Physicochemical investigations on DNA isolated from bacteria after photodynamic inactivation

Abstract

AbstractSpectrophotometric and sedimentation measurements have been used to 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 to 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 performed 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 photo‐dynamic action or secondary lesions induced by alkaline treatment. Further studies at neutral pH conditions are necessary to answer this question.–As a byproduct, evidence is given for the existence of single‐strand interruptions in the control DNA from untreated bacteria.