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R H Doi

Publications and source records attributed to R H Doi.

At least 127 records · Page 7Linked to original sources
PubMed1978

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

Bacillus subtilis↗
PubMed1977

Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase.

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepharose column which bound the beta' subunit. A phosphocellulose column was used to separate the alpha and beta subunits. During purification, the enzyme eluted from the DNA-cellulose column in three separate forms in the order alpha2betabeta'deltaomega1,alpha2betabeta'omega1, and alpha2betabeta'omega1sigma. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as beta. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase.

Bacillus subtilis↗
PubMed1977

Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis.

The Bacillus subtilis DNA-dependent RNA polymerase holoenzyme and core enzyme each contain approximately two atoms of zinc per molecule. When the dissociated subunits of the enzyme are passed through a blue dextran-Sepharose affinity column, only the beta subunit binds to the column. The total zinc content of the enzyme is tightly bound to the beta subunit. Dialysis studies suggest that the two zinc ions differ in the strength of their association with the beta subunit. The presence of zinc in beta is consistent with several other lines of evidence which indicate that this subunit is dirrectly involved in phosphodiester bond formation. The blue dextran-Sepharose column procedure should be useful in future studies of the dissociation and reassociation of the enzyme since the method is rapid and provides excellent recovery of the beta subunit as well as the alpha and beta' subunits of the RNA polymerase.

Bacillus subtilis↗
PubMed1977

Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.

The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.

Bacillus subtilis↗
PubMed1977

Bacillus subtilis ribonucleic acid polymerase mutants conditionally temperature sensitive at various stages of sporulation.

Rifampin-resistant mutants of Bacillus subtilis that are conditionally temperature sensitive during sporulation have been isolated and characterized. The mutants can grow at the same rate as the wild type at the nonpermissive temperature but cannot sporulate. Depending on the mutation, they are blocked at either stage 0 to I, II, II to III, or IV of sporulation. The mutants showed an altered pattern of RNA synthesis after the stage at which they were blocked. The effect of rifampin on the activity of enzymes from mutant vegetative cells and sporulating cells was significantly different, suggesting that the RNA polymerase from sporulating cells was different from the RNA polymerase of vegetative cells. These results suggest that the conformation of the RNA polymerase core plays an important role in determining correct transcription during sporulation.

Bacillus subtilis↗
PubMed1976

Effect of netropsin on the derepression of enzymes during growth and sporulation of Bacillus subtilis.

Netropsin, a polypeptide antibiotic which binds specifically to adenylate-thymidylate-rich regions of deoxyribonucleic acid, inhibitis sporulation at about stage II, but does not inhibit growth of Bacillys subtilis. An analysis of the sporulation-associated enzymes aconitase, alkaline phosphatase, and glucose dehydrogenase revealed that their rates of expression were not affected by the presence of the antibiotic. The derepression of histidase, a vegetatively induced enzyme was stimulated by netropsin. Oxygen utilization by the cells during sporulation was not effected nor was spore germination prevented by the drug. Netropsin, however, did prevent the formation of dipicolinic acid. These and earlier results suggest that netropsin may be affecting the transcription of only select sporulation genes that are particularly rich in adenylate-thymidylate base pairs.

Aconitate Hydratase↗
PubMed1975

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.

Bacillus↗
PubMed1974

Transcription from the complementary deoxyribonucleic acid strands of Bacillus subtilis during various stages of sporulation.

The messenger ribonucleic acid (mRNA) pattern of log-phase and sporulating cells of Bacillus subtilis has been analyzed by deoxyribonucleic acid (DNA)-RNA hybrid studies with the complementary-strand fragments of DNA. Approximately 80% of the mRNA of log-phase and sporulating cells from stages I, III, and IV hybridizes with the heavy DNA fragments, and 20% hybridizes with the light DNA fragments. Hybrid competition studies indicated that there was either a greatly reduced rate of transcription or a turn-off of some log-phase genes during the sporulation stages. However, a significant amount of log-phase gene transcription occurred even at late stages of sporulation. Similar studies indicate a significantly increased rate of transcription or a turn-on of sporulation phase genes during the latter stages of sproulation. There is a sequential increase in the amount of sporulation-specific transcription from both complementary-strand fragments of DNA. These results indicate that the RNA polymerase population in sporulating cells can transcribe both log-phase and sporulation-phase genes.

Bacillus subtilis↗
PubMed1974

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.

Bacillus subtilis↗
PubMed1974

Ultrastructural analysis during germination and outgrowth of Bacillus subtilis spores.

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.

Bacillus subtilis↗
PubMed1973

Ultrastructural studies of sporulation in a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis.

Morphological studies of a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis have revealed that sporulation is inhibited at stage II when the cells are grown at 47.5 C. Growth and sporulation occur normally at 30 C with the mutant. The mutant grows normally at 47.5 C but is prevented from sporulating at the nonpermissive temperature by an abnormal septation during forespore membrane formation which prevents the subsequent engulfment process (stage III). The mutation affects the normal functioning of ribonucleic acid polymerase at the nonpermissive temperature resulting in abortive sporulation.

Bacillus subtilis↗
PubMed1973

Crystal formation by a ribonucleic acid polymerase mutant of Bacillus subtilis.

A crystalline inclusion has been observed in a ribonucleic acid polymerase mutant of Bacillus subtilis which is conditionally temperature sensitive only during sporulation. The crystal is formed at the permissive temperature in 1 to 2% of the sporulating (stage III-IV) cells; about 85% of the cells sporulate normally, while the cells with crystals do not sporulate. The wild type does not form crystals at either the permissive (30 C) or the nonpermissive (47 C) temperatures. The crystal may result from altered transcription during sporulation at 30 C.

Bacillus subtilis↗