In Vivo Aminoacylation of Transfer Ribonucleic Acid in Bacillus subtilis and Evidence for Differential Utilization of Lysine-Isoaccepting Transfer Ribonucleic Acid Species

The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.     

Messenger Ribonucleic Acid of Dormant Spores of Bacillus subtilis

Evidence of the presence of messenger ribonucleic acid (mRNA) in dormant spores of Bacillus subtilis has been obtained. The bulk RNA from spores was isolated and labeled in vitro with tritiated dimethyl sulfate. The spore RNA hybridized to 2.4 to 3.2% of the B. subtilis genome. The RNA hybridized to both the complementary heavy and light fractions of deoxyribonucleic acid (DNA). Bulk RNA from log-phase cells competed with virtually all the spore RNA for the heavy DNA fraction and with part of the spore RNA for the light DNA fraction. Bulk RNA from stage IV cells in sporulation also competed with all of the spore RNA for the heavy DNA fraction and with essentially all the spore RNA for the light DNA fraction. These results indicate that dormant spores contain mRNA species present in both log-phase cells and stage IV cells of sporulation. The RNA polymerase in the developing forespore must be able to recognize promotor sites for both log-phase and sporulation genes.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Chromatographic Differences Between Transfer Ribonucleic Acids from Spores and Cells in Exponential Growth

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Changes in Chromatography of Transfer Ribonucleic Acids Associated with Stage of Development

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.     

Synthesis of Ribosomal Ribonucleic Acid During Sporulation of Bacillus subtilis

The incorporation of radioactive uracil into 50s and 30s ribosomal subunits and ribosomal ribonucleic acid (rRNA) was studied during the growth cycle of different sporogenic and asporogenic strains of Bacillus subtilis. It was found that partially synchronized cultures of the strains examined incorporated labeled uracil into the two ribosomal subunit species and rRNA during sporulation and during the stationary phase of the asporogenic strains. Kinetic studies have shown that, compared to vegetative cells, the percentage of uracil incorporated into the ribosomal subunits of cells taken 30 min after the end of exponential growth was decreased by about 25 to 35%. This decrease, however, appeared to be a general characteristic of stationary-phase cells and seems to depend on the nature of the sporulation medium and to some extent on the nature of the strain but not on the sp(+) or sp(-) phenotype of the strain. Moreover, by use of actinomycin D it was shown that the labeled uracil incorporated, in the presence of the drug, during the sporulation period was located in the ribosomal subunits (stable RNA). Based on these results, we concluded that during sporulation ribosomal genes are transcribed and consequently rRNA continues to be synthesized, although to a lesser extent than during vegetative growth. These results are discussed in the light of those obtained by Hussey et al.     

Transfer Ribonucleic Acid Nucleotidyltransferase and Transfer Ribonucleic Acid in Sendai Virions

Sendai virions contain both transfer ribonucleic acid (tRNA) nucleotidyltransferase and its substrate, tRNA missing its CCA-OH end.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Ribonucleic Acid and Protein Synthesis in a Mutant of Bacillus subtilis Defective in Potassium Retention

A mutant of Bacillus subtilis 168 (strain 168 KL), which had lost its normal capacity to accumulate K(+), was used to explore the interrelationship between protein and ribonucleic acid (RNA) synthesis. In contrast to the wild type, the growth rate of strain 168 KL was markedly dependent on the K(+) concentration in the medium. K(+) uptake in the mutant strain was identical to that in the parent, but the mutant was unable to retain and accumulate K(+). Protein synthesis was markedly dependent on the K(+) concentration in the medium, whereas RNA synthesis was relatively unaffected by changes in the level of K(+). Most of the RNA synthesized during K(+) depletion was ribosomal RNA; it appeared in crude extracts in the form of ribonucleoproteins particles with sedimentation values between 4S and 30S. These particles were converted into mature ribosomes when growth was allowed to resume by the addition of K(+). Simultaneous synthesis of RNA and protein was necessary for the quantitative conversion of the ribonucleoprotein particles into ribosomes. During recovery from K(+) depletion, ribosomal protein was synthesized in preference to the other proteins of the cell.     

Ribonucleic Acid Polymerase in a Thermosensitive Sporulation Mutant (ts-4) of Bacillus subtilis

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try⁻try⁻ (168tt) parental strain were infected with the virulent phage e at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage e deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the β subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.     

Methyl-Deficient Transfer Ribonucleic Acid in Saccharomyces cerevisiae

Methyl-deficient transfer ribonucleic acid (tRNA) is found in certain methionine auxotrophs of Saccharomyces cerevisiae during logarithmic growth (at one generation time before the late growth phase) and during residual growth in the absence of exogenous methionine. The former effect seems to be accounted for by the general increase in RNA synthesis that occurs at the time; there is no specific synthesis of tRNA in the absence of ribosomal RNA synthesis, nor is the methyl group deficiency limited to a single tRNA species. During methionine starvation, all species of tRNA are methyl-deficient, but this occurs only in strains with certain blocks in the methionine pathway. The kinetics of disappearance of the methyl group donor, S-adenosylmethionine, during starvation of D73 (which accumulates methyl-deficient tRNA), do not differ from other strains, but D73 loses the methylase inhibitor, S-adenosylhomocysteine, much more slowly.     

Natural Messenger Ribonucleic Acid-Directed Cell-Free Protein-Synthesizing System of Bacillus subtilis

A very active in vitro protein-synthesizing system has been developed from Bacillus subtilis. High activity in the extracts is dependent upon precautions taken to reduce proteolytic activity. Endogenous, exogenous natural and synthetic messenger ribonucleic acids (RNAs) are translated by the system. The activity of the B. subtilis system has been compared to that of the Escherichia coli system. With either SPO1 RNA or polyuridylic acid, the activities of the two systems were very similar. Electrophoresis of the products synthesized in vitro by the two systems primed with SPO1 RNA yields similar radioactive profiles. The major bands of radioactivity correspond to proteins of molecular weight between 15,000 and 40,000.     

Effects of Leucine Starvation on Control of Ribonucleic Acid Synthesis in Strains of Bacillus subtilis Differing in Deoxyribonucleic Acid Regulation

When starved for leucine, strains of Bacillus subtilis do not complete chromosome replication to the terminus. The amount of deoxyribonucleic acid (DNA) made poststarvation is characteristic of the strain. In this study, four strains differing in their DNA response were examined for ribonucleic acid (RNA) regulation during leucine starvation. Each of the strains was judged to be stringent for RNA control based on the amount of RNA made poststarvation. Sucrose gradient profiles on RNA made with and without leucine starvation support this conclusion. Accumulation of guanosine tetraphosphate during leucine starvation showed no correlation with the amount of DNA synthesized. We concluded that modulation control of DNA synthesis during leucine starvation is independent of RNA control.     

Cytokinins and Transfer Ribonucleic Acids. I. Presence of Cytokinins in Various Amino-acid Specific Transfer Ribonucleic Acids of Baker's Yeast

Soluble ribonucleic acid of baker's yeast was fractionated by countercurrent distribution and assayed for both cytokinin activity, using tobacco pith callus tissue, and for certain specific amino-acid acceptor activities. Two groups of fractions showed cytokinin activity, one of which corresponds to serine and tyrosine transfer ribonucleic acids which are known to contain isopentenyl adenine, while the other corresponds lo undetermined species of ribonucleic acids.     

Synthesis of Bacterial Flagella I. Requirement for Protein and Ribonucleic Acid Synthesis During Flagellar Regeneration in Bacillus subtilis

A relatively simple immunochemical procedure for estimating flagellar protein was developed. This procedure involved measuring the binding of purified, radioactively labeled, antiflagellar antibodies to bacteria. The assay was used to determine the requirements for ribonucleic acid (RNA) and protein synthesis during flagellar regeneration in Bacillus subtilis. Immediate inhibition of flagella development was observed when chloramphenical or puromycin was added to cells. This inhibition indicated the absence of a large pool of flagella precursors that could be assembled in the absence of protein synthesis. When the cells were starved for uracil or treated with actinomycin D to inhibit RNA synthesis, the ability of the cells to regenerate flagella decayed with a half-life of 5.5 min. When B. subtilis auxotrophs were starved for tryptophan, they continued to synthesize flagella, although this process was also inhibited by actinomycin D. On the basis of these results, we concluded that (i) the system involved in flagellar regeneration does not have unusual metabolic stability, (ii) regeneration requires both concomitant protein and RNA syntheses, and (iii) B. subtilis continues to synthesize messenger RNA during tryptophan starvation.     

Characterization of Altered Forms of Glycyl Transfer Ribonucleic Acid Synthetase and the Effects of Such Alterations on Aminoacyl Transfer Ribonucleic Acid Synthesis In Vivo

The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.     

Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.     

Itoic Acid Synthesis in Bacillus subtilis

Under conditions of iron deficiency, strains of Bacillus subtilis produced 2,3-dihydroxybenzoic acid (DHB), 2,3-dihydroxybenzolyglycine (DHBG), or both of these compounds. DHB(G) production [production of DHB(G) refers to the production of DHB, or DHBG, or both] was proportional to the amount of iron present and occurred logarithmically, paralleling growth. Supplementation of media with more than 150 mug of iron per liter at zero-time inhibited DHB accumulation completely. In the presence of DHB, lower levels of iron inhibited DHB(G) production, so that the actual inhibitor of synthesis may involve the Fe(3+):[DHB(G)](3) complex. The strains producing DHBG also produced coproporphyrin III during iron-deficient growth, whereas a strain producing DHB did not produce coproporphyrin III under these conditions. Accumulation of DHB(G) was influenced by the levels of aromatic amino acids and anthranilic acid in the medium. In vivo experiments with strain B-1471 demonstrated that DHB was coupled to added glycine to form DHBG. Metabolism of DHB(G) was observed in two of the strains studied.     

Hyaluronic Acid Production in Bacillus subtilis

The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.     

Polyribosomes from Bacillus subtilis During Amino Acid Starvation in the Presence and in the Absence of Actinomycin

Polysomes were extracted from Bacillus subtilis cells starved for a required amino acid. The monosome peak appeared soon after starvation; no difference in the rate of degradation was detected when the cells were starved for arginine or tryptophan in a double auxotroph. RNA production during starvation was not inhibited by actinomycin, but the molecular weight of the product made in the presence of the antibiotic was much lower. Indications that stable messenger ribonucleic acid is present for up to 90 min after amino acid starvation are also presented.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.