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.     

Characterization of a Salmonella typhimurium hisU mutant defective in tRNA precursor processing.

The DA11 mutant of Salmonella typhimurium, originally isolated as derepressed for the histidine operon, carries a temperature-dependent alteration in a nucleolytic enzyme specifically involved in the maturation of tRNA. As a consequence of this alteration, no detectable synthesis of any mature tRNA species occurs in DA11 upon shift at 43 degrees C, whereas many tRNA precursors, whose sizes range between 80 and 750 nucleotides, do accumulate. Kinetic studies on the synthesis and processing of these maturation intermediates show that these molecules represent different stages in the maturation pathway, most of them being the products of previous nucleolytic events. These RNA molecules are in vivo substrates of methylation and thiolation enzymes and can be cleaved in vitro to 4S RNA by wild-type but not by DA11 cell-free extract. Evidence is presented that DA11 is very probably a ribonuclease P mutant.     

prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase.

The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N.K. Pandey and R.L. Switzer, J. Gen. Microbiol, 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis.     

Characterization of a guanine-sensitive mutant defective in adenylo-succinate synthetase activity

A contingent auxotrophic mutant of CHO-Kl cell is described. This mutant grows in minimal medium. Its growth is inhibited by the exogenous addition of guanine at levels which do not affect the wild type parent. Adenine reverses the guanine effect. This mutant does not complement ade-H (defective in adenylosuccinate synthetase) and has been denoted as ade-HG because of its guanine sensitivity. Some partial revertants of ade-H are found to be also sensitive to guanine, suggesting a close relationship between the ade-H locus and the guanine sensitivity. Studies of 14C-hypoxanthine incorporation into nucleotides indicated that ade-HG has some adenylosuccinate synthetase activity whether it is pre-exposed to guanine or not. Early de novo purine synthesis in ade-HG, however, is greatly inhibited when pre-exposed to guanine. This inhibition of purine synthesis by guanine is reversible and its recovery is facilitated by adenine.     

Characterization of a cold-sensitive hisW mutant of Salmonella typhimurium

Previous studies of hisW mutants of Salmonella typhimurium have led to the suggestion that such strains are defective in tRNA maturation. (J. E. Brenchley and J. Ingraham, J. Bacteriol. 114:528-536, 1973). In this study, we report that one hisW strain is defective in the accumulation of all stable RNA species. Polyacrylamide gel electrophoresis of radiolabeled RNA indicated tha at the nonpermissive temperature (20 degrees C) all stable RNa species in the cold-sensitive hisW3333 mutant were synthesized and rapidly degraded. We propose that the cold sensitivity of this strain is caused by such a restriction in stable RNA accumulation at low temperature. In vitro and in vivo studies demonstrated that the RNA degraded in this strain was synthesized de novo and was not preexisting RNA. Furthermore, physiological and genetic recovery from the cold-sensitive hisW phenotype resulted in relatively normal RNA synthesis and accumulation. Thus, the RNA alterations observed in this strain were not explained by defects in a tRNA modification enzyme. Rather, these findings suggest the existence of defective RNA processing and that a control mechanism for the overall synthesis or accumulation of stable RNA species is altered in the hisW3333 mutant.     

Sulfhydryl chemistry of Salmonella typhimurium phosphoribosylpyrophosphate synthetase: identification of two classes of cysteinyl residues

Sulfhydryl-specific reagents were used to study the reactivities and function of the four cysteinyl residues per subunit present in Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. In the presence of high concentrations of denaturants all four cysteinyl residues reacted with sulfhydryl-specific reagents. In the absence or in the presence of low levels of denaturing agents, two classes of cysteinyl residues were identified. A single sulfhydryl reacted rapidly with iodoacetamide and 5,5'-dithiobis(nitrobenzoic acid) (DTNB) without significant loss of enzymatic activity. This single sulfhydryl was identified as Cys-229 by reaction with iodo[1-14C]acetamide, followed by isolation and sequence analysis of a single radiolabeled peptide. The three remaining sulfhydryls reacted to various extents depending on the conditions and sulfhydryl-specific reagents employed. At low Pi concentrations, these residues reacted fully with DTNB, leading to an 80 to 90% loss of enzymatic activity. ATP and high levels of Pi prevented this reaction. These results, along with studies comparing the S. typhimurium PRPP synthetase sequence with the sequences of PRPP synthetases from other species, suggest that the cysteinyl residues in the Salmonella enzyme are not catalytically essential. That one or more of the three less reactive residues may lie in or near the active site is not excluded.     

Inaccurate protein synthesis in a mutant of Salmonella typhimurium defective in transfer RNA pseudouridylation

Protein synthesis was studied comparatively in a wild-type strain of Salmonella typhimurium and in hisT mutant cells defective in the pseudouridylation of transfer RNA. From a quantitative point of view, no significant differences between the two types of strain was observed when measuring the rate of protein synthesis during either exponential growth or starvation for histidine. In contrast, the qualitative analysis of proteins by two-dimensional gel electrophoresis showed that histidine-starved hisT cells mistranslate the genetic program at a higher frequency than exponentially growing hisT cells or either starved or unstarved hisT+ cells.     

Regulation of histidyl-transfer ribonucleic acid synthetase formation in a histidyl-transfer ribonucleic acid synthetase mutant of Salmonella typhimurium

Control of formation of the histidyl-transfer ribonucleic acid (tRNA) synthetase with an increased K(m) for histidine was studied in a hisS mutant of Salmonella typhimurium. Histidine restriction of both the hisS and hisS(+) strains resulted in a derepression of synthesis of histidyl-tRNA synthetase. When grown in a concentration less than the K(m) (100 mug/ml) of l-histidine, the hisS mutant maintained a higher level of histidyl-tRNA synthetase than the hisS(+) strain. Addition of excess amounts of l-histidine to the growth medium of the hisS mutant culture grown with 100 mug of l-histidine per ml resulted in a repression of histidyl-tRNA synthetase formation to equal that of the hisS(+) strain grown in 100 mug of l-histidine per ml. These data confirm previous findings that histidine tRNA is involved in the repression of synthesis of histidyl-tRNA synthetase.     

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.     

Studies of the quaternary structure and the chemical properties of phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) was purified to virtual homogeneity from Salmonella typhimurium cells by a modification of previously published procedures. The molecular weight of the subunit was determined to be 31,000 +/- 3,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium analysis of the enzyme dissolved in 6 M guanidine hydrochloride. The amino acid composition of the enzyme was determined. Proline was identified as the only NH2-terminal residue. PRPP synthetase is apparently composed of identical or nearly identical subunits. NATIVE PRPP synthetase exists in multiple states of aggregation under all conditions. However, two predominant states were demonstrated under certain conditions. A form with molecular weight of 320,000 +/- 20,000 was found at pH 7.5 in the presence of MgATP. At pH 8.2 to 8.6, with or without MgATP, the predominant form corresponded to a molecular weight of 150,000 to 200,000; sedimentation equilibrium and velocity analysis indicated 160,000 +/- 15,000 as the most reliable molecular weight. More highly aggregated forms were observed at 4 degrees and higher protein concentrations. Removal of inorganic phosphate from PRPP synthetase by dilution or dialysis resulted in disaggregation. The fundamental unit of PRPP synthetase appears to consist of five (or possibly six) subunits, which can associate to form a dimer (10 or 12 subunits) and more highly aggregated forms. A pentameric subunit structure is consistent with the multiple species resolved by electrophoresis of the native enzyme in discontinuous polyacrylamide gel systems. Visualization of PRPP synthetase by negative staining with uranyl acetate and electron microscopy revealed fields of very asymmetric molecules, the dimensions of which corresponded to the M = 160,000 form. Dimers and higher aggregates of this unit were also seen. An unusual model, in which the five subunits are asymmetrically arranged, accounts very well for the electron microscopic appearance of the enzyme. The tendency of the enzyme to aggregate is viewed as a consequence of the unsatisfied bonding regions of the fundamental asymmetric unit.     

The isolation and characterisation of a mutant of Salmonella typhimurium defective in mutation frequency decline

A mutant of Salmonella typhimurium strain trpC3 has been isolated which is defective in mutation frequency decline (MFD) for UV-induced suppressor revertants to tryptophan independence. Several characteristics of this mutant, PW₄, suggest that it is altered in the timing or rate of the general excision repair mechanism. Survival is greater in strain PW₄ when the first post-irradiation cell division is delayed by the inhibition of immediate protein synthesis. Similarly, stationary phase cells, which show an extended lag after irradiation, are more UV-resistant than lag-phase cells, which recover more rapidly. These data are consistent with the hypothesis that, in contrast with the parent strain trpC₃, the time available in the mutant strain for the action of excision repair is critical in the determination of survival after UV treatment. Contransductional analysis of the mutant locus indicates close linkage to metE, a region in which excision repair genes have been located.     

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.     

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.     

Biochemical and genetic characterization of a mutant of Salmonella typhimurium defective in a locus for glutamate dehydrogenase activity

Summary By two consecutive treatments with N-methyl-N-nitro-N-nitrosoguanidine we obtained mutant SM151 of Salmonella typhimurium which differs from the parental LT2 strain in: a) is able to use l-glutamate as carbon source (first mutation), and b) requires that amino acid for growth (second mutation). It was found that the requirement of mutant SM151 for glutamate was due to a very low activity of glutamate dehydrogenase. Both glutamate-oxaloacetate transaminase and aspartase activities were present at normal levels. Glutamate dehydrogenase activity was strongly repressed by glutamate; aspartase activity was under severe catabolite (glucose) repression, while glutamate-oxaloacetate transaminase was partially repressed by glutamate. By conjugation and transduction the locus gdh, responsible for the low activity of the glutamate dehydrogenase of mutant SM151, was located at about minute 128 of the bacterial chromosome and found to be linked to the argC, argF, and metB loci.     

Characterization of a HindIII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium

The glnA gene, encoding glutamine synthetase in Salmonella typhimurium, has been cloned into the plasmid pBR322. One hybrid plasmid, pJB1, containing an 8.5 kb insert generated by a HindIII digest, was analyzed using eleven different restriction enzymes. Evidence that the region controlling glutamine synthetase expression remained on the insert was obtained by showing that the regulation is normal in cells carrying plasmids with the insert in the original and reversed orientation. Several new plasmids derived from pJB1 following SalI and EcoRI digestions were examined for their ability to complement a glnA202 mutation in order to locate the DNA segment needed for glutamine synthetase expression. The results show that cells containing plasmid pJB8, which has a 21 kb deletion, produce and regulate glutamine synthetase normally, whereas cells with a plasmid (pJB11) similar to pJB8, but lacking a 0.25 kb EcoRI fragment, do not exhibit glutamine synthetase activity. The analysis of proteins produced in minicells containing pJB8 and pJB11 show that they both produce a protein that migrates with the glutamine synthetase subunit. Because pJB11 makes an inactive protein of similar size to the glutamine synthetase subunit, the 0.25 kb deletion may encode only the C-terminus of this protein. Consistent with this finding is the presence of a strong RNA polymerase-binding site on pJB8 to the right of the 0.25 kb EcoRI that could correspond to a promoter near the N-terminus of the glnA gene.