Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation.

Abstract

The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected stateresistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.Haemophilus influenzae is a Gram-negative bacterium that can be induced to high levels of competence for transformation under conditions of slowed growth (1). Competent cells efficiently take up homologous donor DNA from the medium and integrate it into the chromosome to yield transformation rates of 1-3% for various genetic markers (for reviews, see refs. 2-5). Although various changes in outer and inner membrane composition have been observed during competence induction (6-9), little is known about the actual biochemistry and mechanics of DNA uptake. Furthermore, it is puzzling how donor DNA, which remains duplex after uptake, escapes restriction and degradation by cellular nucleases while awaiting integration. Recently, Kahn et al. (10) have suggested that specialized membranous extensions on the surface of competent cells might be responsible for capturing and protecting the donor DNA after uptake. By comparative morphological studies, these membranous structures were shown to appear on the surface of H. influenzae and Haemophilus parainfluenzae as they become competent (10, 11). The structures extend "35 nm from the cell, average 20 nm in diameter, are located at points of fusion between the inner and outer membrane, and are composed of a lipid bilayer-predominantly outer membrane proteins (10, 12) and lipopolysaccharide. Pore structures with an opening of 5 nm are localized at points of attachment of the membranous extension to the outer membrane. (An electron micrograph of this structure is seen in Fig. 5 KW22 and KW23 (13), were grown in 2.5% heart infusion broth (Difco), supplemented with 10 ,4g of hemin (Eastman) and 2 ,ug of NAD (Sigma) per ml. Competent cells, routinely able to transform at 3% for chromosomal markers, were prepared in MIV media as described (1). Plasmids pPUP3 (pBR322 containing an 11-basepair uptake site in the Pst I site) (14) and pCML6 were isolated from Escherichia coli strains HB101 (r-, m-, recA) and MM294 (endA hsdR) by the procedure of Birnboim and Doly (15)

Physiological characterization of natural transformation in Acinetobacter calcoaceticus

“…DNA transport across the cytoplasmic membrane in natural transformation occurs in a single-stranded fashion in Bacillus (Piechowska & Fox, 1971 ;DavidoffAbelson & Dubnau, 1973), and Streptococcus (Morrison & Guild, 1973) and possibly also in Haemophilus (Kahn et al, 1983). We have shown here that this is also the case in Acinetobacter.…”

DNA-binding proteins in cells and membrane blebs of Neisseria gonorrhoeae

“…This specific interaction between the gonococcal transformation uptake sequence and a bleb-associated protein further suggests certain similarities between gonococcal blebs and Haemophilus transformasomes (17). Reportedly, transformasomes sequester and later internalize exogenous DNA during transformation (1,17).…”

Generalized gene transfer by virus-like particles from marine bacteria

“…It may stabilize VLP adhesion at a certain receptor site of the recipient cell to promote further gene transfer. Some analogous features of the substance might be membranous materials found during the transformation process in Bacillus (te Riele & Venema 1984), Haemophilus (Kahn et al 1983) and Neissena (Doward et al 1989). …”

“…The gene transfer agent (GTA) of Rhodopseudomonas is reported to be encapsulated (Wall et al 1975, Solioz & Marrs 1977. Transformasomes from Haemophilus are reported to produce membranous material which is capable of encapsulating exogenous DNA in the surroundings of the cell (Kahn et al 1983). However nothing is known about the relationship between those reported membranous encapsulated particles and the particles studied in this study.…”