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.     

Percent Charging of Transfer Ribonucleic Acid and Levels of ppGpp and pppGpp in Dormant and Germinated Spores of Bacillus megaterium

The levels of transfer ribonucleic acids (tRNAs) specific for 14 amino acids were almost identical in dormant spores and in spores germinated from 6 to 75 min. Germinated spore tRNAs specific for all amino acids tested were between 63 and 93% charged, and there was no significant change in this value from 6 to 75 min of germination. In contrast, tRNAs isolated from dormant spores specific for nine different amino acids were almost completely(>93%) uncharged. However, some dormant spore tRNAs, i.e., those for arginine, histidine, isoleucine, and valine, showed significant (21 to 72%) levels of aminoacylation. Dormant spores contained no detectable guanosine penta- (pppGpp), tetra- (ppGpp), or triphosphate (GTP). However, these nucleotides appeared in the first minutes of germination, and thereafter all increased in parallel with a ratio of pppGpp plus ppGpp to GTP of 0.07 to 0.11, which is characteristic of unstarved vegetative cells.     

Dipicolinic Acid Location in Intact Spores of Bacillus megaterium

Beta-attenuation analysis of intact spores of Bacillus megaterium containing tritium-labeled dipicolinic acid has shown that dipicolinic acid is located in the spore protoplast and not in the cortex.     

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.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Absence of Adenine-Rich Ribonucleic Acid from Purified Infectious Reovirus 3

The basic properties of released and cell-associated reovirus are the same. Both contained as their total nucleic acid complement only double-stranded ribonucleic acid (RNA) with an adenine content of 28%. Preparations of purified cell-associated virus, but not released virus, contained adenine-rich RNA which could be separated from the virus with little or no loss of infectivity. These adenine-rich ribonucleic acids were present in the virus preparations either as free RNA or associated with some structures of molecular weight less than 25 x 10(6) daltons. In contrast to our previous report, double-stranded reovirus RNA possessed little or no template activity for the Escherichia coli deoxyribonucleic acid and RNA polymerases.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

The Structure and Function of the Spore Outer Membrane in Dormant and Germinating Spores of Bacillus megaterium

Attempts to demonstrate the presence of the spore outer membrane in mature, dormant spores of a strain of Bacillus megaterium are described. The outer, integument, layers of this organism were found to contain one-third of the total spore cytochrome content, several enzymes of the electron transport chain (specifically NADH oxidase, dehydrogenase, cytochrome c reductase and NADPH dehydrogenase) and a large number of polypeptides extractable with sodium dodecylsulphate in the presence of dithiothreitol and protease inhibitors. These all suggest the presence of a membraneous element. Electron microscopic evidence is presented on the structure of the dormant integument enzymes. Changes in the integument enzymes and in the gel electrophoresis profile of the extractable integument polypeptides which occur during spore gemination, are described and compared with those that take place in the spore inner membrane. The heat sensitivity of the integument enzymes is compared with that of the inner membrane enzymes and the implications for theories of spore heat resistance discussed.     

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.     

Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.     

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.     

Protein Synthesis During Fungal Spore Germination IV. Transfer Ribonucleic Acid from Germinated and Ungerminated Spores

Transfer ribonucleic acid (tRNA) fractions isolated from germinated and ungerminated spores of Botryodiplodia theobromae and Rhizopus stolonifer had acceptor activity for all 20 amino acids commonly found in protein, when tested with an enzyme fraction from germinated spores. Accordingly, it is unlikely that the absence of tRNA for a particular amino acid limits protein synthesis in fungal spores.     

Gene Dosage for 5S Ribosomal Ribonucleic Acid in Escherichia coli and Bacillus megaterium

The number of gene copies for 5S ribosomal ribonucleic acid (rRNA), relative to that for 16 and 23S rRNA, has been determined by deoxyribonucleic acid (DNA)-RNA hybridization for Escherichia coli and Bacillus megaterium. In both cases, the number of 5S rRNA genes equals the number of 16 or 23S rRNA genes. Rapid procedures for preparing extremely highly purified DNA suitable for DNA-RNA hybridization experiments and chemically pure 5S rRNA are described.     

Inhibition of Ribonuclease II of Escherichia coli by Sodium Ions, Adenosine-5′-Triphosphate, and Transfer Ribonucleic Acid

Ribonuclease II action on polyuridylate is competitively inhibited by transfer ribonucleic acid and noncompetitively inhibited by sodium ions. At low substrate levels, adenosine-5'-triphosphate is also inhibitory.     

Presence and Function of Sulfur-containing Transfer Ribonucleic Acid of Bacillus subtilis

Bacillus subtilis transfer ribonucleic acid (tRNA) was analyzed for the occurrence of thionucleotides by in vivo labeling with (35)S and fractionation by methylated albumin kieselguhr column chromatography. Alkaline hydrolysates of tRNA were also examined by column chromatography and paper electrophoresis, and the amino acid-accepting ability of thionucleotide-containing tRNA was tested after iodine oxidation. The results showed that B. subtilis tRNA contains 4-thiouridylate, a second nucleotide with properties similar to 2-thiopyrimidine, and a third unidentified thionucleotide. The amino acid-accepting ability for serine, tyrosine, lysine, and glutamic acid was markedly inhibited after oxidation of the tRNA with iodine, suggesting the presence of thionucleotides in these tRNA species. This inhibition could be reversed by thiosulfate reduction. The iodine treatment totally inactivated all lysine tRNA species, partially inactivated the serine tRNA species, and did not affect the accepting ability for valine. A comparison of tRNA from cells in the log and stationary phases and from spores revealed similar iodine inactivation patterns in all cases. The thionucleotide content in B. subtilis tRNA differed from that in Escherichia coli, both in extent and in distribution. A possible function of the thionucleotides in tRNA is discussed.     

Activation Energy for Glucose-Induced Germination of Bacillus megaterium Spores

The maximum germination rate of Bacillus megaterium QM B1551 spores in glucose increased, and the lag before its attainment decreased, with increasing germination temperature. The activation energy for germination (μ = approximately 20 kcal/mole), based on rate or on lag, was consistent with an enzymatic mechanism.     

Location of Elements in Ashed Spores of Bacillus megaterium

The structure of the skeleton of spores of Bacillus megaterium was examined after ashing in a plasma asher and the elemental composition of the ashed whole spores was determined with an analytical electron microscope. All spores were ashed in situ although they shrank by about 15%. Even P and S, in addition to metals, were recovered well from ashed samples. Ash was rich in the core and the coat, and poor in the cortex. Ca, P, S, and Mg were detected in the core and coat of the spore of B. megaterium QM B1551. Ca in the core was markedly decreased by germination or autoclaving. In the spore of B. megaterium ATCC 19213, almost all of the ash was detected in the core and its elemental composition was similar to that of the core of the strain QM B1551 spore. These results suggest strongly that the core is the site of Ca associated with dipicolinic acid.     

Glutamic Acid Decarboxylase in Spores of Bacillus megaterium and Its Possible Involvement in Spore Germination

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase (GAD). Although dormant spores showed no GAD activity, spores given sonic treatment and heat-activated spores had high activities when assayed for this enzyme. Several parameters of GAD in heat-activated spores were examined. The effects of KCN, NaN(3), 2,4-dinitrophenol, and KF on GAD activity were examined. Only KCN was an effective inhibitor of GAD activity in heated spores and was also shown to be the only effective inhibitor of GAD activity in vegetative bacteria. Similar patterns of inhibition were obtained with GAD activity and with spore germination, KCN being the only effective inhibitor of both, although at different concentrations. Spore GAD activity in heat-activated spores showed a loss with storage at 4 C; on the other hand, storage at 25 C was not accompanied by a loss, but, to the contrary, showed an increase in GAD activity of about 30%. A comparison of GAD activity at different times during germination, growth, and sporulation showed it to be highest in freshly germinated spores. Although vegetative cells contained GAD activity, the level in log-phase cells was approximately one-half the level obtained with freshly germinated spores. Heat-activated mutant spores with a requirement of gamma-aminobutyric acid for germination gave no GAD activity. GAD activity appeared in mutant spores after germination and increased to levels comparable to parent spores after 9 min of germination.     

Termination of Deoxyribonucleic Acid in Escherichia coli by 2′,3′-Dideoxyadenosine

2′,3′-Dideoxyadenosine was previously shown to be lethal to Escherichia coli and to inhibit deoxyribonucleic acid (DNA) synthesis irreversibly in this organism. It was also shown that triphosphate of this analogue terminates DNA chains in an in vitro system. Data presented here show that the nucleoside is relatively insensitive to E. coli adenosine deaminase and is converted intracellularly into the dideoxynucleotide, including the triphosphate. Thymine nucleotide pools were not reduced in inhibited bacteria, nor did preformed DNA break down. Some adenine was liberated from the dideoxyadenosine on incubation, and the latter was incorporated into ribonucleic acid. Nevertheless, about 4,000 molecules of the dideoxynucleoside were incorporated into DNA per cell. The dideoxynucleotide occurred in DNA chains in a terminal position, liberated selectively by venom phosphodiesterase. The possible nature of the lethal event is discussed.