End Of Logarithmic Growth Research Articles

THE STUDY OF PROKARYOTIC GENE EXPRESSION

One of the methods to evaluate the level of gene expression is a real-time quantitative polymerase chain reaction (qPCR). Interest in the study of molecular mechanisms of gene expression and its evaluation in prokaryotes is due to the lack of research on this issue and a number of methodological problems. The paper presents a study of gene expression mechanism in prokaryotes evidence from Aeromonas salmonicida AS1 gyrase B and collagenase genes. As a result of the research, Random primer and oligo (dT) primer (two 3’-terminal nucleotides of the primer complementary to stop codon nucleotides of the transcribed DNA sequence) with anchor and adapter of our own design were tested, which are used in the reaction of reverse transcription. The use of oligo (dT) primer became possible only after polyadenylation of extracted RNA using special poly-A polymerase kit. It is determined that the developed protocol of reverse transcription (RT) using oligo (dT) primer and adapter with certain sequence on its 5’-terminus designed for further annealing of the reverse primer during real-time PCR along with preliminary polyadenylation of RNA excludes specific amplification of the background genomic DNA. This technique may be applied in evaluating the expression level of low-expression genes when high background genomic DNA content is found in the RNA sample, e.g. at the end of logarithmic growth of prokaryotic cells.ContributionAll authors bear responsibility for the work and presented data. All authors made an equal contribution to the work. Minaev M. Yu. developed scientific and methodological approaches to work, determined the scope of research, analyzed the data obtained, performed the narrative and corrected it in final. Makhova A.A. selected research objects, carried out RNA extraction, reverse transcription and PCR analysis, performed the narrative part. The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism.Conflict of interestThe authors declare no conflict of interest.

Production of chlorogenic acid and isoorientin hypoglycemic compounds in Cecropia obtusifolia calli and in cell suspension cultures with nitrate deficiency

The antidiabetic properties of Cecropia obtusifolia are attributed to chlorogenic acid (CGA) and isoorientin (ISO) phenolic compounds; both compounds possess hypoglycemic, hypolipidemic, and antioxidant properties. As a potential strategy for an adequate supply of authentic plant raw material, the aim of this study was to establish in vitro conditions for the development of cell suspension cultures that produce these bioactive compounds. Callus cultures of leaf explants from acclimatized tree and in vitro plantlets were set up using different auxin levels; treatments with 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthalene acetic acid (NAA) to 8.92 µM with 6-benzylaminopurine (BAP) at 2.22 μM stimulate highest callus production. Seedling cotyledon, hypocotyl, leaf, and stem explants developed calli bearing roots with 2,4-D. With NAA, hypocotyl, cotyledon, and leaf explants developed morphogenic calli; 75% of stem explants formed calli, and the remaining calli developed shoots. Determined CGA concentrations in calli were similar to those detected in the leaves of wild trees, and ISO was not produced. Cell suspension cultures were established from leaf explants friable calli with 8.92 μM 2,4-D in combination with 2.22 μM BAP, employing 4 and 5% inocula in fresh weight; CGA levels were maintained and ISO was produced only at the end of logarithmic growth. On diminishing nitrate content in Murashige and Skoog (MS) medium to 8.0 mM, maximum cell biomasses diminished, CGA production is increased and twice with 16.0 and, instead of CGA production is tripled and quadrupled with 16.0 and 8.0 mM nitrates, respectively, and ISO synthesis was induced earlier and for a longer time period, increasing its levels at the end of culture. Two compounds with ultraviolet spectra similar to those of caffeic and ferulic acids were formed. Our results offer a protocol of cell suspension cultures for C. obtusifolia bioactive production and hypoglycemic property conservation.

Pediocin ST18, an anti-listerial bacteriocin produced by Pediococcus pentosaceus ST18 isolated from boza, a traditional cereal beverage from Bulgaria

Lactic acid bacteria isolated from boza, a cereal-fermented beverage from Belogratchik, Bulgaria, were screened for the production of bacteriocins. Thirteen of the 52 strains isolated inhibited the growth of Pediococcus spp., Listeria innocua and Lactobacillus plantarum. One of the strains, identified as Pediococcus pentosaceus ST18, produced pediocin ST18 at 3200 arbitrary units (AU) ml −1 in MRS broth at the end of logarithmic growth (i.e. after 24 h). Concentration by ammonium sulfate precipitation, followed by separation in a Sep-Pack C 18 column and reverse-phase HPLC on a C18 Nucleosil column yielded two active antimicrobial peptides, which suggests that pediocin ST18 may be a two-peptide bacteriocin. The peptide had bacteriostatic action towards L. innocua, with no cell lyses. Pediocin ST18 remained active after 30 min at 121 °C and after 2 h of incubation at pH 2–12. No loss in activity was recorded after treatment with α-amylase, SDS, Tween 20, Tween 80, urea, Triton X-100, N-laurylsarcosin, EDTA and PMSF. Pediocin ST18 does not adhere to the cell surface of the producer strain.

The phosphatidylcholine to phosphatidylethanolamine ratio of Saccharomyces cerevisiae varies with the growth phase.

This study compares the effect of the growth phase on the phospholipid composition and the activity of several phospholipid biosynthetic enzymes in a wild-type yeast grown in fermentable (glucose) and non-fermentable (lactate) semi-synthetic and complete synthetic media. Several distinct differences as well as similarities were found. The cellular phosphatidylcholine: phosphatidylethanolamine (PC:PE) ratio was found to vary with the growth phase, with increases in PC levels at the expense of PE during the transition to stationary phase. The variation was most pronounced in semi-synthetic lactate medium, which is routinely used for the isolation of mitochondria, where the PC:PE ratio changed from 0.9 to 2.2 during this transition. Similar growth phase-dependent changes in PC and PE content were observed in isolated organelles such as mitochondria, mitochondria-associated membranes and microsomes. Phosphatidylinositol (PI) levels were much higher in cells grown on lactate compared to cells grown on glucose (20% vs. 5-10%). Irrespective of the medium, PI levels increased upon entering stationary phase. The activities of the phospholipid biosynthetic enzymes phosphatidylserine synthase and the phospholipid-N-methyltransferases were found to be maximal at the end of logarithmic growth and to decrease upon entering stationary phase in all media. Cells grown on lactate displayed a significantly higher phospholipid:protein ratio than cells grown on glucose. The results are discussed in terms of regulation of phospholipid biosynthesis and membrane biogenesis in response to growth phase and carbon source.

Immunocytochemical localization of a calmodulinlike protein in Bacillus subtilis cells.

To determine possible functions of the calmodulinlike protein of Bacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [(45)Ca]CaCl(2), ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from B. subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles of B. subtilis also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.

COCCOLITH PRODUCTION AND DETACHMENT BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

ABSTRACTLaboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. 14C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. 14C‐labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a 14C‐based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell−1·d−1 while net detachment rates were as high as 15 coccoliths· cell−1·d−1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.

Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum)

Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth.

Stage-specific in vitro expression of the Bacillus subtilis spoVE gene.

Synthesis of the spo VE gene product (a 32 kilodalton protein) of Bacillus subtilis was studied using a DNA-directed protein-synthesizing system from B. subtilis. The spo VE gene product was synthesized specifically by T2 (2 hours after the end of logarithmic growth) cell extracts but not by vegetative cell extracts. A dot blot hybridization experiment with a spoVE-specific probe showed that the spoVE mRNA was synthesized by vegetative as well as by T2-cell extracts, and was present in most spo0 mutants. These results suggest that the transcription of the spoVE gene is not dependent on the spo0 gene function and the synthesis of the SpoVE protein is somehow controlled at the level of translation. Heterologous combination experiments using supernatant fractions from the vegetative and T2 cells, and ribosomes from the vegetative and T2 cells showed that T2-specific synthesis of the SpoVE protein is dependent on the T2-cell supernatant. The addition of the T2-cell supernatant to the vegetative cell extract i...

Emendation of the Genus Thermobacteroides: Thermobacteroides proteolyticus sp. nov., a Proteolytic Acetogen from a Methanogenic Enrichment

Thermobacteroides proteolyticus sp. nov. was isolated from a methanogenic enrichment culture inoculated from a thermophilic digestor (55°C) that was fermenting tannery wastes and cattle manure. The cells were anaerobic, gram-negative, nonsporeforming, nonmotile rods that were 0.5 μm wide and 1 to 6 μm long. At the end of logarithmic growth, they were pleomorphic, with some filamentous cells. The deoxyribonucleic acid base composition was 45 mol% guanine plus cytosine. The temperature optimum was 63°C (growth range 35, to 75°C); the pH optimum was 7.5 (growth range, pH 5.0 to 8.5). The growth substrates used included yeast extract, peptone, casein, gelatin, and Trypticase peptone. Fructose, glucose, maltose, sucrose, and mannose were weakly used as growth substrates; however, addition of yeast extract and either rumen fluid or Trypticase peptone stimulated utilization of these carbohydrates. Acetate, H2, and CO2 were the major products of growth in medium containing gelatin or glucose. The cells were resistant to kanamycin. The type strain is strain BT (= ATCC 35242).

Changes in membrane-associated proteins during sporulation in Bacillus subtilis.

Membrane proteins from vegetative and sporulating cells of Bacillus subtilis were separated by the two-dimensional gel electrophoresis system using isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (O'Farrell technique). Membrane proteins were isolated according to published procedures. The gels were stained with Coomassie blue. Three different concentrations of proteins were analyzed to detect even minor constituents. Over two hundred different membrane proteins were identified in vegetative cells by their isoelectric point (pI) and molecular weight (Mr). Analysis of membrane proteins from cells harvested during and at the end of logarithmic growth (A600 approximately equal to 0.8; T0) and every hour thereafter until T4 showed that in the wild-type strain 55 proteins are degraded mostly at the beginning or sporulation. Many others (76 proteins) are newly synthesized during sporulation. About 16 proteins are synthesized at times during sporulation but again degraded within 1 h or less. Others (uncertain proteins, 65) are degraded and resynthesized again. This observation is in agreement with experiments previously published by Andreoli et al. [Andreoli, A. J., Kao, M., Chui, R., Cabrera, J., and Wong, S. K. S (1981) in Sporulation and Germination (Levinson, H. S., Sonenshein, A. L., and Tipper, D. J., eds) pp. 168-173, American Society for Microbiology, Washington] using Bacillus cereus. Experiments with the early blocked asporogenous mutant JH 649 (spoOF) showed that few proteins (40%) are degraded and even fewer (30%) are newly synthesized between A600 approximately equal to 0.8 and T4. Protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline) have no effect on the protein patterns. The experiments presented here show that proteins involved in differentiation in B. subtilis can be identified by the two-dimensional gel electrophoresis system and with the aid of asporogenous mutants. In order to assure that no cytoplasmic proteins are contaminating the membrane preparations, several cytoplasmic enzyme activities have been measured. Their concentration was found to be always below 0.005% of total protein, which is below the level of detection by Coomassie blue staining.

Production by Bacillis subtilis of brown sporulation-associated pigments.

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.

Lipid variation of the green alga Fritschiella tuberosa during growth in axenic batch culture

The lipid and pigment pattern of the chaetophoralean green alga Fritschiella tuberosa was studied during different growth periods. Four phases in axenic batch culture are observed. The first phase commences at the end of logarithmic growth and results in a decrease of phosphatidyl inositol and hexadecatetraenoic acid.The second phase coincides with the end of linear growth and is characterized by a decrease in glycolipids, phospholipids and α-linolenic acid, and an increase of linoleic and oleic acid. The third phase starts with the beginning of the stationary phase. During this time glycolipids, phospholipids, and α-linolenic acid decrease and oleic acid, linoleic acid and triacylglycerol increase. The fourth phase begins about one week after the onset of the stationary growth phase. A marked accumulation of triacylglycerol took place, secondary carotenoids were detectable and the long filamentous cells of Fritschiella changed into slightly rotund short cells.

Benzeneboronic acid selectively inhibits sporulation of Bacillis subtilis.

m-Aminobenzeneboronic acid at levels of 0.2 mM in nutrient broth medium selectively inhibited sporulation without appreciably altering vegetative growth. Significant inhibitory effects were seen even when it was added as late as 6 h after the end of logarithmic growth. The pH changes associated with growth and sporulation of Bacillus subtilis in nutrient broth were not significantly altered by the inhibitor. When it was present in cultures of actively growing cells, its inhibitory effect could not be reversed by simple dilution. The compound caused extensive clumping, of cells, which appeared not to be related to the ability of boronates to esterify to diols.

Studies on the Control of Development

Ribosomes from vegetative cells (vegetative ribosomes) of Bacillus subtilis synthesize guanosine 3′(2′)‐diphosphate 5′‐diphosphate (p2Gp2) and guanosine 3′(2′)‐diphosphate 5′‐triphosphate (p3Gp2). In contrast, ribosomes isolated from sporulating cells (sporulation ribosomes) synthesize adenosine 3′(2′)‐diphosphate 5′‐diphosphate (p2Ap2) and adenosine 3′(2′)‐diphosphate 5′‐triphosphate (p3Ap2). The products obtained with the aid of vegetative ribosomes were characterized by comparison with authentic substances in two chromatographic systems. The reaction mechanism is identical with that found in Escherichia coli, since [3H]GTP or GDP is pyrophosphorylated by ATP to form p2Gp2 and p3Gp2. Omission of ATP completely stops the formation of any reaction products. Furthermore, no reaction products are observed when GTP or GDP are omitted in a reaction mixture containing vegetative ribosomes. In contrast, incubation of sporulation ribosomes with ATP alone or ADP and ATP results in the formation of two products identified as p2Ap2 and p3Ap2 by their mechanism of synthesis as well as chemical and enzymatic degradation. Sporulation ribosomes (isolated at least an hour after the end of logarithmic growth) synthesize drastically less (by a factor of 100) p2Gp2 and p3Gp2. Vegetative ribosomes, on the other hand, synthesize only 1/100 the amount of p2Ap2 and p3Ap2 compared to sporulation ribosomes when isolated at least an hour before the end of logarithmic growth. By using washed ribosomes and ribosomal wash from vegetative and sporulation ribosomes, we were able to show that changes in the composition of ribosomes (probably in ribosomal proteins and/or ribosome‐associated factors) must occur during the transition from vegetative to sporulating cells. Washed ribosomes from vegetative cells cannot replace washed ribosomes from sporulating cells to synthesize p2Ap2 and p3Ap2 in the presence of ribosomal wash from sporulating cells and vice versa. During the transition from one type of ribosomes to the other, as far as the unusual nucleotide‐synthesizing activity is concerned, both types of nucleotides can be synthesized. While the ability to synthesize p2Gp2 and p3Gp2 decreases approximately at the end of logarithmic growth, the ability to synthesize p2Ap2 and p3Ap2 increases at the same time. The reason behind this obvious change in ribosomal structure and associated factors is discussed in the light of our finding that these changes are not observed in some early blocked asporogenous mutants.The observation that ribosomes from sporulating cells synthesize traces of p2Gp2 and p3Gp2 (by a factor of almost 100 less than vegetative ribosomes) is probably due to the presence of some non‐sporulating cells in any B. subtilis culture. Likewise, there are always some spores contained in a vegetatively growing culture, which are probably responsible for the observed small amounts of p2Ap2 and p3Ap2 synthesized.

Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

Synthesis of S-100 protein in monolayer cultures of rat-glial cells.

Synthesis and degradation of the brain‐specific S‐100 protein were measured in monolayer cultures of clonal rat glial cells C 6. The cells synthesize a small but definite amount of S‐100 protein in logarithmic growth. The increase in accumulation of S‐100 protein which occurs at the end of logarithmic growth is brought about by changes in synthesis.

Studies on the control of development. Accumulation of guanosine tetraphosphate and pentaphosphate in response to inhibition of protein synthesis in Bacillus subtilis.

Bacillus subtilis cells accumulate unusual phosphorylated substances at the end of logarithmic growth in a semi-synthetic medium. Two of these substances are guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'(2')-diphosphate 5'-triohosphate (pppGpp) which, in contrast to amino-acid-starved Escherichia coli cells, are not degraded in sporulating cells of B. subtilis after the addition of chloramphenicol. Moreover, inhibition of protein synthesis in growing cells of B. subtilis causes accumulation of ppGpp and pppGpp, which is also in contrast to E. coli. This was shown by isolation and characterization of substances produced in these cells after the addition of chloramphenicol. Other inhibitors of protein synthesis acting at the ribosomal level also cause the accumulation of ppGpp and pppGpp. There is no difference between the action of antibiotics affecting 50-S and/or 30-S ribosomal subunits, since chloramphenicol, tetracycline erythromycin and neomycin cause the accumulation of almost equal amounts of these nucleotides. This apparently resolves the close connection between ppGpp accumulation and the rate of stable RNA synthesis, which was believed to exist also in B. subtilis because of the stringent response observed after amino acid starvation coupled with ppGpp accumulation. Antibiotics which inhibit protein synthesis differently than by affecting the ribosomes (puromycin) or which inhibit RNA (rifampicin) or DNA (nalidixic acid) synthesis do not cause ppGpp accumulation. The accumulation of ppGpp and pppGpp in the presence of charged tRNA provided by chloramphenicol treatment suggests that the signal for the synthesis of unusual nucleotides is an inhibition of the binding of tRNA (charged or uncharged) to the acceptor site of the ribosome. This activates the rel gene product which forms ppGpp and pppGpp from GTP and ATP. Sporulating cells of B. subtilis without chloramphenicol treatment produce besides ppGpp and pppGpp other unusual substances, which are likely to be highly phosphorylated nucleotides contained adenine as base moiety.

The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis.

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.

Regulation of Transcription during Sporulation of Bacillus cereus T. DNA-Dependent RNA Polymerase from Vegetative and Sporulating Cells

DNA-dependent RNA polymerase from vegetative cells of Bacillus cereus T was purified to homogeneity by poly(ethyleneimine) precipitation, elution and DNA affinity chromatography. The purified enzyme shows high activity on the DNA of B. cereus phage oCL. The subunit composition (ββ′α2)σ, corresponds to the RNA polymerase of other prokaryotes. The molecular weights of the subunits are: β 140000, β′ 130000, α 43000, σ 56000. The σ factor can be separated from core enzyme by phosphocellulose chromatography. Both isolated σ factor, and the ασ fraction isolated from complete enzyme by cellogel electrophoresis, stimulate the activity of core enzyme on oCL DNA. Enzymes isolated from sporulating cells at 1.5, 3 and 12 h after the end of logarithmic growth did not differ from vegetative enzyme in their subunit patterns and no change in template specificity could be found.

Synthesis of ribosomal RNA during sporulation in Bacillus subtilis.

SPORULATION in Bacillus subtilis is associated with specific changes in RNA metabolism required for the expression of sporulation genes. Although net RNA synthesis stops abruptly at the end of logarithmic growth (T0), active turnover and differential transcription of the genome have been demonstrated1–3. Losick and Sonnenshein4 suggested that modifications of RNA polymerase may play a primary role in the regulation of gene expression during the sporulation process. The expression of ribosomal RNA genes during this period has been subject to conflicting reports1,5–7. The reported turn-off of these genes at the onset of sporulation5,6 has not been observed in similar experiments7. The latter, and the reported changes in template specificity of RNA polymerase4,8 were shown to be strongly dependent on the nature of the sporulation medium and to a lesser extent on the phenotype (sp+ or sp−) of the strain7,8. We have analysed the pattern of synthesis and turnover of stable RNA components in B. subtilis induced to sporulate in the highly defined resuspension medium, SM (ref. 9). We report that rRNA synthesis does continue during sporulation, although at a reduced rate, resulting in the gradual replacement of vegetative rRNA components by similar newly synthesised sporulation species.