Molecular basis for the transformation defects in mutants of Haemophilus influenzae

Abstract

To determine the molecular basis of transformation defects in Haemophilus influenzae, the fate of genetically marked, ³²P-labeled, heavy deoxyribonucleic acid (DNA) was examined in three mutant strains (rec₁^-, rec₂^-, and KB6) and in wild type having ³H-labeled DNA and a second genetic marker. Transforming cells upon lysis with digitonin followed by low-speed centrifugation are separable into the supernatant fraction, containing mainly the unintegrated donor DNA, and the pellet, containing most of the resident DNA along with integrated donor DNA. Electron micrographs of digitonin-treated cells also indicate that the resident DNA is trapped inside a cellular structure but that cytoplasmic elements such as ribosomes are extensively released. DNA synthesis in digitonin-treated cells is immediately blocked, as is any further integration of donor DNA into the resident genome. Isopycnic and sedimentation analysis of supernatant fluids and pellets revealed that in strains rec₂^- and KB6 there is little or no association between donor and resident DNA, and thus there is negligible transfer of donor DNA genetic information. In these strains, the donor DNA is not broken into pieces of lower molecular weight as it is in strain rec₁^- and in the wild type, both of which show association between donor and recipient DNA. In strain rec₁^-, although some donor DNA atoms become covalently linked to resident DNA, the incorporated material does not have the donor DNA transforming activity.