Effect of nalidixic acid on the activation of RNA synthesis in outgrowing spores of Bacillus subtilis

Outgrowth of B. subtilis spores depends on the action of DNA gyrase (comp. Matsuda and Kameyama 1980). Application of nalidixic acid (100 micrograms/ml) to dormant spores of Bacillus subtilis prevents the outgrowth. Application of nalidixic acid (100 micrograms/ml) during the early outgrowth phase (after a 20 min germination period) does not prevent, but only delay spore outgrowth. Germination of spores is not influenced. Nalidixic acid is an effective inhibitor of RNA synthesis in outgrowing spores, whereas vegetative cells are more resistant. Spores can grow out inspite of a remarkably reduced intensity of RNA synthesis. Nalidixic acid particularly inhibits the synthesis of stable RNA, probably that of ribosomal RNA. We suggest that DNA gyrase-catalyzed alterations in DNA structure are involved in the regulation of the gene expressional program of outgrowing B. subtilis spores.     

Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis.

Nucleic acid synthesis was studied during germination and outgrowth of normal spores of Bacillus subtilis, as well as of spores carrying the genome of phage phie. In a system in which development was restricted to the spore-darkening phase, synthesis of ribonucleic acid (RNA), but not deoxyribonucleic acid (DNA), was detected. The extent of RNA synthesis and turnover, during this phase was similar for the two types of spores. In a partially darkened population of spores of either type, there was little RNA degradation, whereas there was considerable turnover in a fully darkened population. The DNA-dependent RNA polymerase of dormant or dark spores was not active in vitro with phi DNA as template, although a sigma-like factor could be separated from the polymerizing activity by zone centrifugation. Within 40 min after resuspension of dark spores in a medium that allows outgrowth, the enzyme acquired the ability to transcribe the phage DNA efficiently. During outgrowth, both normal and carrier spores synthesized DNA, but in carrier spores this DNA was almost entirely phage specific. The pattern of RNA accumulation in normal spores was in two distinct phase (0 to 60 min and 90 to 180 min). The second phase was absent in outgrowing carrier spores. The burst of phage in carrier spores occurred at 160 to 180 min.     

Mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucoside by germinating and outgrowing spores of Bacillus species

AIMS: To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species. METHODS AND RESULTS: Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold during spore outgrowth in the presence of beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. CONCLUSIONS: Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus. The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.     

Independence of Bacillus subtilis spore outgrowth from DNA synthesis.

The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidoxic acid and 6-(p-hydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhbition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors.     

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.     

Synthesis of poly(A)-containing RNA by isolated spinach chloroplasts.

Chloroplasts were isolated from spinach leaves and the intact chloroplasts separated by centrifugation on gradients of silica sol. Chloroplasts prepared in this way were almost completely free of cytoplasmic rRNA. The purified chloroplasts were incubated with 32PO4 in the light. The nucleic acids were then extracted and the RNA was fractionated into poly(A)-lacking RNA and poly(A)-containing RNA (poly(A)-RNA) via oligo(dT)-cellulose chromatography. The poly(A)-RNA had a mean size of approximately 18--20 S as determined by polyacrylamide gel electrophoresis. The poly(A)-RNA was digested with RNase A and RNase T1, and the resulting poly(A) segments were subjected to electrophoresis on a 10% w/v polyacrylamide gel 98% v/v formamide). Radioactivity was incorporated into both poly(A)-RNA and poly(A)-lacking RNA and into the poly(A) segments themselves. The poly(A) segments were between 10 and 45 residues long and alkaline hydrolysis of poly(A) segments followed by descending paper chromatography showed that they were composed primarily of adenine residues. There was no 32PO4 incorporation into acid-insoluble material in the dark. We conclude that isolated chloroplasts are capable of synthesizing poly(A)-RNA.     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.     

Bacillus subtilis 168 genetic transformation mediated by outgrowing spores: necessity for cell contact.

Transforming activity released in sequential genetic order during the first synchronous cycle of DNA replication during outgrowth of spores of Bacillus subtilis 168 was investigated. A transformation assay was used consisting of outgrowing spores as DNA donors and multiply marked competent cells as recipients. DNA synthesis inhibitors known to stop DNA release were used during and subsequent to DNA transfer to recipient cells. The released DNA sedimented with the outgrowing cells after low-speed centrifugation, and it was discovered that markers released both early and late were resistant to up to 500 microgram of deoxyribonuclease per ml under conditions in which the transforming capacity of purified DNA was eliminated by 5 microgram of the nuclease per ml. Inaccessibility to deoxyribonuclease was increased and maintained during the transformation event while detergents and proteolytic attack did not expose the released chromosome to nuclease action. The results indicate that tight physical contact between outgrowing spores and competent cells is required for transformation in this system.     

Isolation of genes preferentially expressed during Bacillis subtilis spore outgrowth.

From the Charon 4A library of Ferrari et al (J. Bacteriol. 146:430-432, 1981) we isolated three genes involved in Bacillus subtilis spore outgrowth by screening the library by hybridization with labeled RNA from outgrowing spores in the presence of an excess of unlabeled vegetative RNA. Hybridization competition experiments with purified clones showed that the clones contained sequences that were transcribed only during spore outgrowth or preferentially during spore outgrowth. Fragments of the cloned DNAs were subcloned in plasmid pJH101, and by using plasmid integration and PBS1 transduction the chromosomal loci were mapped. The three loci which we mapped are outG and outH, which are located between polC and dnaA, and outI, which is located near pycA. Using the cloned DNAs and derived plasmids in dot hybridization experiments with RNA extracted from cells at different developmental stages, we defined for two clones a region that is transcribed only during the outgrowth phase.     

Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells.

Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.     

Degradation of maternal poly(A)-containing RNA during early embryogenesis of an insect (Smittia spec., chironomidae,diptera)

Summary Eggs of the chironomid midgeSmittia spec. were shown to contain maternal rRNA, tRNA and poly(A)-containing RNA. The ribonucleoprotein spectrum consisted of monosomes, ribosomal subunits, and subribosomal particles, whereas polysomes could be detected only in small amounts. Poly(A)-containing RNA was found in different regions of the RNP spectrum, mainly between 15 S and 60 S. After labelling maternal RNA by feeding tritiated uridine to the larvae, the radioactivity associated with poly(A)-containing RNA accounted for about 4% of the label in the total RNA extracted from newly deposited eggs. About half of the radioactivity in the poly(A)-containing RNA was lost between egg deposition and an advanced blastoderm stage. The loss was accompanied by both a decrease in the size of the poly(A)-containing RNA molecules and a shift of poly(A)-containing RNP particles to less dense regions in sucrose gradients. Comparison with poly(A)-containing RNA synthesized by the embryo indicates that the reduction in size of maternal poly(A)-containing RNA is not artifactual but reflects its degradation after the formation of blastoderm.     

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.     

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.     

Transfer ribonucleic acid synthesis during sporulation and spore outgrowth in Bacillus subtilis studied by two-dimensional polyacrylamide gel electrophoresis.

The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.     

Mutual relationship between antibiotics and resting spores of Bacillus subtilis: morphological changes and macromolecular synthesis after germination of spores treated with cyclic polypeptide and aminoglycoside antibiotics

Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin. Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetable cells, some mesosomes and DNA fibrils. On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B- and gramicidin S-treated resting spores. With gramicidin S-treated germinated spores a few vacuoles were formed in the cytoplasm. Both polymyxin B- and gramicidin S-treated germinated spores showed little or no synthesis of DNA, RNA, and protein. The vegetative cells derived from streptomycin-treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early. Their DNA and RNA synthesis was normal, whereas protein synthesis was low. In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin-treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled D-alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal. The outgrowing cells from gentamicin-treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled D-alanine. Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited. These results coincide with the growth curves of individual antibiotic-treated resting spores.     

Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells.

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after 'step-down' rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after 'step-down'. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.