RECOVERY OF HAEMOPHILUS INFLUENZAE FROM ULTRAVIOLET AND X-RAY DAMAGE

Abstract— Results of experiments on reactivation of ultraviolet (u.v.)-irradiated Haemophilus influenzae and cellular reactivation of u.v.-damaged transforming deoxyribonucleic acid (DNA) and bacteriophage are reported. Liquid-holding recovery (LHR) is small for the u.v.-sensitive mutant BC100 which, relative to the wild type, also has greatly reduced host-cell reactivation (HCR) of u.v.-inactivated phage, and competent cultures show reduced competent cell reactivation (CCR) of u.v.-inactivated transforming DNA. BC100 cells can be transformed with DNA isolated from the wild type strain Rd to a u.v. resistance similar to that of Rd, and irradiation of the DNA reduces the transformation frequency for this marker (uvr). The u.v.-resistant mutant BC200 displays very little LHR under the usual conditions where reactivation occurs after plating. The colony-forming ability (cfa) of irradiated BC200 is greater than that of Rd, but HCR and CCR are the same on this mutant as on the wild type. The major difference between Rd and BC200 is the enhanced u.v. survival of cfa of the latter. It was determined that this difference reflects cell lysis of irradiated Rd and lack of lysis in BC200 cultures. That lysis is closely correlated with damage to the bacterial chromosome is suggested by the finding that the lytic response of Rd (as determined turbidimetrically) can be negated by the liquid-holding procedure, but lysis of BC100 (which lacks comparable DNA-repair ability) can be only partially inhibited by this procedure. LHR occurs when post-plating dark recovery is incomplete, is temperature-sensitive, and occurs unimpeded when post-u.v. protein synthesis is inhibited by chloramphenicol. It is suggested that enzymatically catalyzed reactivation of DNA occurs or is initiated during liquid-holding of u.v.-irradiated H. influenzae Rd and that the necessary enzyme(s) exists prior to appearance of u.v. lesions in the DNA. Results are reported for X-ray inactivation of transforming DNA as assayed on BC100, Rd and BC200 and of the cfa of the three strains.