Genetic basis for alkaline activation of germination in Bacillus thuringiensis subsp. israelensis.

Differences in activation between spores from strains of Bacillus thuringiensis subsp. israelensis with and without the toxin-encoding plasmid pBtoxis are demonstrated. Following alkaline activation, the strain bearing pBtoxis shows a significantly greater germination rate. Expression of just three genes constituting a previously identified, putative ger operon from this plasmid is sufficient to produce the same phenotype and characterizes this operon as a genetic determinant of alkaline activation.

Isolation and characterization of 2,3-dichloro-1-propanol-degrading rhizobia.

2,3-Dichloro-1-propanol is more chemically stable than its isomer, 1, 3-dichloro-2-propanol, and is therefore more difficult to degrade. The isolation of bacteria capable of complete mineralization of 2, 3-dichloro-1-propanol was successful only from enrichments at high pH. The bacteria thus isolated were found to be members of the alpha division of the Proteobacteria in the Rhizobium subdivision, most likely Agrobacterium sp. They could utilize both dihaloalcohol substrates and 2-chloropropionic acid. The growth of these strains in the presence of 2,3-dichloro-1-propanol was strongly affected by the pH and buffer strength of the medium. Under certain conditions, a ladder of four active dehalogenase bands could be visualized from this strain in activity gels. The enzyme involved in the complete mineralization of 2,3-dichloro-1-propanol was shown to have a native molecular weight of 114,000 and consisted of four subunits of similar molecular weights.

Non-variable sources of pure water and the germination and outgrowth of Bacillus subtilis spores.

Variable germination and outgrowth occurred when Bacillus subtilis NCTC 8236 spores were inoculated into nutrient broth prepared with distilled water. More reproducible findings were achieved when the medium was prepared with Elgastat water and the greatest reproducibility occurred with Elgastat water as vehicle combined with a rigorous acid-washing of all glassware. This combined procedure also produced optimum and reproducible results for the synchronous growth of two B. subtilis 168 strains in casein medium supplemented with appropriate amino acids, a technique of value in monitoring the development of resistance to antibacterial agents during sporulation. The levels of aluminium in distilled water were higher than those of other elements; however, the incorporation of aluminium sulphate into broth prepared with Elgastat water had no effect on germination, and outgrowth was reduced (but not eliminated) only at high concentrations of this salt.

Transformation of vegetative cells of Bacillus anthracis with plasmid DNA.

Methods have been developed for chemical transformation and electro-transformation (electroporation) of vegetative cells of Bacillus anthracis with supercoiled plasmid DNA. Chemical transformation was dependent on incubation in Tris/HCl with osmotic support and transformation with plasmid DNA was effected by treatment with polyethylene glycol 3350. Maximum transformation frequencies were 3.8 x 10(-5) transformant c.f.u. per viable c.f.u. (1 x 10(3) c.f.u. per micrograms DNA). Optimal frequencies were pH dependent and were affected by growth-medium composition. Transformation was not observed with linear or multimeric plasmid DNA. Electro-transformation of B. anthracis using high field intensity electroporation was dependent on the composition of both the growth medium and the electroporation buffer. Maximum electro-transformation frequencies were 5.3 x 10(-4) c.f.u. per viable c.f.u. (2.6 x 10(4) c.f.u. per micrograms DNA). The use of early exponential phase cells was critical to both procedures and the maximum efficiency (c.f.u. per micrograms DNA) of each system was strain dependent under the conditions described.

Possible mechanisms for the revival of glutaraldehyde-treated spores of Bacillus subtilis NCTC 8236.

Spores of Bacillus subtilis NCTC 8236 were exposed to 2% alkaline glutaraldehyde and subsequently subjected to various treatments in an attempt to revive injured spores. Treatment with alkali (sodium or potassium hydroxide or, to a lesser extent, sodium bicarbonate) proved to be most successful. Some revival was achieved after thermal treatment. No revival was obtained with lysozyme or with various types of coat-removing agents. Experiments designed to distinguish between germination and outgrowth in the revival process established that sodium hydroxide (optimum concentration, 20 mmol/l) added to glutaraldehyde-treated spores increased the potential for germination. In contrast, spores which had been allowed to germinate before exposure to low concentrations of glutaraldehyde and then to sodium hydroxide were inhibited at the outgrowth phase to a much greater extent than germinated spores treated with the dialdehyde without subsequent alkali exposure. The results overall are discussed in terms of the possible mechanism and site of action of glutaraldehyde and the practical implications and significance of its use as a sporicide.

Alkali-induced revival of Bacillus spores after inactivation by glutaraldehyde.

Spores of Bacillus subtilis 168 were apparently fully inactivated by exposure to 2% (w/v) glutaraldehyde for 20 h but a few spores could be revived by further treatment with 10-100 mM NaOH. A similar effect was found with spores from a range of Bacillus species. A minimum concentration of 5% (w/v) glutaraldehyde was required to prevent the alkali-induced reactivation. The implications of these results for the use of glutaraldehyde as a sporicidal agent are discussed.

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains.

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.

A note on a membrane filtration method for the concentration and enumeration of bacteriophages from water.

A membrane filtration method is described for the recovery and enumeration of bacteriophage from water. The method is conveniently used in the field and requires no complex or expensive equipment.

Transfer of plasmids among bacilli.

Plasmids were transferred from Staphylococcus aureus to Bacillus subtilis and between B. subtilis and several other species of Bacillus by protoplast fusion and regeneration. The plasmids replicate and express themselves normally in all bacilli examined.

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.

Phosphatidylseryl-transfer RNA in Bacillus licheniformis 749/C.

Phosphatidylserine has been found in extracts of Bacillus licheniformis made under alkaline conditions but not under neutral or acidic ones and was derived from the tRNA fraction. In tRNA preparations kept below neutrality during purification, phosphatidylserine was the only phospholipid released when the pH was raised to 9.0. The amount of bound phosphatidylserine could be increased by incubating tRNA from B. licheniformis or Escherichia coli with CTP and phosphatidic acid in the presence of an S-30 extract from either organism. The tRNA carrying phosphatidylserine has been separated from the bulk of the tRNA by DEAE-Sephadex chromatography in the presence of a detergent. On deaminoacylation of this material and rechromatography on DEAE-Sephadex, a number of peaks were found, indicating that this behavior is not confined to a single isoaccepting species.

Production and possible function of serine protease during sporulation of Bacillus subtilis.

The production of extracellular protease during sporulation in Bacillus subtilis 168 was investigated. Two proteases are produced, an alkaline serine protease and a neutral metalloprotease. In vivo inhibition of the serine protease with phenylmethylsulfonylfluoride indicated that the metalloprotease was degraded by the serine protease during sporulation. The experiments with phenylmethylsulfonylfluoride also show that the serine protease is necessary for the sequential process of sporulation and that it is required continuously for the first 2 to 3 h of the 8-h process.

Criteria for categorizing early biochemical events occurring during sporulation of Bacillus subtilis.

Two criteria are suggested for assessing the relevance of biochemical events occurring early in sporulation. The first is thymidine starvation, a condition known to inhibit sporulation. This also inhibits the production of metalloprotease, serine protease, and ribonuclease; alpha-amylase production, however, is unaffected. The second is the effect of a regulator mutation which increases the production of the proteases. In the mutant, ribonuclease is produced in correspondingly large quantities whereas alpha-amylase production is unaffected. We conclude that, whereas the serine protease is part of the main sequence of events leading to formation of the spore, the metalloprotease is a side effect, i.e., connected with the main sequence but not part of it. Ribonuclease could, on present evidence, be either in the main sequence or a side effect associated with it. Amylase, however, seems to be separately regulated and neither directly nor indirectly connected with the sporulation sequence.