapping , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics
2.
J Bacteriol ; 171(1): 205-12, 1989 Jan.
Article in English | MEDLINE | ID: mdl-2464576

ABSTRACT

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.


Subject(s)
Carboxy-Lyases/genetics , Escherichia coli/genetics , Genes, Bacterial , Genes , Operon , Amino Acid Sequence , Base Sequence , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Escherichia coli/enzymology , Molecular Sequence Data , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Messenger/genetics , Restriction Mapping , Sequence Homology, Nucleic Acid , Species Specificity , Transcription, Genetic
3.
Gene ; 44(1): 55-62, 1986.
Article in English | MEDLINE | ID: mdl-3021590

ABSTRACT

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.


Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Operon , DNA Restriction Enzymes , DNA-Directed RNA Polymerases/metabolism , Genes , Genes, Bacterial , Genetic Complementation Test , Genetic Vectors , Plasmids , Promoter Regions, Genetic , beta-Galactosidase/genetics
4.
J Bacteriol ; 164(3): 1288-93, 1985 Dec.
Article in English | MEDLINE | ID: mdl-2999079

ABSTRACT

The guaC gene encodes GMP reductase, which converts GMP to inosine monophosphate. Regulation of guaC expression was examined by use of guaC-lac fusions created by Mu d1(lac). In these strains, beta-galactosidase is induced by guanine derivatives, and this induction is prevented by adenine. Our previous implication that glutamine acts as a negative effector of transcription was confirmed by showing that glutamine analogs (diazo-oxo-norleucine and methionine sulfoximine) can also induce beta-galactosidase. GMP was implicated as a likely candidate for the in vivo inducer by introducing a gpt block to prevent the conversion of guanine to GMP and a deoD block to prevent the interconversion of guanine and guanosine. Regulatory mutants were isolated by growth on lactose plus adenine. Though these showed high constitutive levels of beta-galactosidase, they were normal for the regulation of GMP reductase when the fusion was corrected by transduction to guaC+ or when guaC+ was introduced by plasmid complementation. The regulatory mutants were linked to guaC.


Subject(s)
Escherichia coli/genetics , Gene Expression Regulation , NADH, NADPH Oxidoreductases/genetics , GMP Reductase , Glutamine/analogs & derivatives , Glutamine/pharmacology , Guanosine Monophosphate/metabolism , Inosine Monophosphate/metabolism , Mutation , Purines/pharmacology , beta-Galactosidase/genetics
5.
J Gen Microbiol ; 131(2): 245-52, 1985 Feb.
Article in English | MEDLINE | ID: mdl-2580045

ABSTRACT

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.


Subject(s)
Phosphotransferases/deficiency , Ribose-Phosphate Pyrophosphokinase/deficiency , Salmonella typhimurium/enzymology , GMP Reductase , Genes , Genetic Markers , Mutation , NADH, NADPH Oxidoreductases/metabolism , Nucleotides/metabolism , Phosphoribosyl Pyrophosphate/metabolism , Ribonucleotides/biosynthesis , Ribose-Phosphate Pyrophosphokinase/genetics , Ribose-Phosphate Pyrophosphokinase/metabolism , Salmonella typhimurium/genetics , Salmonella typhimurium/growth & development , ortho-Aminobenzoates/biosynthesis
6.
J Bacteriol ; 151(1): 153-61, 1982 Jul.
Article in English | MEDLINE | ID: mdl-7045073

ABSTRACT

Expression of the purE operon of Salmonella typhimurium was analyzed by using an Escherichia coli F' episome containing a purE-lac fusion. The fusion removes the lacOP and part of the lacZ genes of the lac operon and places the intact lacY and lacA genes under control of the purE operon as shown by inhibition of growth on melibiose (lacY) and repression of thiogalactoside transacetylase (lacA) by various purines. Two classes of regulatory-deficient mutants were found among those resistant to inhibition by purines. One class was trans active (chromosomal) and corresponded to previously described purR mutants involving a deficient cytoplasmic repressor substance. These were also altered in the expression of the purF, purD, purG amd purI genes as evidenced by loss of repressibility of the synthesis of glycinamide ribotide and aminoimidazole ribotide. The other class was cis active (episomal), specific for only purE expression, and thus corresponded to an altered purE operon signal (operator or promoter). The metabolic requirements for the expression of purE were also monitored by measuring repression of the transacetylase in strains with various genetically altered metabolic backgrounds. Repression by guanine required an intact guanine phosphorbosyltransferase (gpt) and repression by adenine and all nucleosides required purine nucleoside phosphorylase (deoD). Synthesis of cyclic AMP (cya) and its receptor protein (crp) were no longer required for the expression of the lac genes under purE control.


Subject(s)
Mutation , Purines/metabolism , Salmonella typhimurium/genetics , Acetyltransferases/genetics , Operon , Salmonella typhimurium/isolation & purification , Salmonella typhimurium/metabolism , Species Specificity
7.
J Gen Microbiol ; 121(2): 357-64, 1980 Dec.
Article in English | MEDLINE | ID: mdl-7021762

ABSTRACT

Two mutants of Salmonella typhimurium were isolated which differ from their respective parental strains in their growth responses to guanine and xanthine. Both mutants had purine phosphoribosyltransferase activities similar to their parental strains. One mutant, CB-3, had a lower guanine uptake rate apparently caused by a genetic lesion in a specific gene (designated guaP) responsible for facilitating the transport of guanine. This gene mapped at 3.5 min in the sequence azi-guaP-nadC. The second mutant, GP103, had a purine carrier molecule with altered specificity, as demonstrated by a competition between hypoxanthine and xanthine for uptake.


Subject(s)
Pentosyltransferases/metabolism , Purines/metabolism , Salmonella typhimurium/enzymology , Chromosome Mapping , Guanine/metabolism , Hypoxanthines/metabolism , Mutation , Salmonella typhimurium/genetics , Xanthines/metabolism
8.
J Biol Chem ; 255(20): 9594-8, 1980 Oct 25.
Article in English | MEDLINE | ID: mdl-7000760

ABSTRACT

A DNA-binding protein was isolated from Escherichia coli using a procedure designed for selective enrichment of regulatory proteins. In this procedure, a multicopy bacterial plasmid carrying a purE operon was used to sequester and mobilize the putative regulatory protein for pur genes. Purification of the protein from a plasmid-enriched lysate was obtained by precipitation with polyethylene glycol, elution from the precipitate with high salt and fractionation by phosphocellulose, and AMP affinity chromatography. Analysis of DNA binding by nitrocellulose filter assays showed that binding of the protein required the presence of pur genes and was either ATP dependent for some genes (purE, purA) or GTP dependent for others (purF, purI). Plasmid DNA carrying the guaAB operon did not bind the protein.


Subject(s)
Carrier Proteins/isolation & purification , DNA, Bacterial/metabolism , DNA/isolation & purification , Escherichia coli/metabolism , Carrier Proteins/metabolism , DNA/metabolism , DNA-Binding Proteins , Genes , Guanosine Triphosphate/pharmacology , Kinetics , Plasmids , Protein Binding
9.
J Bacteriol ; 143(3): 1156-64, 1980 Sep.
Article in English | MEDLINE | ID: mdl-6447689

ABSTRACT

The purF locus codes for the first enzyme, glutamine phosphoribosylpyrophosphate amidotransferase, of the purine biosynthetic pathway. A strain of Escherichia coli K-12 was isolated in which the lac structural genes were fused to the control region of the purF locus. This purF-lac fusion was shown to respond to purine-specific regulatory signals. A plaque-forming lambda transducing phage bearing this purF-lac fusion was isolated. This phage was used to genetically determine the direction of transcription for the pufF locus by two independent means. Results from both methods agreed that the direction of transcription of the purF locus was clockwise on the standard Escherichia coli K-12 genetic map.


Subject(s)
Amidophosphoribosyltransferase/genetics , Escherichia coli/genetics , Genes, Regulator , Genes , Pentosyltransferases/genetics , Transcription, Genetic , Bacteriophage lambda , Chromosomes, Bacterial , Lac Operon , Recombination, Genetic , Transduction, Genetic
10.
J Bacteriol ; 143(2): 864-71, 1980 Aug.
Article in English | MEDLINE | ID: mdl-6782081

ABSTRACT

The pathway for the utilization of 2,6-diaminopurine (DAP) as an exogenous purine source in Salmonella typhimurium was examined. In strains able to use DAP as a purine source, mutant derivatives lacking either purine nucleoside phosphorylase or adenosine deaminase activity lost the ability to do so. The implied pathway of DAP utilization was via its conversion to DAP ribonucleoside by purine nucleoside phosphorylase, followed by deamination to guanosine by adenosine deaminase. Guanosine can then enter the established purine salvage pathways. In the course of defining this pathway, purine auxotrophs able to utilize DAP as sole purine source were isolated and partially characterized. These mutants fell into several classes, including (i) strains that only required an exogenous source of guanine nucleotides (e.g., guaA and guaB strains); (ii) strains that had a purF genetic lesion (i.e., were defective in alpha-5-phosphoribosyl 1-pyrophosphate amidotransferase activity); and (iii) strains that had constitutive levels of purine nucleoside phosphorylase. Selection among purine auxotrophs blocked in the de novo synthesis of inosine 5'-monophosphate, for efficient growth on DAP as sole source of purine nucleotides, readily yielded mutants which were defective in the regulation of their deoxyribonucleoside-catabolizing enzymes (e.g., deoR mutants).


Subject(s)
2-Aminopurine/analogs & derivatives , Adenine/analogs & derivatives , Salmonella typhimurium/metabolism , 2-Aminopurine/metabolism , Adenosine Deaminase/metabolism , Guanine/metabolism , Mutation , Purine-Nucleoside Phosphorylase/metabolism , Purines/metabolism
12.
J Bacteriol ; 143(1): 105-11, 1980 Jul.
Article in English | MEDLINE | ID: mdl-6249786

ABSTRACT

The addition of a glutamine analog, 6-diazo-5-oxo-L-norleucine, or an inhibitor of glutamine synthetase, L-methionine-dl-sulfoximine, to the growth media of most Salmonella typhimurium strains resulted in a marked elevation of guanosine monophosphate reductase levels. The elevation caused by either compound required protein synthesis and could be antagonized by exogenous glutamine. In addition, when glutamine auxotrophs were grown in suboptimal concentrations of glutamine, the guanosine monophosphate reductase levels were increased. It is postulated that glutamine or a product of its metabolism may function under normal conditions as a negative regulatory element in the control of guanosine monophosphate reductase and that decreased effective intracellular levels of glutamine result in an increase in the level of the enzyme.


Subject(s)
Azo Compounds/pharmacology , Diazooxonorleucine/pharmacology , Glutamine/physiology , Guanosine/metabolism , NADH, NADPH Oxidoreductases/metabolism , Salmonella typhimurium/enzymology , Bacterial Proteins/biosynthesis , Enzyme Induction , GMP Reductase , Guanosine Monophosphate/metabolism , Methionine Sulfoximine/pharmacology
14.
Ciba Found Symp ; (48): 23-41, 1977.
Article in English | MEDLINE | ID: mdl-204462

ABSTRACT

Bacterial systems have been used to identify and characterize the organization of the genetic units and the regulatory elements that control purine metabolism. An analysis of 13 genes that control the biosynthesis of AMP and GMP has revealed three multigenic operons. These show properties of gene contiguity, promoter sites, coordinate expression and polarity effects. The unit controlling the formation of IMP is one operon (pur JHD) consisting of three genes which together control the formation of phosphoribosylglycinamide synthetase (EC 6.3.4.13), an early enzyme in the biosynthetic pathway, and a terminal bifunctional complex (IMP cyclohydrolase--formyltransferase). Regulatory mutants were isolated and characterized by several methods including the use of a unique fusion of two unrelated operons. Both operator constitutive and repressor type (purR) mutations have been identified. The purR product functions in the common control of several genetically distinct enzymes that participate before the formation of IMP. Plasmid DNA enriched for the purE operon has been isolated and used in the study of the role of nucleotide effectors in the binding of repressor-like proteins. AMP but not GMP is needed for binding, and purR mutants are deficient in the binding substance. Mutants with differential blocks in the salvage and interconverting reactions have been used to characterize the regulatory elements of the formation and the activity of guanosine kinase, GMP reductase (EC 1.6.6.8), and purine nucleoside phosphorylase (EC 2.4.2.1). Two structural gene products (purF) and (purG) have been implicated as possible regulatory elements for the use of guanosine, and a role for glutamine in the induction of GMP reductase has been revealed.


Subject(s)
Escherichia coli/metabolism , Purine Nucleotides/metabolism , Salmonella typhimurium/metabolism , Adenosine Monophosphate/biosynthesis , DNA Helicases/metabolism , DNA, Bacterial/metabolism , Escherichia coli/genetics , Genes, Regulator , Glycine , Guanosine Monophosphate/biosynthesis , Ligases/metabolism , Mutation , Nucleotide Deaminases/metabolism , Operon , Plasmids , Ribosemonophosphates , Salmonella typhimurium/genetics
15.
Mol Gen Genet ; 145(1): 31-6, 1976 Apr 23.
Article in English | MEDLINE | ID: mdl-775307

ABSTRACT

A defect in the repression of the de novo purine biosynthetic enzymes was detected among purA mutants of Salmonella typhimurium. We suggest that the defect is caused by an altered purine regulation gene (purR) which affects the response level of at least five of the de novo enzymes to repression by excess adenine. Thus the unlinked genes controlling these enzymes constitute a regulation controlled wholly or in part by a purR gene product. The regulation of the guanine operon is regulated by some other mechanism independent of purR.


Subject(s)
Genes, Regulator , Mutation , Purine Nucleotides/biosynthesis , Salmonella typhimurium , Adenine/metabolism , Genetic Complementation Test , Genetic Linkage , Guanine/metabolism , Phenotype
16.
Biochim Biophys Acta ; 403(1): 47-57, 1975 Sep 22.
Article in English | MEDLINE | ID: mdl-169910

ABSTRACT

The levels of guanosine 5'-phosphate reductase (EC 1.6.6.8) in Salmonella typhimurium appear to be modulated by changes in the ratio of the adenine and guanine nucleotide pools. Alterations of this ratio may be induced by high levels of guanosine in the culture medium or by genetic lesions in one of several purine interconversion enzymes, such as pur A or pur B mutants. The induction of the reductase requires transcription and translation processes and, in contrast to earlier observation with Escherichia coli, is not dependent on cyclic adenosine 3',5'-phosphate or the cyclic adenosine 3',5'-phosphate receptor protein.


Subject(s)
NADH, NADPH Oxidoreductases/metabolism , Salmonella typhimurium/enzymology , Cyclic AMP/pharmacology , Enzyme Induction/drug effects , Genotype , Guanine Nucleotides , Guanosine/pharmacology , Histidine/pharmacology , Hypoxanthines/pharmacology , Protein Biosynthesis/drug effects , Purines/pharmacology , Salmonella typhimurium/drug effects , Species Specificity
17.
J Bacteriol ; 121(1): 77-82, 1975 Jan.
Article in English | MEDLINE | ID: mdl-1090579

ABSTRACT

Salmonella typhimurium strain GP660 (proAB-gpt deletion, purE) lacks guanine phosphoribosyltransferase and hence cannot utilize guanine as a purine source and is resistant to inhibition by 8-azaguanine. Strain GP660 was mutagenized and a derivative strain (GP36) was isolated for utilization of guanine and hypoxanthine, but not xanthine, as purine sources. This alteration was designated sug. The strain was then sensitive to inhibition by 8-azaguanine. Column chromatographic analysis revealed the altered phosphoribosyltransferase peaks for both hypoxanthine and guanine to be located together, in the same position as hypoxanthine phosphoribosyltransferase (hpt gene product) of the wild-type strain. Genetic analysis showed the sug mutation to be allelic with hpt. Therefore sug represented a modification of the substrate specificity of the hpt gene product.


Subject(s)
Guanine/metabolism , Hypoxanthines/metabolism , Mutation , Pentosyltransferases/metabolism , Salmonella typhimurium/enzymology , Alleles , Azaguanine/pharmacology , Cell-Free System , Chromatography, Ion Exchange , Genes , Mutagens , Nitrosoguanidines , Phenotype , Recombination, Genetic , Salmonella typhimurium/metabolism , Transduction, Genetic , Xanthines/metabolism
20.
J Bacteriol ; 112(2): 910-6, 1972 Nov.
Article in English | MEDLINE | ID: mdl-4563984

ABSTRACT

Certain proAB deletion mutants of Salmonella typhimurium were found to be simultaneously deleted in a gene required for the utilization of guanine and xanthine (designated gxu). These mutants were resistant to 8-azaguanine and when carrying an additional pur mutation were unable to use guanine or xanthine as a purine source. The defect was correlated with deficiencies in the uptake and phosphoribosyltransferase activities for guanine and xanthine. Hypoxanthine and adenine activities were unaltered. The deficiency was restored to normal by transduction to pro(+) and in F' merodiploids.


Subject(s)
Genes, Regulator , Mutation , Pentosyltransferases/metabolism , Salmonella typhimurium/enzymology , Adenine/metabolism , Azaguanine/pharmacology , Carbon Isotopes , Cell-Free System , Chromosome Mapping , Culture Media , Drug Resistance, Microbial , Genetics, Microbial , Guanine/metabolism , Guanine Nucleotides , Hypoxanthines/metabolism , Oxidoreductases/metabolism , Salmonella typhimurium/drug effects , Salmonella typhimurium/growth & development , Salmonella typhimurium/metabolism , Xanthines/metabolism
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