Physicochemical investigations on DNA isolated from bacteria after photodynamic inactivation

Abstract

Spectrophotometric and sedimentation measurements have been used t o investigate the influence of photodynamic in vivo action on the DNA from Proteus mirabilis. After photodynamic treatment of bacteria using thiopyronine as sensitizer the DNA was isolated under equal conditions both from the illuminated suspension and the corresponding control and dark suspension. The results obtained with various control samples show the high reproducibility of the DNA preparation technique used.The photodynamic effect leads to a deterioration of cell lysis and t o a considerable lowering of DNA yield. Photodynamic inactivation of bacteria results in several changes in the UV-spectrum of DNA which indicate a loss of guanine bases.From the sedimentation experiments peIformed on native and alkaline denatured DNA molecular weight distributions, number average molecular weights, and average numbers of single-and double-strand breaks have been evaluated. The results show that DNA from photodynamically treated bacteria exhibitsa strong degradation of the single polynucleotide chains which considerably increases with light dose. On the basis of our result it cannot be decided whether the single-strand scissions observed represent primary lesions of photodynamic action or secondary lesions induced by alkaline treatment. Further studies a t neutral p H conditions are necessary to answer this question. 208H.-E. JACOB, EVA SARFERT and H. TRIEBEL of photodynamic action on the DNA of bacteria. The DNA investigated was isolated from the bacteria after photodynamic treatment. Material and methodsBacterial strains and cultivation: The Proteus rnirabilis strains were isolated in the Zentralinstitut fur Genetik und Kulturpflansenforschung Gatersleben. For photodynamic treatment and isolation of DNA the two strains PG 273 and PG 672 were used. Strain PG 273 (thr-, uvr+, rec') represents the wild-type and strain PG 672 (thr-, uvr-, rec-) is the sensible mutant with small repair ability (BOHME 1967(BOHME , 1968. The experiments with strain PG 672 were performed in order to reduce repair processes possibly occurring during the time interval between start of illumination and begin of DNA extraction.Stationary phase cultures were prepared in vessels containing 100 ml meat-peptone broth at 37 "C for 16 hrs. Three litres of this culture gave approx. 30 g wet bacteria mass by centrifugation.Photodynamic treatment: The wet bacteria mass was resuspended in 2100 ml NaCl solution (0.9%) and divided in three parts: (A) Control sample -bacteria; (B) Dark sample -bacteria + dye; (C) Illuminated sample -bacteria + dye + light.I n suspensions with high cell numbers -approx. 1 x 101O cells/ml -the oxygen is quickly removed by respiration. Oxygen, however, is a component of the photodynamic action (it is necessary, e.g., for oxidation of the reduced dye radical). Therefore we have aerated the suspension with oxygen using a G3-filter (VEB Jenaer Glaswerke) for producing very small bubbles. The suspension was placed in a glass tube (length 45 om, diameter 6 cm). The ...