Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

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.     

Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.     

Mutant strains of Salmonella typhimurium with defective phosphoribosylpyrophosphate synthetase activity

A mutant of Salmonella typhimurium with undetectable phosphoribosylpyrophosphate (PRPP) synthetase activity in vitro and abnormally low PRPP pools in vivo was identified by screening temperature-sensitive isolates by an autoradiographic procedure. The lack of PRPP synthetase activity in vitro and temperature-sensitive growth were shown to result from separate, but closely linked mutations mapping at 47 units on the Salmonella chromosome. Mutant cell extracts prepared by a variety of methods did not show any detectable PRPP synthetase activity, but material that was immunochemically cross-reactive with PRPP synthetase was detected by complement fixation analysis. A second mutant, isolated by localized mutagenesis, contained about half the PRPP synthetase and cross-reacting material of the parental strain.     

Isolation and partial characterization of an argR mutant of Salmonella typhimurium.

An arginine regulatory mutant (i.e., mutated in the argR gene) has been isolated from a strain of Salmonella typhimurium LT2. The argR mutant was found to excrete arginine into the growth medium with glycerol but not glucose as carbon source. Constitutive synthesis of arginine biosynthetic enzymes was observed. Whereas previous results (A. T. Abd-E1-A1 and J. L. Ingraham, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K169, p. 175) have shown constitutive synthesis of carbamyl phosphate synthetase in the argR mutant, the regulation of the synthesis of the last five enzymes of the pyrimidine pathway was unaffected. However, in pyrH mutants, known to exhibit derepressed synthesis of the pyrimidine enzymes, a 10-fold derepression of ornithine transcarbamylase was observed.     

pepM is an essential gene in Salmonella typhimurium.

The pepM gene of Salmonella typhimurium codes for a methionine-specific aminopeptidase that removes N-terminal methionine residues from proteins. This gene was inactivated in vitro by the insertion of a DNA fragment coding for kanamycin resistance. The inactivated gene could not replace the wild-type chromosomal pepM gene unless another functional copy was present in the cell. The lethal effect of the pepM insertion was not a result of polarity on any gene downstream, nor was it affected by the presence or absence of other peptidases.     

Structural gene for NAD synthetase in Salmonella typhimurium.

We have identified the structural gene for NAD synthetase, which catalyzes the final metabolic step in NAD biosynthesis. This gene, designated nadE, is located between gdh and nit at 27 min on the Salmonella typhimurium chromosome. Mutants of nadE include those with a temperature-sensitive lethal phenotype; these strains accumulate large internal pools of nicotinic acid adenine dinucleotide, the substrate for NAD synthetase. Native gel electrophoresis experiments suggest that NAD synthetase is a multimeric enzyme of at least two subunits and that subunits from Escherichia coli and S. typhimurium interact to form an active heteromultimer.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.     

Molecular characterization of the Salmonella typhimurium parE gene.

The DNA sequence of the wild type S. typhimurium parE gene was determined. The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S. typhimurium gene. Subclones of the S. typhimurium parE gene localized the sites of four heat sensitive mutations within parE. The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB. Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro). All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.     

Binding of the substrates and the allosteric inhibitor adenosine 5'-diphosphate to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium

The binding of the substrates, ATP and ribose-5-P, and the most effective inhibitor, ADP, to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium was characterized using equilibrium dialysis of these compounds labeled with 32P. In the absence of ribose-5-P, ATP, ADP, and the ATP analogue alpha,beta-methylene ATP each bind cooperatively with half-saturation at 50 to 90 microM and Hill coefficients of 1.5 to 2. We propose that all three compounds bind at the same set of sites, which are presumably the active sites. When ribose-5-P was added, methylene ATP and ADP binding at these sites became tighter (Kd approximately 3 to 6 microM at 10 mM ribose-5-P) and lost its cooperativity. In the presence of ribose-5-P, ADP, but not methylene ATP, bound to a second site with half-saturation at approximately 150 microM and a Hill coefficient greater than 3. This result confirms the existence of an allosteric ADP site, which was previously postulated from kinetic studies (Switzer, R. L., and Sogin, D. C. (1973) J. Biol. Chem. 248, 1063-1073). Binding of ribose-5-P could not be detected in the absence of nucleotides, but it was readily measured in their presence. The apparent Kd of ribose-5-P varied from greater than 1 mM to approximately 5 microM as the concentration of either ADP or methylene ATP was increased from 0 to 2 mM. Inhibition of the enzyme by action of ADP at both active and allosteric sites could be observed kinetically.     

Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.

The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.