Deoxyribonucleic acid-binding properties and membrane protein composition of a competence-deficient mutant of Haemophilus influenzae

Abstract

A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry ofheterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.

Uptake of heterologous DNA by Haemophilus influenzae

“…influenzae DNA in competition with H. influenzae DNA. The DNA binding in the low-pH system appears to involve different receptor sites (16,21). Both receptor mechanisms are dependent on competence induction, since noncompetent cells do not bind appreciable amounts of either native or denatured DNAs in the neutralor low-pH systems.…”

Role of the electrochemical proton gradient in genetic transformation of Haemophilus influenzae

“…Below pH 5.5 another transformation system becomes operational. This system is not specific for homologous DNA and is also able to translocate single-stranded DNA (14). In this paper only the properties of the first system have been studied.…”

“…A surprising observation made in this paper is the role of the proton motive force in the development of competence. In the initial phase of competence development, the proton motive force remains constant, and competence-specific proteins and special cell wall structures (2,3,10,14,26) are synthesized. In the second phase, the proton motive force and especially the electrical potential increases, and this increase leads to an increase of the degree of competence.…”

“…The medium pH was 8.0. The times (in minutes) at which samples were taken during competence development are indicated on the figure. the generation on the cell surface of membrane extensions (2,3,10,14,26). In conditions in which cells lose their ability to take up DNA, the cells shed these structures as free vesicles (3), suggesting that loss of the competent state of the cell is an irreversible process.…”