Mechanism of additive genetic transformation in Haemophilus influenzae

Abstract

Transforming deoxyribonucleic acid (DNA) preparations from Haemophilus influenzae Rd strains carrying a chromosomally integrated, conjugative, antibiotic resistance transfer (R) plasmid were exposed to ultraviolet radiation and then assayed for antibiotic resistance transfer on sensitive wild-type Rd competent suspensions and on similar suspensions of a uvr-1 mutant unable to excise pyrimidine dimers. No host cell reactivation of resistance transfer (DNA repair) was observed. Parallel experiments with ethanol-precipitated, heated, free R plasmid DNA preparations gave much higher survival when assayed on the wildtype strain compared to the survival on the uvr-1 strain. These observations indicate that additive genetic transformation (in this case, the addition of the integrated R plasmid to the recipient genome) involves single-strand insertion. Additive genetic transformation describes the addition of long segments of donor DNA to the recipient cell genome through the process of DNA uptake by genetically competent cells. It was first reported for Haemophilus influenzae by Bendler (4), who studied the transfer of ampicillin resistance from some hospital isolates to strain Rd. He speculated that the donor strains could have possessed a chromosomally integrated plasmid. It was recently demonstrated

REPAIR OF ULTRAVIOLET‐IRRADIATED TRANSFORMING DNA IN A recA MUTANT OF Haemophilus influenzae

“…The absence of an effect by recombination repair is explained by the inability of the rec system to transfer these long nonhomologous DNA segments efficiently (Stuy and Walter, 1981). Excision repair would be expected to eliminate a long nonhomologous marker upon reaching it, since in our hypothesis the mechanism of marker addition involves looping out of the nonhomologous region as a single strand (Stuy, 1980;Stuy and Walter, 1981;Walter and Stuy, 1982a). In this scheme excision repair of proximal lesions would contribute to lower marker survival, just as is postulated for point mutation markers.…”

“…We have recently described the phenomenon of additive transformation (Stuy and Walter, 1981;Stuy, 1980). In this phenomenon the donor DNA carried long nonhomologous plasmid-derived DNA sequences (conferring resistance to either chloramphenicol or tetracycline) which had been spliced into the left Eco RI-sensitive site in their HP1 prophages.…”

ULTRAVIOLET SENSITIVITY OF THE ADDITION, DELETION and REPLACEMENT OF LONG NONHOMOLOGOUS DNA SEGMENTS BY GENETIC TRANSFORMATION OF HAEMOPHILUS INFLUENZAE

“…we have proposed a model in which the insert-flanking regions form double helical structures with the remaining recipient strand and in which the insert loops out as a single strand. The transfer of chromosomally integrated conjugative R plasmids by transformation (Stuy, 1980a) and the transfection by DNA from HPI lysogens (Stuy, 1980b) have been explained by this model. We speculated that the looped-out single strand inserts are susceptible to endonucleases.…”

Homology and Repair of UV-Irradiated Plasmid DNA in Haemophilus influenzae

“…Survival of the tetracycline marker of p2265 probably occurred by recombination of the damaged plasmid with the undamaged, partly homologous, plasmid pamp90383 of the recipient cell. The lack of excision repair might then be attributed to the vulnerability of a single-strand loop in a recombination intermediate, as suggested by Stuy (19). However, the novobiocin transformation of irradiated pNovl, which is affected by excision, also involves recombination with chromosomal DNA that is only partly homologous with the plasmid.…”

“…Figure 1A also shows that the inactivation of the ability of irradiated excision-proficient rec-2 cells containing p2265 to donate the plasmid in conjugation was the same as the inactivation of the transforming ability of p2265 irradiated outside the cell. The fact that the irradiated plasmid DNA is apparently also not subject to excision repair although there is no partly homologous DNA inside the cell to form a recombination intermediate is evidence against the Stuy (19) explanation for lack of excision repair. Figure 2 shows that transformation to ampicillin resistance by pNovl in Rec-strains was more resistant than in strains Rd and BC200.…”