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Temperature-pH effect upon germination of bacterial spores.

Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed.

Bacillus subtilis

Two components in the radiation sensitization of bacterial spores by p-nitroacetophenone: the -OH component.

p-Nitroacetophenone (PNAP) sensitizes Bacillus megaterium spores under anoxic conditions to the lethal effects of 50 kVp X-rays. Concentrations between approximately 5 X 10(-4) M and 3-8 X 10(-3) M produce the maximum effect, an increase of about 30 per cent over the anoxic response when the spores are irradiated in water. Compounds that scavenge -OH decrease, but cannot completely eliminate, this maximum amount of sensitization. These results indicate that PNAP acts to increase spores' radiation sensitivity through two separable types of chemical reactions: one which involves -OH and one which does not. Possible mechanisms responsible for these two components of damage are discussed. In these experiments 1/15 M phosphate buffer acts in several unexpected ways. This concentration itself increased the anoxic spore response by about 9 per cent (relative to the anoxic response in water). In addition, although the maximum amounts of sensitization were the same, the amounts of sensitization from lower PNAP concentrations differed when the suspending fluid was buffer instead of water. An interaction was also seen during the PNAP-t-butanol experiments; again, the responses at low PNAP concentrations were different in buffer and in water. No mechanisms for these actions of this buffer were suggested, although somewhat similar effects may occur with other organisms. Clearly, such effects must be recognized and evaluated before quantitative analyses of the actions of sensitivity-modifying agents can be completed.

Acetophenones

Effects of some -OH scavengers on the radiation sensitization of bacterial spores by p-nitroacetophenone and O2 in suspension.

Spores of Bacillus megaterium were irradiated in suspension with 50 kVp X-rays under three reference conditions: in anoxia (i.e., 100 per cent N2); in anoxia with 2mM p-nitroacetophenone (PNAP), a concentration that shows the maximum amount of sensitization by this agent; and in air. The responses were then measured when different concentrations of an hydroxyl radical scavenger were also present. Allyl alcohol, t-amyl alcohol, t-butanol, ethanol, glycerol, the formate ion, and methanol were the -OH scavengers that were used. A comparison of the effects these additives have on the three reference responses provides indirect, comparative information on the sensitizing processes of PNAP and O2. Neither t-amyl alcohol nor t-butanol affects the response in air or N2 alone. Both these additives, however, can reduce the sensitization from PNAP slightly. The other additives have significant protective effects under the three conditions. In general, the additives have similar effects in air and in 100 per cent N2 but much greater protective effects in PNAP. Although the sensitizing mechanisms are not identified by these experiments, the effects of O2 and PNAP, as judged by the desensitizing actions of these additives, appear quite different.

Acetophenones

The effect of transition metal ions on the resistance of bacterial spores to hydrogen peroxide and to heat.

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.

Bacillus

Polarized relationship of bacterial spore loci to the "old" and "new" ends of sporangia.

The frequency of association of spore loci with the "old" and "new" ends of rod-shaped sporangia in batch cultures of Bacillus megaterium ATCC 19213 was estimated by phase contrast microscopy. The analysis was facilitated by (i) the association of most of the sporangia into chains of two to five sporangia and (ii) the occurrence of two types of cross wall distinguishable by their degree of splitting. It was concluded that a newly formed spore is located at the "old" end of a sporangium. By inference, the sporulation division septum locus is distal to the ultimate normal cell division septum, i.e., proximal to the "old" pole of the B. megaterium sporangium. This result is discussed in relation to deoxyribonucleic acid segregation during sporulation.

Bacillus megaterium