Chromosome Replication in Sporulating Cells of Bacillus subtilis

A method of specifically labeling the chromosomal terminus of Bacillus subtilis is described. When sporulating cultures were pulse-labeled with [³H]thymidine and then treated with 6-(p-hydroxyphenylazo)uracil, a drug which inhibits deoxyribonucleic acid (DNA) synthesis rapidly and completely, the only labeled spores formed were those that had completed replication during the pulse period. DNA-mediated transformation was used to show that the DNA of spores formed in the presence of 6-(p-hydroxyphenylazo)uracil had the same ratio of origin to terminus markers as DNA from untreated spores. Furthermore, spores formed in the presence of 6-(p-hydroxyphenylazo)uracil had the same DNA content as untreated spores. These two observations indicated that spores formed in the presence of 6-(hydroxyphenylazo)uracil contained completed chromosomes. The rate of termination of chromosomes destined to be packaged into spores was determined by this method, using the Sterlini-Mandelstam replacement system and a single medium exhaustion system for inducing sporulation. With both systems the rate of termination reached a broad peak 2 h after the start of sporogenesis. This was measured from the time of resuspension by using the replacement system and from the point where exponential growth ceased in the exhaustion system. The amount of spore DNA synthesized in the Sterlini-Mandelstam sporulation-inducing medium was very close to one-half the amount of the DNA present in mature spores. This suggests that chromosomes destined to be packaged into spores were replicated from close to the origin and possibly initiated in the sporulation-inducing medium. A method was devised for estimating the time taken to complete replication of the chromosomes destined to be packaged into spores. This was probably no more than 50 min. Whereas starvation must have occurred almost simultaneously in most cells in the population, the chromosome replication that was essential for sporogenesis was distributed over a wide time span. Thus, in some cells, replication started within 10 min of the nutritional step-down, but the peak rate was not reached for 1 h; thereafter replication continued at a substantial rate.     

Microbial Cells as Biosorbents for Heavy Metals: Accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa

Uranium accumulated extracellularly on the surfaces of Saccharomyces cerevisiae cells. The rate and extent of accumulation were subject to environmental parameters, such as pH, temperature, and interference by certain anions and cations. Uranium accumulation by Pseudomonas aeruginosa occurred intracellularly and was extremely rapid (<10 s), and no response to environmental parameters could be detected. Metabolism was not required for metal uptake by either organism. Cell-bound uranium reached a concentration of 10 to 15% of the dry cell weight, but only 32% of the S. cerevisiae cells and 44% of the P. aeruginosa cells within a given population possessed visible uranium deposits when examined by electron microscopy. Rates of uranium uptake by S. cerevisiae were increased by chemical pretreatment of the cells. Uranium could be removed chemically from S. cerevisiae cells, and the cells could then be reused as a biosorbent.     

Cell-Bound Serine Proteinase of Sporulating Cells of Bacillus subtilis

2-Deuterio-2-cyclohexen-l-one, 3-deuterio-2-cyclohexen-l-one and 2-methyl-2-cyclohexen-1-one were reduced by Clostridium La 1 giving a single bioconversion product resulting from reduction of the carbon-carbon double bond. Stereochemistry of the reaction was studied.     

Termination of Bacillus subtilis chromosome replication as visualized by autoradiography

A mutant of Bacillus subtilis W23 thy his, temperature sensitive for the initiation of rounds of chromosome replication, has been used to investigate the manner in which the two growing points on the replicating circular chromosome approach one another during termination (completion) of the round in progress.The mutant, growing exponentially at 34 °C, was transferred to 45 °C and [³H]thymidine added shortly afterwards. After a further short interval, the specific radioactivity was lowered by a factor of three and rounds of replication were allowed to complete under these conditions. The pattern of heavy and light grain density regions in chromosomal structures made visible by autoradiography was examined, and many with both ends more heavily labelled than their internal region were found. From an analysis of the relative frequency and size of these structures it is concluded that the two growing points in a single chromosome can continue to move at similar rates (within a factor of 2) as they approach one another to within < 10% of the length of the whole circular chromosome in the vicinity of the terminus. The data obtained do not prove that all chromosomes behave in this manner, but are consistent with such an interpretation.     

Macromolecular Synthesis in Newly Transformed Cells of Bacillus subtilis

The capacity of newly transformed cells of Bacillus subtilis to synthesize deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein has been determined by following the kinetics of suicide after their exposure to tritiated precursors of each of these macromolecules. Competent cells, whether transformed or not, are heterogeneous with respect to DNA synthesis. About 40 to 50% are latent in DNA synthesis. This latency may persist for 2.5 to 3 hr since transformants are resistant to thymineless death for this period after DNA addition. The remainder of the transformants synthesize DNA at one-half the rate of the cells of the total population. Synthesis of stable RNA does not occur at an appreciable rate in newly transformed cells. Newly transformed cells, however, do synthesize protein extensively, as demonstrated by the lethality of incorporated tritiated amino acids. Either chloramphenicol or actinomycin D treatment during the time of exposure to the tritiated amino acid prevented the suicide of transformants.     

Lysis of Bacillus subtilis Cells by Glycerol and Sucrose Esters of Fatty Acids

The lytic action of glycerol and sucrose esters of fatty acids with different carbon chain lengths on the exponentially growing cells of Bacillus subtilis 168 was investigated. Of each series of esters, glycerol dodecanoate and sucrose hexadecanoate were the most active. Lysis at 1 h after the addition of 0.1 mM glycerol dodecanoate or 20 μg of sucrose hexadecanoate per ml was 81 or 79%, respectively, as evaluated by the reduction in optical density. During this treatment a great loss of viability occurred that preceded lysis. The results that were obtained suggest that autolysis is induced by these esters. The esters caused morphological changes in the cells, but a seeming adaptation of the cells to esters was seen.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

Anaerobic growth of Bacillus mojavensis and Bacillus subtilis requires deoxyribonucleosides or DNA

Bacillus mojavensis strains JF-2 (ATCC 39307), ROB2, and ABO21191(T) and Bacillus subtilis strains 168 (ATCC 23857) and ATCC 12332 required four deoxyribonucleosides or DNA for growth under strict anaerobic conditions. Bacillus licheniformis strains L89-11 and L87-11, Bacillus sonorensis strain TG8-8, and Bacillus cereus (ATCC 14579) did not require DNA for anaerobic growth. The requirement for the deoxyribonucleosides or DNA did not occur under aerobic growth conditions. The addition of a mixture of five nucleic acid bases, four ribonucleotides, or four ribonucleosides to the basal medium did not replace the requirement of B. mojavensis JF-2 for the four deoxyribonucleosides. However, the addition of salmon sperm DNA, herring sperm DNA, Escherichia coli DNA, or synthetic DNA (single or double stranded) to the basal medium supported anaerobic growth. The addition of four deoxyribonucleosides to the basal medium allowed aerobic growth of B. mojavensis JF-2 in the presence of hydroxyurea. B. mojavensis did not grow in DNA-supplemented basal medium that lacked sucrose as the energy source. These data provide strong evidence that externally supplied deoxyribonucleosides can be used to maintain a balanced deoxyribonucleotide pool for DNA synthesis and suggest that ribonucleotide reductases may not be essential to the bacterial cell cycle nor are they necessarily part of a minimal bacterial genome.     

A Complex Pattern of Traveling Stripes Is Produced by Swimming Cells of Bacillus subtilis

Motile cells of Bacillus subtilis inadvertently escaped from the surface of an agar disk that was surrounded by a fluid growth medium and formed a migrating population in the fluid. When viewed from above, the population appeared as a cloud advancing unidirectionally into the fresh medium. The cell population became spontaneously organized into a series of stripes in a region behind the advancing cloud front. The number of stripes increased progressively until a saturation value of stripe density per unit area was reached. New stripes arose at a fixed distance behind the cloud front and also between stripes. The spacing between stripes underwent changes with time as stripes migrated towards and away from the cloud front. The global pattern appeared to be stretched by the advancing cloud front. At a time corresponding to approximately two cell doublings after pattern formation, the pattern decayed, suggesting that there is a maximum number of cells that can be maintained within the pattern. Stripes appear to consist of high concentrations of cells organized in sinking columns that are part of a bioconvection system. Their behavior reveals an interplay between bacterial swimming, bioconvection-driven fluid motion, and cell concentration. A mathematical model that reproduces the development and dynamics of the stripe pattern has been developed.     

Experimental Measurement of Monovalent Cation Adsorption onto Bacillus subtilis Cells

Typically, the effects of ionic strength on metal adsorption to geosorbents are accounted for by models of the surface electric field, assuming a planar surface. However, bacterial cell walls are not two-dimensional surfaces. Furthermore, electric field model parameters for complex systems are difficult to determine and apply. We propose an alternative approach to electric field models of ionic strength effects by explicitly accounting for monovalent cation adsorption onto specific bacterial binding sites. We calculate stability constants for monovalent metal-bacterial surface complexes, and use them to determine the magnitude of correction needed for a previously determined stability constant for a Cd-bacterial surface complex.     

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.     

Production of sedoheptulose by Bacillus subtilis

Wild type strains of Bacillus subtilis produced sedoheptulose from d-ribose but not from d-glucose, B. subtilis mutants deficient in transketolase produced sedoheptulose when d-glucose was used as a carbon source. The addition of d-ribose to the culture medium increased the amount of sedoheptulose accumulated, reaching about 20 mg/ml of culture broth. The mutant strains reverted to wild type strains at a high frequency during cell growth, and therefore the accumulation of sedoheptulose was caused by the genetic instability of the mutant: d-ribose formed from d-glucose by the mutant strain was converted into sedoheptulose by revertant cells that appeared during cultivation.     

Transformation of Bacillus subtilis by electroporation

Summary Optimal conditions for the transformation of Bacillus subtilis by electroporation were achieved. Frequencies of greater than 10⁵ transformants/&#x03BC;g of plasmid DNA were obtained for a number of strains and plasmids. Increased transformation efficiency of mini-prep DNA from B. subtilis and Escherichia coli was obtained after microdialysis.