To determine the molecular basis of transformation defects in Haemophilus influenzae, the fate of genetically marked, (32)P-labeled, heavy deoxyribonucleic acid (DNA) was examined in three mutant strains (rec(1) (-), rec(2) (-), and KB6) and in wild type having (3)H-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 rec(2) (-) 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 rec(1) (-) and in the wild type, both of which show association between donor and recipient DNA. In strain rec(1) (-), although some donor DNA atoms become covalently linked to resident DNA, the incorporated material does not have the donor DNA transforming activity.

Bacteriolysis↗

PubMed1972

Molecular events accompanying the fixation of genetic information in Haemophilus heterospecific transformation.

Heterospecific transformation between Haemophilus influenzae and H. parainfluenzae was investigated by isopycnic analysis of deoxyribonucleic acid (DNA) extracts of (3)H-labeled transforming cells that had been exposed to (32)P-labeled, heavy transforming DNA. The density distribution of genetic markers from the resident DNA and from the donor DNA was determined by transformation assay of fractions from CsCl gradients, both species being used as recipients. About 50% of the (32)P atoms in H. parainfluenzae donor DNA taken up by H. influenzae cells were transferred to resident DNA, and only a small amount of the label was lost under conditions of little cell growth. There was less transfer in the reciprocal cross, and almost half of the donor label was lost. In both crosses, the transferred donor material transformed for the donor marker considerably more efficiently when assayed on the donor species than on the recipient species, indicating that at least some of the associated (32)P atoms are contained in relatively long stretches of donor DNA. When the transformed cultures were incubated under growth conditions, the donor marker associated with recipient DNA transformed the donor species with progressively decreasing efficiency. The data indicate that the low heterospecific transformation between H. influenzae and H. parainfluenzae may be due partly to events occurring before association of donor and resident DNA but results mostly from events that occur after the association of the two DNA preparations.

Bacteriolysis↗

PubMedDate not supplied

Prospects for genetic engineering.

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Genetic Engineering↗