Cell Wall Protein in Bacillus subtilis

The cell wall of Bacillus subtilis 168 contains protein that is refractory to removal by salts, detergents, and denaturants.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

Membrane association of a Staphylococcus aureus plasmid in Bacillus subtilis.

The Staphylococcus aureus plasmid pUB110 was found to be enriched in deoxyribonucleic acid-membrane complexes isolated from Bacillus subtilis containing pUB110.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis.

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

Genetic Regulation of Cell Division Initiation in Bacillus subtilis

The growth and division properties of a temperature-sensitive mutant of Bacillus subtilis defective in the initiation of cell division have been studied. Log-phase cells transferred from 30 to 45 C continue to increase in length but fail to initiate new divisions. Deoxyribonucleic acid synthesis continues at 45 C, and genomes are segregated along the filament length. When filaments are returned to 30 C, division initiation resumes, and the long multinucleate clones are partitioned into normal-size cells. Occasionally, multiple cross walls initiate in close proximity, resulting in tiny cells, some of which are anucleate. Division resumption is sensitive to protein synthesis inhibitors, suggesting there is a new protein required for the initiation of division in filaments.     

Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis.

We have observed a connection between cell wall synthesis and the initiation of chromosome replication in Bacillus subtilis. Initiation of chromosome replication was prevented in synchronous cultures in the presence of the cell wall synthesis inhibitor vancomycin. When vancomycin was added to the cultures after initiation of chromosome replication, one round of replication was completed but no reinitiation occurred. Similar results were obtained when cell wall synthesis was inhibited by ristocetin, cycloserine, cloxacillin, or cephaloridine. When sucrose was added to the medium, initiation of deoxyribonucleic acid replication occurred in the presence of vancomycin, to an extent which allowed replication of no more than approximately one-half of the deoxyribonucleic acid of the culture. The same was found in cultures of spheroplasts of B. subtilis. However, initiation of chromosome replication in spheroplasts was completely insensitive to cloxacillin.     

Cell division suppression in the Bacillus subtilis div IC-A1 minicell-producing mutant.

Growth and division patterns of Bacillus subtilis wild-type (div IV-A1+) and minicell-producing mutant (div IV-A1) clones were studied after spore germination during microcolony development in chambers that facilitate continuous observation with a phase contrast microscope. Data obtained from 13 div IV-A1+ clones were used to derive the equation DE equals [(mum minus 17.6)/8.8], which expresses the relationship of cell divisions present in clones of various lengths. This equation was used to determine the number of divisions expected in div IV-A1 clones if the mutant clones were able to divide as often as wild-type clones. The observed number of divisions present in mutant clones was found to be only 25.27% of the number expected on the basis of this equation. Although individual div IV-A1 clones varied in the percentage of division equivalents expressed, there appeared to be no correlation between the overall clone growth rate and the number of divisions expressed. Culturing div IV-A1+ and div IV-A1 clones together in the same growth chamber revealed that there were no diffusible interactions influencing the division phenotypes of either mutant or wild-type cells. At later stages of growth, mixed microcolonies containing cells of both genotypes were formed. A length analysis of individual cells in these populations indicated that the relative division suppression of mutant compared with wild-type cells characteristic of the initial stages of clone development was maintained. It is likely, therefore, that the excessive length of minicell-producing cells (div IV-A1) is a reflection primarily of division suppression in the mutant and not simply of mislocation of division along cell length.     

Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis.

Although exponential growth of Bacillus subtilis 168 in a phosphate-limited medium halted with the exhaustion of inorganic phosphate, the bacteria continued to grow at a slower rate for a further 3 to 4 h at 37 degrees C. This postexponential growth in the absence of an exogenous phosphate supply was accompanied by a loss of teichoic acid from the cell walls of the bacteria. Quantitative analysis of walls and culture fluids showed that the phosphate loss from the walls could not be accounted for by an increase in phosphate-containing compounds in the medium, which implied that the cells were using their own wall teichoic acids to supply phosphate necessary for growth. Addition of exogenous teichoic acid to phosphate-starved cultures resulted in stimulation of growth and in the simultaneous disappearance of teichoic acid phosphate from the medium. It is proposed that teichoic acids, which can contain more than 30% of the total phosphorus of exponential-phase cells, can be used as a reserve phosphate source when the bacteria are starved for inorganic phosphate.     

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.     

Deoxyribonucleic Acid Distribution in Bacillus subtilis Independent of Cell Elongation

A temperature-sensitive DNA(-) mutant of Bacillus subtilis has been studied during the resumption of deoxyribonucleic acid (DNA) synthesis following a 45 to 30 C temperature shift. For several hours after return to 30 C, DNA synthesis proceeds although the cells fail to elongate appreciably. Autoradiographs of cell populations synthesizing DNA during the recovery period demonstrate that DNA can become distributed to previously unoccupied regions along the cell length. By varying the labeling regime, newly synthesized DNA as well as DNA present at the time of transfer from 45 to 30 C were followed independently. Measurements of the percent of cell length covered by grains ((3)H-thymine in DNA) demonstrate the progressive refilling of DNA-vacant cell regions by both newly synthesized and original DNA. These data indicate that cell surface growth is not an absolute requirement for segregation of bacterial DNA.     

Minicell Yield and Cell Division Suppression in Bacillus subtilis Mutants

Minicell yield is determined by the probability of a minicell-producing division and the relationship of growth to division in Bacillus subtilis mutants.     

Fructose transport in Bacillus subtilis.

The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems. The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.     

Cell physiology and protein secretion of Bacillus licheniformis compared to Bacillus subtilis

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species.     

Chromosomal-DNA amplification in Bacillus subtilis.

Tetracycline-resistant (Tetr) mutants RAD1, RAD2, RAD6, and RAD7 were isolated from Bacillus subtilis BC92 after protoplasting, polyethylene glycol treatment, and regeneration on a medium containing tetracycline. The Tetr phenotype in RAD1, RAD2, and RAD6 was very stable with less than 5% loss of resistance after 30 generations of growth in the absence of selection. Of the four isolates, three contained amplified chromosomal DNA closely associated with the Tetr phenotype. The intensity of restriction fragments present in HindIII and EcoRI digests of chromosomal DNA from RAD1, RAD6, and RAD7 indicated the presence of tandemly duplicated DNA. Disparity in the size and number of amplified fragments suggested that the tandemly duplicated DNA is different in all three isolates. The sizes of the duplicated DNA present in RAD1, RAD6, and RAD7 were estimated to be 10, 19, and 20 kilobases, respectively. No amplified DNA was detected in RAD2. Results of transductional-mapping studies with PBS1 showed that the tetracycline resistance (tet) loci of RAD1, RAD2, and RAD6 all mapped near the origin of chromosomal replication and close to the guaA locus. Amplified DNA characteristic of RAD1 and RAD6 was cotransduced with the tet locus. Cotransfer of amplified DNA with the guaA locus or other nearby loci in the absence of tet was not observed. In every case, loss of Tetr was accompanied by loss of amplified DNA. A possible explanation for the occurrence of the amplified DNA is presented.     

Multiple catalases in Bacillus subtilis.

Vegetative cells of Bacillus subtilis in logarithmic growth phase produced one catalase, labeled catalase 1, with a nondenatured molecular weight of 205,000. As growth progressed, other activity bands with slower electrophoretic mobilities on polyacrylamide gels appeared, including a series of bands with a common nondenatured molecular weight of 261,000, collectively labeled catalase 2, and a minor band, with a molecular weight of 387,000, labeled catalase 3. Purified spores contained only catalase 2, and it was not produced in spo0A- or spo0F-containing mutants. Strains deficient in catalase 1 or catalase 2 or both were selected after mutagenesis. Sensitivities of the two main catalases to NaCN, NaN3, hydroxylamine, and temperature were similar, but the apparent Kms for H2O2 differed, being 36.6 and 64.4 mM, respectively, for catalase 1 and catalase 2. The levels of catalase 1 increased 15-fold during growth into stationary phase and could be increased 30-fold by the addition of H2O2 to the medium. Catalase 2, which was not affected by H2O2, appeared only after the cells had reached stationary phase, and the maximum levels were only half of the basal level of catalase 1.