Joseph S. Gots

Alterations in purine nucleotide pyrophosphorylases and resistance to purine analogues

Abstract Resistance to purine analogues in mutants isolated from Salmonella typhimurium , strain LT-2 , resulted in the loss of specific purine nucleotide pyrophosphorylases. Thus, mutants resistant to 2,6-diaminopurine, 6-mercaptopurine, and 8-azaguanine lacked adenylic, inosinic and guanylic pyrophosphorylases, respectively. The mutants resistant to 2,6-diaminopurine were of two types. One of them, dap-r-3 , was deficient in adenylic pyrophosphorylase, whereas in the other mutant, dap-r-6 , activity of this enzyme was unaffected. However, both the mutants were unable to make corresponding nucleotides from 2,6-diaminopurine. Resistance to 6-mercaptopurine also resulted in two types of mutations. One type, mp-r-I , was deficient only in inosinic pyrophosphorylase; the other, mp-r-2 , in both inosinic and guanylic pyrophosphorylases. Resistance to 8-azaguanine in the mutant, azg-r , resulted in loss of only guanylic pyrophosphorylase activity. All the mutants retained xanthylic pyrophosphorylase activity. Results indicated the existence of at least three (possible four) purine nucleotide pyrophosphorylases. Resistance to all the purine analogues was characterized by the inability of the resistant strains to make nucleotides from the analogues. These observations suggested that the nucleotides were probably the active inhibitory forms of the analogues, and that a deficiency in activating enzymes led to the resistance in the above mutants.

Production of extracellular penicillin-inactivating substances associated with penicillin resistance in Staphylococcus aureus.

Summary A potent penicillin-inactivating substance is produced by penicillin-resistant Staphylococcus aureus but not by penicillin-sensitive strains. The substance is soluble and diffuses readily into both liquid and solid culture medium. The variation of susceptibility to penicillin among various strains of Staphylococcus aureus appears to be associated with a difference in this enzyme formation.

Purine analogs as feedback inhibitors.

Summary Accumulation of ribotide of 5-amino-4-imidazole carboxamide (AICAR), excreted in the riboside form by a purine-requiring mutant of Escherichia coli, was inhibited by a number of structural analogs of purines. The most potent inhibitors were 6-thioguanine, 6-mercaptopurine, and 2,6-diaminopurine. The type of inhibition obtained suggests that the antimetabolites sufficiently resemble natural purines in structure to act as feedback inhibitors in biosynthetic control.

Genetic Alteration of Adenylic Pyrophosphorylase in Salmonella

A mutant of Salmonella typhimurium, resistant to inhibition by 2, 6-diaminopurine, differs from its sensitive wild-type parent by an altered adenylic acid pyrophosphorylase. The altered enzyme, though still active with adenine as substrate, is inactive in the conversion of diaminopurine to its nucleotide. It also differs from the wildtype enzyme in a number of physical and chemical properties.

Further studies on genetically altered purine nucleotide pyrophosphorylases of Salmonella.

Summary Studies on the genetic alteration of purine nucleotide pyrophosphorylases in Salmonella typhimurium has been extended to an analysis of a genetically altered IMP-GMP pyrophosphorylase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8). The altered enzyme was isolated from a mutant resistant to growth inhibition by 8-azaguanine. It differed from the wild type enzyme in its (1) inability to use 8-azaguanine as substrate, (2) elution pattern from DEAE-cellulose columns, (3) resistance to inhibition by p -chloromercuribenzoate, and (4) substrate affinities for hypoxanthine and phosphoribosyl pyrophosphate. The mutant enzyme was not altered in its ability to bind 8-azaguanine since the analogue was still active in inhibiting the conversion of natural substrates. A genetically altered AMP-pyrophosphorylase showed similar characteristics, in that inhibition of the conversion of adenine by the mutant enzyme was inhibited by 2, 6-diaminopurine even though the analogue could no longer serve as substrate.

Specific Action of Adenine as a Feedback Inhibitor of Purine Biosynthesis

Purines can prevent the formation of aminoimidazole precursors which are accumulated by bacterial mutants genetically blocked in purine biosynthesis. If the block does not interfere with interconversions among adenine, guanine, hypoxanthine and xanthine, then any of the purines can act as a feedback inhibitor. If conversion of the other purines to adenine is prevented, then adenine becomes a specific requirement for inhibition; this indicates that feedback control operates at a level involving adenine or one of its congeners.

Mechanism of resistance to 2,6-diaminopurine in Salmonella typhimurium.

Abstract 2,6-Diaminopurine-resistant mutants derived from Salmonella typhimurium strain LT -2 showed a decreased capacity to incorporate 2,6-diaminopurine. However, they differed in their ability to take up adenine from the medium. The mutant, dap - r -6, which showed increased sensiivity to adenine inhibition, incorporated adenine at the same rate as LT -2. On the other hand, dap - r -3, which was resistant to adenine inhibition, showed a reduced incorporation of this purine. Incorporation of hypoxanthine, guanine, and xanthine were unaffected in this mutant. The 5-amino-4-imidazole carboxamide, an intermediate in purine biosynthesis, was incorporated poorly by both LT -2 and dap - r -3. Examination of cell-free extracts revealed that the resistant mutants were unable to catalyze a reaction between 2,6-diaminopurine and 5-phosphoribosyl-1-pyrophosphate to form the corresponding nucleotide. Adenylic pyrophosphorylase activity in the extracts of dap - r -6 was the same as in LT -2, but was greatly reduced in dap - r -3. The inosinic, guanylic, and xanthylic pyrophosphorylase activities were unaffected in both mutants. Possible mechanisms of resistance to 2,6-diaminopurine in the resistant mutants are discussed in light of the above observations.

Nitrofurans as electron acceptors for certain respiratory enzymes

Abstract 1. 1. Three isolated respiratory enzymes have been investigated as to their ability to reduce nitrofurans. One of these enzymes diaphorase, a flavoprotein, was found to act as a nitrofuran reductase. Triosephosphate dehydrogenase, a sulfhydryl enzyme; and cytochrome oxidase were not affected by furacin. 2. 2. The inhibition of the reduction of certain hydrogen acceptors (oxygen, methylene blue, and 2,3,5-triphenyltetrazolium) in the presence of furacin can be ascribed to the preferential reduction of furacin by diaphorase.

Requirement of cyclic AMP for induction of GMP reductase in Escherichia coli.

Abstract Guanosine-5′-monophosphate reductase is induced in Escherichia coli by growth of the bacteria in the presence of guanine or guanosine. In mutants that lack adenyl cyclase this induction does not occur unless cyclic 3′,5′-AMP is present. GMP reductase is not subject to catabolite repression.

Tryptophan Metabolism and its relation to Phage Resistance in Escherichia coli

SUMMARY: A scries of phage-susceptible tryptophan-requiring mutants of Escherichia coli, strain B, were found to fall into five distinct phenotypes as determined by their alternate growth response to anthranilic acid and indole, and by the substances which they accumulated in the culture fluids. An unidentified substance partially characterized by colorimetric reactions and absorption spectrum accumulated in the culture fluid of two of these types. Tryptophan-requiring T1 phage resistant mutants (B/1, trp) which were readily obtained by phage selection could not be isolated by the penicillin method. This mutant represents a unique tryptophanless phenotype, not only in terms of its resistance to T1 phage, but also on the basis of: (1) accumulation of a new metabolic product; (2) growth stimulation by histidine and tyrosine; (3) suppression of accumulation of certain metabolic products by the other tryptophan auxotrophs; (4) epistatic expression of phenotype when superimposed on the tryptophan auxotrophs by secondary mutation.