Characterization of Bacillus subtilis mutants temperature-sensitive in the synthesis of ribonucleic acid

Two mutants of Bacillus subtilis temperature-sensitive in RNA synthesis were isolated. One mutation (rna-20) was demonstrated to be an allele of a previously identified gene (Riva et al., 1976). The other mutation (rna-16) identified a different gene and was mapped near aroI. The rna-16 mutation at the permissive temperature affected the spore outgrowth process. Purified RNA polymerase from rna-16 did not show any temperature sensitivity or structural defect.     

Stringent control of ribonucleic acid synthesis in Bacillus subtilis treated with granaticin.

The antibiotic granaticin interferes in Bacillus subtilis with the charging process of tRNALeu causing both the arrest of protein synthesis and bacteriostasis [A. Ogilvie, K. Wiebauer & W. Kersten (1975) Biochem. J. 152, 511-515]. A concomitant inhibition of RNA synthesis is observed. This inhibition was studied with mutant strains of B. subtilis. 2. Granaticin inhibits protein and RNA synthesis in stringently controlled B. subtilis (rel+) to about the same extent. In a relaxed mutant strain (rel-) of B. subtilis, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug. 3. Chloramphenicol, which is known to abolish the stringent control mechanism, added simultaneously with granaticin, allows the synthesis of RNA to proceed in the stringent strain. 4. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate in granaticin-treated stringently controlled B. subtilis but not in the rel- mutant. 5. It is concluded that the inhibition of RNA synthesis granaticin can adequately be explained as a stringent response caused by the interference by the drug with leucyl-tRNA synthetase.     

Mutant of Bacillus subtilis with a temperature-sensitive lesion in ribonucleic acid synthesis during germination.

We have isolated a mutant of Baccillus subtilis with a temperature-sensitive lesion in the process of spore germination. The temperature-sensitive mutation affects only germination and outgrowth, and the earliest defect observed is an early block of ribonucleic acid synthesis during germination at 46 C. Upon return to 35 C there is a complete repair of the impaired function, even in the absence of protein synthesis. Protein synthesis inhibition during germination of the mutant spores at 46 C has the effect of increasing the amount of ribonucleic acid made. The temperature-sensitive mutation is located near aroI.     

Possible inhibitory effect of teichoic acid on Bacillus subtilis transfer ribonucleic acid

1. tRNA of Bacillus subtilis was found to be variably contaminated with membrane teichoic acid. 2. Samples with high contents of teichoic acid showed no accepting activity for tRNA(Phe) and tRNA(Tyr). 3. Removal of teichoic acid restored accepting activity and fractions containing teichoic acid, separated on Sephadex G-150, inhibited the charging of tRNA(Tyr). 4. The presence of teichoic acid did not inhibit the charging of tRNA(His).     

Genetic analysis of ribonucleic acid polymerase mutants of Bacillus subtilis

Deoxyribonucleic acid-dependent ribonucleic acid polymerase mutants of Bacillus subtilis strain Marburg were isolated after mutagenesis of spores with ethyl methane sulfonate. Genetic analysis by PBS1-mediated transduction and by transformation indicated that mutations responsible for all of the four phenotypic classes studied (rifampin resistance, streptovaricin resistance, streptolydigin resistance, and temperature sensitivity) were clustered close to the cysA14 locus. Three-factor transformation analysis has indicated the most probable marker order as follows: Rif(R)(Stv)(R)-Std(R)-Ts(418)-Ts(427). In addition, further characterization of the classical group I reference marker, cysA14, is reported.     

Ribosomal ribonucleic acid and ribosomal precursor ribonucleic acid in Anacystis nidulans

The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.     

Organization of transfer and ribosomal ribonucleic acid genes in Bacillus subtilis.

The structural relationship between the transfer ribonucleic acid (tRNA) and the ribosomal RNA (rRNA) genes of Bacillus subtilis has been studied by restriction endonuclease analysis of total chromosomal deoxyribonucleic acid (DNA) and characterization of DNA fragments cloned in Escherichia coli. The DNA sequences encoding rRNA and tRNA were assayed by hybridization to radioactive RNA. The results support the conclusion that the tRNA genes are interspersed between and closely linked to the rRNA genes of B. subtilis. They probably do not appear between the 16S and 23S rRNA genes as in E. coli.     

New transfer ribonucleic acid species during sporulation of Bacillus subtilis.

The transfer ribonucleic acid (tRNA) populations from log-phase cells, sporulating cells (stage III), and dormant spores were compared by tRNA-deoxyribonucleic acid hybridization techniques. New tRNA species not found in log-phase cells were observed in stage III cells. Some of the tRNA made during sporulation were also present in dormant spores. Although the role and function of these new tRNA species cannot be ascribed directly to the sporulation process, their presence indicates that new tRNA genes can be transcribed during sporulation and suggests that translational control may be exerted during sporulation by tRNA.     

Properties of 5-fluorouracil-containing ribonucleic acid and ribosomes from Bacillus subtilis

Growth of a strain of Bacillus subtilis that requires uracil, thymine, adenine, and tryptophan in the presence of 5-fluorouracil (FU) results in the synthesis of ribonucleic acid (RNA) and ribosomes in which 55 to 65% of the RNA uracil has been replaced by the fluorine derivative. Examination of analogue-containing ribosomes by sucrose density gradient centrifugation and thermal denaturation studies suggests that, as far as the size, shape, and packing structure are concerned, extensive FU substitution has little or no effect. FU appears to replace uracil in RNA without selectivity for one RNA class over another, as determined by methylated albumin-kieselguhr column chromatography and sucrose density gradient centrifugation. The total amino acid content of the cells is markedly affected by growth in the presence of FU. The possibility of an FU effect on genetic translation is discussed.