Recombination During Transformation in Hemophilus Influenzae

Abstract

In bacterial transformation, the irreversible uptake by a cell population of DNA extracted from a variant strain is followed by integration and expression of the variant "information" carried by the transforming DNA. Little is known of the details or mechanism of transformation after uptake of the DNA by a cell, i.e., of the process of integration which results in a permanent hereditary change. One must conclude from studies on linked transforming factors, however, that some process of recombination is involved.1 3 The studies reported in this paper were designed to determine the time and some of the conditions for recombination during transformation.Goodgal and Herriott have shown that a population of cells which has irreversibly taken up transforming DNA releases a large part of this activity when lysed shortly after uptake.4 In the experiments to be described, the intracellular fate of transforming DNA has been followed quantitatively by this technique in the case of two linked markers located on the same DNA molecule. Goodgal (3) has shown that in Hemophilus influenzae the two factors responsible for resistance or sensitivity to the antibiotics streptomycin and cathomycin (Novobiocin) are linked. When DNA is extracted from donor cells resistant to both cathomycin and streptomycin and is used to transform cells sensitive to both antibiotics, some 20 per cent of the resistant clones are endowed genetically with resistance to both antibiotics. The remainder of the transformations are to streptomycin resistance alone and to cathomycin resistance alone. The degree of linkage obtained depends to some extent on the method of preparation of the DNA and the conditions of transformation, but for material treated in the same way, the fraction of linked transformations is the same. The failure to retain complete linkage may be attributed to loss or inactivation of one or the other marker in the process of extracting the DNA from the donor cells, in the process of transformation itself, or in both processes.One can transform to streptomycin or cathomycin sensitivity as well as to resistance. The frequency of transformation to sensitivity is the same as to resistance, and the same linkage relationships obtain.3 Resistance and sensitivity thus may be considered as alleles of the same locus, the frequency of genetic incorporation of either or both factors being a function of the spatial relationships between the loci and not of the particular forms utilized in the test.3 When, in the course of transformation, both streptomycin and cathomycin loci are genetically incorporated, linked transformations are obtained. When only one locus is genetically incorporated, transformations result 'which are recombinant with respect to the two markers in question, one marker supplied by the donor population and the other by the recipient population. This is the restricted manner in which the term recombination will be used in this paper, i.e., recombination between two linked loci on a single DNA molecule. 505

On the Nature of Recombinants Formed during Transformation in Hemophilus influenzae

“…It is believed that this model of the transformation process explains an apparent paradox which was observed earlier when it was found that the synthesis of transforming DNA was initiated relatively soon after DNA uptake, approximately 15 min, whereas replication of transforming cells did not occur until some two generations later (1). A one generation lag was ascribed to the fact that H. influenzae appears as chains of two in a growing culture, whereas the other generation lag could possibly be due to the need for an additional round of replication.…”

Directional transport and integration of donor DNA in Haemophilus influenzae transformation.

“…4B, (8,9) and, through unknown intermediate steps, only a single strand integrated into the chromosome (10,11).…”

“…The internal singlestranded DNA then undergoes homologous integration over a period of about 15 min (7). In gram-negative Haemophilus influenzae, cells take up double-stranded DNA (8,9), and there is no detectable single-stranded DNA intermediate, although only a single strand is thought to integrate (10,11). We have demonstrated that donor DNA is first "captured" within outermembrane extensions on the cell surface (12) (transformasomes), where it is in a protected state, untouched by external DNase or internal restriction and modification enzymes (13).…”

Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum

“…DNA eclipse is observed in the grampositive organisms Streptococcus pneumoniae and Bacillus subtilis (10,11,35), in which it has been demonstrated that the internalized donor DNA is single stranded after uptake (14,21). This phenomenon is not observed, however, in the gram-negative organisms Haemophilus influenzae and N. gonorrhoeae (3,31,36). In the case of H. influenzae, it has been shown that double-stranded donor DNA is first taken into membrane-bound vesicles called transformasomes, where it is resistant to externally added DNase (17,18).…”