Uv-Resistance of Transforming Dna in Germinating Spores of Bacillus Subtilis

Abstract

Resistance of bacterial spores to ultraviolet ray (UV) increases temporarily in the course of germination (1-4). STAFFORD and DONNELLAN (3) showed that this increase of UV-resistance in germinating spores was correlated with a decrease in the production of thymine-containing photoproducts. The experiments described below were carried out to see if this increase in UV-resistance could be explained in terms of the resistance of DNA in the spores as measured by transforming ability. Spores of Bacillus subtilis Marburg 160 try-arg-were prepared as described by TANOOKA (5). To facilitate the extraction of DNA from spores (6), it was necessary to sensitize the spores to lysozyme (7) prior to germination and UV-irradiation.The sensitization to lysozyme was performed by treating the dormant spores (about 109 spores/ml) at 37° for 90 min with 10% thioglycolic acid in the presence of 7.2 M urea and 0.5 mM dithiothreitol (8). The spores thus treated were centrifuged, washed twice with 0.1 M phosphate buffer (pH 7.8), and suspended in distilled water. At this stage, the spores were highly dormant and required heat-shock for germination.After being heat-shocked at 70° for 2 hr, the spores (about 109 spores/ ml) were germinated at 37° in 10 mM Tris buffer (pH 7.0) containing 10 mM L-alanine. As shown in Fig. 1, these spores exhibited an unusually long lag period (30 min or more at this spore concentration) before commencement of germination as measured by optical density.This lag period tended to increase with spore concentration.Despite these abnormal behaviors caused by the thioglycolic acid treatment, germination of these spores was considered to be physiological in nature because (1) they were fully viable and (2) no appreciable change in optical density occurred in the absence of alanine.For the examination of UV-resistance of DNA in spores, an aliquot (5 ml) removed from the germinating spore suspension was chilled quickly to below 10°, transferred to an aluminium dish (76 mm in diameter) and sub-181

Role of Water in Ultraviolet-Resistance of Germinating Bacterial Spores

“…Photochemical (3) and transformation (4) studies showed that resistance of the spores at this transient stage (U-stage, 5) is explained by the specific physical state, or conformation of DNA in vivo.…”