Study on the cell cycle of Bacillus subtilis using temperature-sensitive mutants

Summary Among temperature-sensitive mutants which were defective in septum formation and formed nonseptate filaments at nonpermissive temperatures three (ts31, ts341, ts526) were identified among 434 temperature-sensitive mutants isolated at random from a mutagenized population of Bacillus subtilis 168. The results of morphological observations and characterization of these mutants showed that ts31 and ts341 were septum-initiation mutants and that ts526 was a DNA elongation mutant. The above mutations, and other mutations affecting septum initiation (div355 and tms12) were mapped by PBS1-mediated transduction on the chromosome in three separate regions as follows: pur A16-ts526-div355-cysA14; metC3-(ts31, tms12)-pyrD1-recA1; ebr-2-ts341-uvrA1-hisA1-cysB3. Our results suggest that the initiation process of septum formation requires at least four kinds of gene product. In addition, the sesult obtained with ts526 suggests an intimate connection between septum initiation and DNA replication.     

Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

Alkaline phosphatase production of Bacillus subtilis

Since alkaline phosphate activity increases in sporulation medium during the developmental period, in spite of the presence of inorganic phosphate, the uptake and intracellular concentration of phosphate were measured. While the uptake of inorganic phosphate decreases and the concentration of acid-soluble organic phosphate remains constant, the intracellular concentration of inorganic phosphate increases to about 30 mM after the end of growth. Some compound other than inorganic phosphate must therefore repress alkaline phosphatase. Other experiments showed that addition of glucose delays both the alkaline phosphatase increase and sporulation by about the same time.     

枯草芽胞杆菌降解毒死蜱特性研究

研究生物量、pH、毒死蜱浓度和温度对枯草芽胞杆菌3374菌株(编号为GU086422)在水溶液中降解毒死蜱特性,考察该菌株对白菜上毒死蜱残留的降解特性。结果表明,在毒死蜱质量浓度为240 mg/L、pH7.0、温度30℃的适宜条件下,枯草芽胞杆菌3374菌株对毒死蜱的降解率达到92.48%。该菌株能够有效提高白菜叶面上毒死蜱残留的降解速度,表明其在白菜上具有有效降解毒死蜱的能力,在无公害农产品生产中具有广阔的应用潜力。     

Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

Background  

Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.     

Defects in the nutrient-dependent methylation of a membrane-associated protein in spo mutants of Bacillus subtilis

Summary Methylation of a membrane-associated protein with an apparent molecular mass of 40000 daltons has been observed in Bacillus subtilis. The methylation was nutrient dependent and occurred with a doubling time of 4 ± 1 min. In wild-type strains, the half-life of turnover of the methyl group(s) was 17 ± 6 min. Several isogenic strains of B. subtilis containing spo0 mutations (spo0A and spo0H) were found to be normal in glutamate-dependent methylation of the protein and turnover of the methyl group(s). In strains containing spo0B and spo0E mutations, the methyl group(s) were incorporated in response to glutamate addition but turnover was not at a normal rate. The half-life of methyl group turnover was extended to 45 ± 3 min in these strains. In a spo0K mutant and in spoILI and spoIIF mutants, the protein was not significantly methylated. The methylation of a 40000 dalton protein was also found to be dependent on phosphate. This methylation was observed in wild-type and spo0A and spo0H strains with a doubling time of 4 ± 1 min and a half-life of turnover of the methyl group(s) of 11 + 3 min. In strains containing spo0B, spo0E, and spo0F mutations, the phosphate-dependent incorporation of the methyl group(s) was normal (5 ± 1 min) but the turnover half-life was extended to 46 ± 8 min. It is not known whether the nitrogen-dependent and phosphate-dependent systems methylated the same protein. The spo0 mutants are defective in the initial stages of sporulation, and it has been proposed that the spo0 gene products may play a role in nutrient sensing. The discovery of defects in the methylation of the 40 kDa protein in some of these spo0 mutants supports the proposal that the protein methylation may be part of a nutrient sensing system for the control of growth and sporulation in Bacillus species.     

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.     

Stimulation of Bacillus subtilis transformation by spermidine

Summary Addition of spermidine in millimolar concentrations to Bacillus subtilis cells during competence development increases transformability. The spermidine must be added at least 30 min before DNA for maximum stimulation. An incubation period of about 30 minutes is also required for the maximum uptake of labeled spermidine. The amount of DNA initially attached and the rate of DNA uptake are increased to the same extent as transformation. The rate of protein synthesis is also equivalently increased. These observations are consistent with an increase in the number of competent cells in the cell population; this increase is mediated by a spermidine-stimulated protein synthesis.     

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.     

Peptide syntheses mediated by Bacillus subtilis protease

Bacillus subtilis protease (Amano protease N) was examined as a catalyst for peptide bond formation via both the kinetically and thermodynamically controlled approaches. In general, the latter approach proved to be superior to the former, and a series of dipeptide syntheses and several segment condensations were achieved in good to high yields using the immobilized enzyme on Celite in acetonitrile with low water content.     

A Useful Cloning Vector for Bacillus subtilis

We have constructed plasmid pDN1050 a new small cloning vector for Bacillus subtilis . pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.     

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.     

Thiostrepton-resistant mutants of Bacillus subtilis: Localization of resistance to the 50S subunit

Summary Sexual crosses were studied between mutants ofPhycomyces blakesleeanus with abnormal phototropism (phenotypemad). Recombination frequencies were determined among five genesmadA tomadE. No clear evidence was found for linkage between any of the genes. Inconsistent results in crosses involvingmadC are attributed to nonisogenicity between the particular strains used. Onemad strain was discovered to be a double mutant. A new gene, tentatively designatedmadG, was segregated from a cross involving that strain.     

RNA polymerase subunit biosynthesis in Bacillus subtilis

Summary The relative rates of RNA polymerase biosynthesis in Bacillus subtilis has been examined under steady-state growth conditions. The synthesis of RNA polymerase subunits (, , , ) has been followed by subunit fractionation of immunoprecipitated [³H]-labelled samples on SDS-polyacrylamide gels. The stoichiometries of ::: subunits have been determined from cultures pulse-labelled during steady-state growth. The results suggest that an unassembled pool of the -subunit exists from which the holoenzyme is formed.Upon shift-up from acetate to glycerol containing medium, a rapid rise in the differential rate of core enzyme synthesis was observed, while the rate of synthesis of the -subunit was not stimulated. During shift-down, a concomitant reduction in the rate of synthesis of all subunits occurred for the first 20 min after the shift; thereafter, a rate of synthesis characteristic of the new growth rate was established.As cultures enter sporulation, an immediate reduction in the rate of -subunit synthesis was demonstrated.     

Intramolecular homologous recombination in Bacillus subtilis 168

Summary Ribosomal protein synthesis is regulated by controlling the fraction of mRNA associated with polysomes. It is known that this value changes in different developmental stages during Xenopus embryogenesis or, more generally, with changing cell growth conditions. We present here an analysis of the proportion of mRNA loaded on polysomes, carried out with probes for five different ribosomal proteins on several batches of Xenopus embryos obtained from different individuals. The results obtained indicate the existence of probe-dependent and individual differences, which reflect genetic variations in the cis- and trans-acting regulatory elements responsible for translational regulation. The fraction of ribosomal protein mRNA loaded onto polysomes can be used as an index of an individual's capacity for ribosome production.     

W-mutagenesis in competent cells of Bacillus subtilis

Summary The relative yield (N _m/N) of fluorescent mutants Ind^- after the transformation of Bacillus subtilis cells by means of UV-irradiated DNA is much higher in an uvr ^- recipient than in an uvr ⁺ strain, when compared at equal fluence, but practically identical at equal survival. Ind^- mutations are induced by UV-irradiation of separated single strands of transforming DNA. The H-strand is much more sensitive to the mutagenic action of UV light. Preliminary irradiation of competent recipient cells by moderate UV fluences increases the survival of UV-or -irradiated transforming DNA (W-reactivation) and the frequency of Ind^- mutations (W-mutagenesis). During transfection of B. subtilis cells by UV-irradiated prophage DNA isolated from lysogenic cells B. subtilis (Ø105 c ⁺) c-mutants of the phage are obtained in high yield only in conditions of W-mutagenesis, i.e. in UV-irradiated recipient cells. These data show that there is no substantial spontaneous induction of error-prone SOS-repair system in the competent cells of B. subtilis.     

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g⁻¹ h⁻¹. Composition of mixture of peptides was dependent on pH, concentrations of Na⁺ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.