Bacterial Sporulation and Regulation of Dihydrodipicolinate Synthase in Ribonucleic Acid Polymerase Mutants of Bacillus subtilis

The activity of dihydrodipicolinate synthase increased late in sporulation in Bacillus subtilis. Mutants blocked at several stages of sporulation due to having an altered ribonucleic acid polymerase failed to exhibit this increase.     

Inhibition of deoxyribonucleic acid replication in Bacillus brevis by ribonucleic acid polymerase inhibitors.

The incorporation of [3H]thymidine into deoxyribonucleic acid by exponentially growing cells of Bacillus brevis was inhibited by streptolydigin and rifampin in the same concentration range in which these drugs inhibit ribonucleic acid synthesis. Complete inhibition occurred within one-third generation time after drug addition, suggesting an effect on deoxyribonucleic acid chain elongation.     

Electron Microscopy of the Altered Spore Morphology of a Ribonucleic Acid Polymerase Mutant of Bacillus subtilis

Electron microscopy was used to analyze sporulating cells and spores of Bacillus subtilis mutants (Rif(r)) which are resistant to rifampin, an inhibitor of ribonucleic acid polymerase. The spores of Rif-18 are pleomorphic and frequently exhibit terminal knobs. These knobs first occur during late stage IV and early stage V of sporulation and are extensions of the inner and outer spore coats. Since the rifampin resistance and altered spore morphology of Rif-18 are 100% cotransformable, these data suggest that the altered spore morphology is the result of an alteration in ribonucleic acid polymerase genes. The morphology and physical dimensions are also reported for spores from Rif-11, Rif-15, and Rif-21. Significant differences in size from the wild type were observed for these mutants.     

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.     

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 Synthesis and Maturation in the Blue-Green Alga Anacystis nidulans

Methods are described for preparation of pulse-labeled ribonucleic acid (RNA) from the blue-green alga Anacystis nidulans. Synthesis of labeled RNA was found to be in part dependent on concurrent photosynthesis and was inhibited by the antibiotic streptolydigin. Mature 23S ribosomal RNA (rRNA) appeared before mature 16S rRNA. Formation of either molecule was inhibited by chloramphenicol, and RNA species of lesser mobility accumulated. These species may be precursors of the mature forms. Maturation of 16S rRNA was also inhibited by streptolydigin. (The effect of this antibiotic on 23S rRNA maturation was not examined). In many respects, ribosomal RNA synthesis and maturation in this blue-green alga appear to follow the pattern already established for bacteria.     

NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication.

The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrHl65/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.     

Degree of Completion of 3′-Terminus of Transfer Ribonucleic Acids of Bacillus subtilis 168 at Various Developmental Stages and Asporogenous Mutants

Transfer ribonucleic acids from sporulating cells, spores, sporangia, or stationary-phase asporogenous mutants of Bacillus subtilis all showed a deficiency in the 3′-terminal adenosine moiety.     

Template Specificity of Ribonucleic Acid Polymerase in Asporogenous Mutants of Bacillus subtilis

Asporogenous mutants of Bacillus subtilis were examined for the change in template specificity of ribonucleic acid (RNA) polymerase characteristic of wild-type cells undergoing sporulation. Mutants blocked at stages II, III, and IV showed a changed specificity of the enzyme after the end of growth and were in this respect indistinguishable from the wild type. The RNA polymerase of eight stage-zero mutants (out of nine tested) which possess mutations that map at six distinct loci retained the template specificity of vegetative cells.     

RNA polymerase is required for DNA initiation in vitro

Summary We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA plymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnoA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.     

Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation.

Inhibition of ribonucleic acid synthesis in Escherichia coli 15 TAU bar with rifampin or streptolydigin leads to large increases in the sizes of cellular ribonucleoside and deoxyribonucleoside triphosphate pools. Inhibition of protein synthesis leads to increases in the sizes of all nucleoside triphosphate pools except the guanosine triphosphate and deoxyguanosine triphosphate pools; a decrease in the size of the latter pool may be responsible for the slowing of deoxyribonucleic acid replication fork movement observed in this strain in the absence of protein synthesis. Analysis of the kinetics of incorporation of labeled precursors into deoxyribonucleic acid and into cellular pools suggests that functional compartmentation of nucleotide pools exists, allowing the incorporation of exogenously supplied precursors into deoxyribonucleic acid without prior equilibration with the cellular pools.     

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.     

Transcription After Bacteriophage SPP1 Infection in Bacillus subtilis

The role of the host polymerase in Bacillus subtilis infected with phage SPP1 was studied in vivo with regard to production of phage-specific and host-specific ribonucleic acid (RNA) and to phage yield. Evidence is presented that the subunit(s) of B. subtilis RNA polymerase which is sensitive to rifampin and streptolydigin is necessary at all times during infection for phage production. The synthesis of phage RNA and the phage yield in strains resistant to either antibiotic were unaffected by the drug. Host RNA synthesis continued throughout infection; phage-specific RNA never accounted for more than 20% of pulse-labeled RNA at any time during infection.     

Evidence for the involvement of ribonucleic acid in the production of F pili.

The effects of rifampin and streptolydigin, inhibitors of ribonucleic acid (RNA) synthesis, on the production of F pili by Escherichia coli were studied by electron microscopy. The inhibition of RNA synthesis reduces the number of new pili produced by depiliated cells, but does not affect their length or the number of pili present at the time of inhibition or the retraction of pili. We suggest that the rifampin-sensitive step may be linked to the establishement of a site for pili production. Evidence is provided that chloramphenicol inhibits retraction. We suggest that retraction requires some protein whose pool size is limited.     

Investigation of proteomic responses of Streptomyces lydicus to pitching ratios for improving streptolydigin production

Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.     

Metabolic analysis reveals the amino acid responses of Streptomyces lydicus to pitching ratios during improving streptolydigin production

Pitching ratio has been reported to impact not only on the primary metabolism, but also the secondary metabolism. Comparative metabolomics was used to explore the metabolic responses of Streptomyces lydicus E9 to pitching ratios (1, 10, and 30 %, v/v). We identified more than 120 metabolites involved in glycolysis, tricarboxylic acid cycle, and amino acid and secondary metabolism, of which there are significant differences in the quantified 32 metabolites under different pitching ratios by gas chromatography coupled to time-of-flight mass spectrometry. The intracellular levels of most amino acids (e.g., valine, alanine, and isoleucine) declined with the increases of pitching ratios. Especially, the relative abundances of glutamate and proline were not only decreased with the increases of pitching rations, but also had much low level at stages II and III, which might be related to the significant enhancement in streptolydigin of S. lydicus E9 under 30 % high pitching ratio. Moreover, principal component analysis revealed that eight metabolites, including glucopyranoside, maltose, cAMP, glycine, proline, lysine, isoleucine, and valine, were considered as potential biomarkers to distinguish the influences of pitching ratios on streptolydigin production. Further investigations demonstrated that the additions of exogenous glutamate and proline (100 mg?L^?1) enhanced significantly the accumulation of streptolydigin, indicating that glutamate was the synthetic precursor of streptolydigin, while proline in S. lydicus E9 was converted into glutamate and consequently improved streptolydigin biosynthesis. Therefore, these findings provide new insights into the amino acid responses of S. lydicus E9 to pitching ratios and provide potential strategies to improve streptolydigin production.     

Dependence of Diaminopurine Utilization on the Mutational Site of Purine Auxotrophy in Bacillus subtilis II. Tracer Experiments

Tracer experiments were carried out in an attempt to explain why guanineless auxotrophs can use diaminopurine as a guanine replacement but nonexacting purine auxotrophs cannot do so. Cell suspensions of the nonexacting purineless Bacillus subtilis MB-1356 incorporated more radioactivity from diaminopurine-2-¹⁴C into nucleic acid than did guanineless B. subtilis MB-1517. The radioactivity in MB-1356 ribonucleic acid (RNA) was distributed in both adenine and guanine nucleotides, thus eliminating the possibility that the deamination of diaminopurine to guanine occurred predominantly on the level of nucleoside di- or triphosphates. Strain MB-1517 incorporated adenine-8-¹⁴C into nucleic acids extremely poorly. This correlated with results obtained with cell-free extracts; strain MB-1517 showed much less adenosine monophosphate (AMP) pyrophosphorylase activity than did MB-1356. Likewise, guanineless MB-1517 converted diaminopurine to its nucleotide much more slowly than did the nonexacting purine auxotroph. The results indicated that the lack of growth of nonexacting auxotrophs on diaminopurine alone is due not to an inability to convert the analogue to nucleic acid adenine but to the greater capacity of the nonexacting auxotrophs to convert diaminopurine to its 5′-ribonucleotide. Presumably, this compound, or a coenzyme analogue produced from it, inhibits growth of mutants which cannot make AMP de novo and only when the medium is devoid of adenine.