Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.     

Biochemical characterization of a mutant isoleucyl-transfer ribonucleic acid synthetase from Escherichia coli K-12

Isoleucyl-transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5; IRS] was partially purified from Escherichia coli K-12 and from an ileS mutant that appears to be altered in IRS. The half-life of wild-type IRS, incubated at 60.5 C, is 69 min, whereas that of mutant IRS is 8 min. Mutant IRS shows about a 100-fold lower affinity than wild-type IRS for isoleucine, dl-valine, thiaisoleucine, and O-methyl-dl-threonine, both in the pyrophosphate exchange assay and in the assay of isoleucyl-tRNA formation. The affinity of the mutant enzyme for adenosine triphosphate in the assay of isoleucyl-tRNA formation is 15-fold lower than that of the wild-type enzyme. The affinity of mutant IRS for tRNA is not changed as compared with wild-type IRS. These data show that mutant IRS has an altered structure and clearly confirm that ileS is the structural gene for IRS.     

Identification of a temperature-sensitive asparaginyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

A temperature-sensitive mutant of Escherichia coli K-12 isolated previously (H. Ohsawa and B. Maruo, J. Bacteriol. 127:1157-1166, 1976) was found to have an alteration in asparaginyl-transfer ribonucleic acid synthetase. This alteration can account for the temperature-sensitive phenotype of the mutant. No evidence was obtained to support the previous suggestion that ribosomal protein S1 is altered in this mutant. Combined with the previous genetic studies, we conclude that the newly defined genetic locus, asnS, for the asparaginyl-transfer ribonucleic acid synthetase maps near pyrD at 21 min on the E. coli chromosome.     

Substrate specificity of a mutant alanyl-transfer ribonucleic acid synthetase of Escherichia coli

The correlation between the in vivo functioning and the in vitro behavior of the thermolabile alanyl-transfer ribonucleic acid (tRNA) synthetase (ARS) of Escherichia coli strain BM113 is presented. As a measure for the ARS activity inside the cell, the amount of acylated tRNA(ala) in vivo was determined. The rapid drop of the per cent tRNA(ala) charged which was observed upon shifting a culture of BM113 to the nonpermissive temperature indicates that in vivo acylation of tRNA(ala) might be the growth-limiting step at high temperature. Since neither growth nor the in vivo charging level of tRNA(ala) was affected by the addition of high l-alanine concentrations to the medium, one may infer that impaired functioning of the mutant enzyme at 40 C seems not to be due to reduced affinity of the enzyme for the amino acid. Separation of bulk tRNA of E. coli and of yeast on benzoylated diethylaminoethyl cellulose and charging of the fractions of the column by wild-type and mutant ARS reveal that only those tRNA species aminoacylated by the wild-type enzyme are also charged by the mutant ARS. Determination of the K(m) values of wild-type and mutant ARS for the three isoaccepting tRNA(ala) species of E. coli shows a ca. 10-fold increase of the apparent K(m) values of the mutant enzyme for all three species. Thus, the mutation proportionally reduces the apparent affinity for tRNA(ala) without causing any detectable recognition errors. Investigation of heat inactivation kinetics of wild-type and mutant ARS without and in the presence of substrates provides further evidence that only the transfer site of the ARS is altered by the mutation. Moreover, whereas both enzymes possess the same pH optimum of the relative maximal velocity, their pH dependence of the K(m) values for tRNA is different. The K(m) of the wild-type enzyme decreases at pH values below 7.0 and that of the mutant enzyme shows the inverse tendency; this again indicates an alteration of the tRNA binding site.     

Biochemical and genetic characterization of a carbamyl phosphate synthetase mutant of Escherichia coli K12.

An unusual Escherichia coli K12 mutant for carbamyl phosphate synthetase is described. The mutation was generated by bacteriophage MUI insertion and left a 5% residual activity of the enzyme using either ammonia or glutamine as donors. The mutation is recessive to the wild-type allele and maps at or near the pyrA gene, but the mutant requires only arginine and not uracil for growth. By a second block in the pyrB gene it was possible to shift the accumulated carbamyl phosphate to arginine biosynthesis. The Km values and the levels of ornithine activation and inhibition by UMP were normal in the mutant enzyme.     

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.     

Characterization of a temperature-sensitive Escherichia coli mutant and revertants with altered seryl-tRNA synthetase activity.

A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.     

Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli

The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.     

Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation.

A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Böck and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.