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W Steinberg

Publications and source records attributed to W Steinberg.

50 records · Page 3Linked to original sources
PubMed1974

Temperature-induced derepression of tryptophan biosynthesis in a tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis.

A tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (l-tryptophan: tRNA ligase adenosine monophosphate, EC 6.1.1.2) mutant (trpS1) of Bacillus subtilis is derepressed for enzymes of the tryptophan biosynthetic pathway at temperatures which reduce the growth rate but still allow exponential growth. Derepression of anthranilate synthase in a tryptophan-supplemented medium (50 mug/ml) is maximal at 36 C, and the differential rate of synthesis is 600- to 2,000-fold greater than that of the wild-type strain or trpS1 revertants. A study of the derepression pattern in the mutant and its revertants indicates that the 5-fluorotryptophan recognition site of the tryptophanyl-tRNA synthetase is an integral part of the repression mechanism. Evidence for a second locus, unlinked to the trpS1 locus, which functions in the repression of tryptophan biosynthetic enzymes is presented.

Amino Acyl-tRNA Synthetases↗
PubMed1974

Properties and developmental roles of the lysyl- and tryptophanyl-transfer ribonucleic acid synthetases of Bacillus subtilis: common genetic origin of the corresponding spore and vegetative enzymes.

The lysyl-transfer ribonucleic acid synthetase (LRS) and tryptophanyl-transfer ribonucleic acid synthetases (TRS) (l-lysine:tRNA ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) have been purified 60- and 100-fold, respectively, from vegetative cells and spores of Bacillus subtilis. There are no significant differences between the corresponding spore and vegetative enzymes with respect to their elution characteristics from columns of phosphocellulose or hydroxylapatite, their molecular weight (~130,000 for LRS and ~87,000 for TRS as determined by gel filtration), their kinetic constants for substrates (in the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reaction), and the kinetics of inactivation by heat and by antibody. The Mg(2+) requirement for optimal enzyme activity of the corresponding spore and vegetative enzyme differ slightly. Mutants having defective (temperature sensitive) vegetative LRS or TRS activities produce spores in which these enzymes are also defective. The mutant spores are more heat sensitive than the parental type, but contain normal levels of dipicolinic acid. They germinate normally at the restrictive temperature (43 C), but are blocked at specific developmental stages in outgrowth. No modification in temperature sensitivity phenotype occurs during outgrowth, nor is there a change in molecular weight of the two enzymes. The implication is that the LRS and TRS activities of the vegetative and spore stages are each coded (at least in part) by the same structural gene. The temperature sensitivity of mutant spores is discussed with respect to those factors which are involved in the formation of the heat-resistant state.

Amino Acyl-tRNA Synthetases↗
PubMed1974

Defects of two temperature-sensitive lysyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis.

Two temperature-sensitive mutants (lysS1 and lysS2) of the lysyl-transfer ribonucleic acid synthetase (l-lysine:tRNA ligase [adenosine 5'-monophosphate], EC 6.1.1.6) of Bacillus subtilis have been isolated. Although protein synthesis is inhibited in both mutants at the restrictive temperature (42 to 45 C), the mutants remain viable in a minimal medium. In comparison with the wild-type lysyl-tRNA synthetase, the l-lysine-dependent exchange of [(32)P]pyrophosphate with adenosine 5'-triphosphate (ATP) for both mutant enzymes is decreased. The lysS1 enzyme is completely defective in the ATP-dependent attachment of l-lysine to tRNA, whereas the lysS2 enzyme has 3- to 10-fold reduced levels of this activity. Temperature-resistant transformants have wild-type enzyme levels, whereas partial revertants to temperature resistance have varied levels of enzyme activity. The attachment and exchange activities of the lysS2 enzyme are more heat labile in vitro than the wild-type enzyme, as is the attachment activity of a partial revertant of the lysS1 mutant. The lysS1 and the lysS2 lysyl-tRNA synthetases have higher apparent K(m) values for lysine and ATP, in both the activation and the attachment reactions. The lysS2 enzyme has a V(max) for tRNA(lys) one-third that of the wild-type enzyme. Molecular weights of approximately 150,000 for the wild-type and lysS2 enzymes and approximately 76,000 for the lysS1 enzyme were estimated from sedimentation positions in sucrose density gradients assayed by the ATP-pyrophosphate exchange activity. We propose that the two mutations (lysS1 and lysS2) directly affect the sites for exchange activity, but indirectly alter attachment activity as a consequence of defective subunit association.

Adenosine Triphosphate↗
PubMed1974

Genetic location of two mutations affecting the lysyl-transfer ribonucleic acid synthetase of Bacillus subtilis.

Two mutations (lysS1 and lysS2), each independently resulting in a thermosensitive, lysyl-transfer RNA synthetase (l-lysine: tRNA ligase [adenosine 5'-monophosphate] EC 6.1.1.6), have been mapped on the Bacillus subtilis chromosome between purA16 (adenine requirement) and sul (sulfanilamide resistance). They are linked by transformation with sul (70 to 74% cotransfer) in the order purA16-lysS1-lysS2-sul. The mutant loci are either in the same gene or in two closely linked genes. They are not linked to the tryptophanyl-tRNA synthetase structural gene or to the lys-1 locus.

Adenine↗
PubMed1974

Thermal death of temperature-sensitive lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis: effect of culture medium and developmental stage.

The growth of thermosensitive Bacillus subtilis lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants (lysS1 and trypS1) (l-lysine:transfer ribonucleic acid [tRNA] ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) was terminated when exponential phase cells were shifted from 30 to 43 C in a rich medium. Under these conditions, the temperature-inhibited cells undergo thermal death; they rapidly lose their ability to form colonies at 30 C. Another lysyl-tRNA synthetase mutant (lysS2) is refractory to thermal death even though its growth is inhibited at 43 C. The thermal death response of the lysS1 mutant is affected by the stage of cell development. At periods in spore outgrowth and sporogenesis these cells become refractory to thermal death. The refractory state does not result from the production of an inhibitor, or from the degradation of an activator of thermal death. However, culture medium composition does modify the thermal death response. Rich media enhance the effect, and no thermal death occurs in the lysS1 strain grown in a minimal medium. Temperature-sensitive cells can grow in a lysine- (0.25 mM) or tryptophan- (0.25 mM) supplemented minimal medium at 43 C, but amino acid concentrations of 25 mM only transiently protect trypS1 and lysS1 cells from thermal death in a rich medium. Osmotic agents such as sucrose (0.5 M) and NaCl (0.34 M) completely prevent thermal death in the lysS1 strain, although growth is still arrested. On solid media, sucrose stabilized lysS1 cells can form colonies at the restrictive temperature, but neither sucrose (0.5 M) nor NaCl (0.34 M) stabilized the lysS1 enzyme in vitro. Chloramiphenicol increased the rate of thermal death of the lysS1 strain but decreased the thermal death response of the trypS1 mutant. Considering the nature of the enzyme defect in the lysS1 strain, the common genetic origin of the spore and vegetative lysyl-tRNA synthetase, and the protective effects exerted by lysine and osmotic agents, it is tentatively concluded that thermal death results from irreversible inactivation of the mutant gene product. According to this hypothesis, either the lysS1 enzyme is altered during sporogenesis or some physiological or structural aspect of this developmental phase can stabilize the mutant phenotype and thereby rescue cells from thermal death.

Alkaline Phosphatase↗
PubMed1974

Bacillus subtilis bacteriophage SP01, SP82, and phi e require host lysyl- and tryptophanyl-tRNA synthetases for phage development.

Infection of Bacillus subtilis mutants having temperature-sensitive lysyl- or tryptophanyl-tRNA synthetases with bacteriophage SP01, SP82, or phie indicates that both host enzymes are essential for phage development. No apparent modification of the temperature-sensitive phenotype of the mutant host enzymes occurs during phage infection.

Amino Acyl-tRNA Synthetases↗
PubMed1973

On the conservation of aminoacyl-transfer ribonucleic acid synthetase genes in Bacillus.

None of the heterologous deoxyribonucleic acid from various bacilli was capable of transforming lysyl- and tryptophanyl-transfer ribonucleic acid (tRNA) synthetase mutants of Bacillus subtilis to wild type. It was concluded that there is little conservation of the aminoacyl-tRNA synthetases even though the tRNA cistrons are conserved genetic functions.

Alleles↗
PubMed1971

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.

Adenosine Triphosphate↗
PubMed1968

Timing of enzyme synthesis during outgrowth of spores of Bacillus cereus. I. Ordered enzyme synthesis.

During outgrowth of spores of Bacillus cereus T, the pattern of enzymes synthesized varied with respect to time. The periods of synthesis of alpha-glucosidase, l-alanine dehydrogenase, and histidase were ordered; each began at a specific time and synthesis continued for only a brief period. An examination of the timing of induced alpha-glucosidase and induced histidase was made to determine whether specific regions of the genome were continually available for transcription and regulation during this period. Several observations indicated that inducers could function during outgrowth, but for only a limited time interval. The period of induced enzyme synthesis occurred over the same interval as that observed for uninduced and catabolically repressed cultures. When inducer was added partway through the period of gene expression, the level of enzyme induction was diminished. Addition of inducer at a time after the period of gene expression had no significant effect. Since messenger ribonucleic acid formed during outgrowth had a half-life of only a few minutes, it was concluded that ordered enzyme synthesis was a result of ordered transcription of the corresponding portion of the genome. An examination of the timing of induced alpha-glucosidase and histidase synthesized in the presence of actinomycin D supported this conclusion.

Amino Acid Oxidoreductases↗
PubMed1968

Timing of enzyme synthesis during outgrowth of spores of Bacillus cereus. II. Relationship between ordered enzyme synthesis and deoxyribonucleic acid replication.

Experiments were carried out to determine whether, during outgrowth of bacterial spores, deoxyribonucleic acid (DNA) replication provided the basis by which ordered transcription was controlled. During outgrowth, significant DNA synthesis does not occur until just prior to the onset of cell division. However, incorporation of radioactive DNA precursors into DNA is observed within 5 to 10 min after the initiation of germination. By employing a thymine-requiring auxotroph and (3)H-bromodeoxyuridine, this incorporation appears to be a result of DNA replication and not repair synthesis. For the following reasons it was concluded that, during outgrowth, transcriptional processes were not ordered by DNA replication. (i) In a thymine auxotroph, thymine addition did not alter the periodicity of induced alpha-glucosidase and histidase synthesis during outgrowth. (ii) DNA synthesis was inhibited 80% by 5-fluoro-2'-deoxyuridine (FUdR), and, after a 5-min lag, completely by mitomycin C, but these inhibitors exerted a differential effect on induced histidase synthesis. Enzyme synthesis was insensitive to FUdR but was inhibited by mitomycin C, presumably as a result of cross-linking of the complementary DNA strands.

Adenine↗
PubMed1968

Relationship between protein and ribonucleic acid synthesis during outgrowth of spores of Bacillus cereus.

The rate of protein and ribonucleic acid (RNA) synthesis was examined during the outgrowth of spores of Bacillus cereus T in a chemically defined medium. RNA synthesis started 2.5 min after the initiation of germination, and protein synthesis after 4 min. Addition of a complete amino acid supplement and uracil supported high rates of RNA and protein synthesis throughout outgrowth. To determine the relationship between the rate of protein (k) and RNA synthesis, the kinetics of formation of various classes of RNA were followed during outgrowth. Ribosomal RNA (rRNA) comprised a relatively constant fraction of the total RNA throughout outgrowth (71 to 78%). The classes of RNA synthesized during this period were determined by germinating spores in radioactive uracil and then at intervals following their stability to actinomycin D. Initially, labile RNA comprised the largest fraction of newly formed RNA (DeltaRNA), and this proportion decreased during outgrowth. The ratio of k/rRNA or k/Delta stable RNA varied considerably during outgrowth, whereas the ratio of k/labile RNA remained constant. The data suggest that the rate of protein synthesis is not rigidly coupled to either total or newly synthesized rRNA (ribosomes) during the early stages of outgrowth.

Amino Acids↗