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

Abstract

DNA transport and integration in Haemophilus influenzae transformation was studied with a plasmid clone of homologous DNA (pCML6). Our results indicate that: (i) donor DNA enters ialized membranous extensions on the cell surface, which we have termed "transformasomes"; (ii) linear DNA undergoes degradation upon exiting transformasomes; and (iii) DNA without a free end remains within transformasomes and is not degraded. By comparing the fate of label from uniformly labeled versus middle-labeled DNA, it appears that donor DNA undergoes degradation from an end prior to recombining with the chromosome. Using donor DNA with covalently closed termini (hairpin ends) prevents efficient exit from transformasomes. When one hairpin is removed, exit of donor DNA is shown to be directional from the free end, with preferential homologous integration of the 3' strand from that end.The transport and efficient integration of donor DNA is a fundamental process that has evolved in several transformable bacteria (for reviews, see refs. 1-4). In gram-positive Streptococcus pneumoniae, double-stranded DNA is bound to cells, and a membrane-bound endonuclease degrades one strand while the other strand is internalized (2,5,6 (14) was grown in heart infusion broth (Difco) sup-plemented with hemin at 10 j&g/ml and NAD' at 2 ,zg/ml (Sigma). Escherichia coli strains HB101 (hsdS recA), and MM294 (endA hsdR) containing plasmids were grown in L broth (15). Plasmids were prepared by the alkaline procedure of Birnboim and Doly (16) and further purified by one or two CsCl/ethidium bromide centrifugations (17). Plasmid pCML6 contains a 10-kilobase (kb) insert of H. influenzae Rd DNA in the BamHI site of a pBR322 derivative (13, 18).Preparation of 3P-Labeled DNA. DNA was nick-translated with Micrococcus lueus polymerase, and nicks were sealed with T4 ligase to a specific activity of 2 x 1 cpm/pyg as described 1 Ci = 37 GBq). After 45 min at 37TC, ATP was added to 1 mM, and incubation was continued an additional 2 min at 37TC, after which the kinase was heat inactivated (630C for 5 min) (see structure II in Fig. 4C). The volume was then increased to 40 p1 by addition of buffer containing 1 mM ATP and 3 units (Weiss) of T4 ligase (New England BioLabs). Ligation to form circles and multimers proceeded at 230C for 2 hr and was terminated by heat inactivation. Monomeric, linear middle-labeled DNA was obtained by cleaving the above multimers with 16 units of Cla I (Boehringer Mannheim) at 37C for 30 min (Fig. 4C, structure EI). For construction of hairpins, 10 ,ug of poly(dGdC) (about 400 bases, Boehringer Mannheim) was heat-denatured, chilled at -500C for 3 min, and phosphorylated with kinase (New England BioLabs). Hairpin ends (5 ug = 90-fold excess) were ligated to middle-labeled DNA (2 pug) in the presence of Cla I by addition of 3 units of ligase (1 hr at 230C) in the above buffer. Gaps and nicks were sealed with 0.7 unit of polymerase I large fragment (New England BioLabs) in the presence of 250 ,M of all four dNTPs (230C for 15 min) and 0...

Plasmid establishment in competent Haemophilus influenzae occurs by illegitimate transformation

“…Second, previous work from this laboratory showed that a free end is required for efficient translocation out of transformasomes; CCCs remain in the protected state (2). From the data in Table 2, however, it is obvious that CCC molecules are able to carry out both marker rescue and plasmid establishment as efficiently as topological forms which do have a free end.…”

“…First, it is known that extensive degradation of donor DNA ordinarily accompanies transformation. One strand of an entering molecule is completely degraded and the other stand is at least partially degraded during entry into the cytoplasm (2,20,22). Yet recovery of plasmids from cells transformed by linear DNA reveals that no information is lost from the ends of linear molecules during plasmid establishment.…”

“…In contrast, the development of natural competence involves the induction of a number of cellular functions for the active uptake, translocation, and integration of exogenous DNA. Donor DNA entering these cells is subject to extensive degradation (2,25), which results in a linear single strand of less than unit length entering the cytoplasm (22 …”

Evolutionary stability of DNA uptake signal sequences in the Pasteurellaceae

“…But H. influenzae and Neisseria spp. transport double-stranded DNA across their outer membranes, and in H. influenzae this transport is known to not require a free end (i.e., closed circular molecules are efficiently transported without nicking or cleavage) (40). At the scale of the cell surface, double-stranded DNA is a stiff and very hydrophilic rod [persistence length Ϸ50 nm (41)], and transport across the outer membrane likely requires both tight binding to a transport protein and the ability to create a kink in the DNA rod, perhaps by inducing local strand separation.…”

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…Transformation probably involves binding of DNA to the cell surface and the formation of linear DNA in the periplasm that is then transported into the cytoplasm and recombined with the host genome. In H. influenzae there is evidence that double-stranded DNA is first taken up into structures known as "transformasomes" and that a single strand of DNA is transported across the inner membrane into the cytoplasm in a 3' to 5' direction, while the other strand is degraded (Concino and Goodgal, 1982;Barany et al, 1983;Dubnau, 1999).…”