With the recent success of the heterologous expression of mycobacterial antigens in corynebacteria, in addition to the importance of these bacteria in biotechnology and medicine, a better understanding of the structure of their cell envelopes was needed. A combination of molecular compositional analysis, ultrastructural appearance and freeze-etch electron microscopy study was used to arrive at a chemical model, unique to corynebacteria but consistent with their phylogenetic relatedness to mycobacteria and other members of the distinctive suprageneric actinomycete taxon. Transmission electron microscopy and chemical analyses showed that the cell envelopes of the representative strains of corynebacteria examined consisted of (i) an outer layer composed of polysaccharides (primarily a high-molecular-mass glucan and arabinomannans), proteins, which include the mycoloyltransferase PS1, and lipids ; (ii) a cell wall glycan core of peptidoglycan-arabinogalactan which may contain other sugar residues and was usually esterified by corynomycolic acids ; and (iii) a typical plasma membrane bilayer. Freeze-etch electron microscopy showed that most corynomycolate-containing strains exhibited a main fracture plane in their cell wall and contained low-molecular-mass porins, while the fracture occurred within the plasma membrane of strains devoid of both corynomycolate and pore-forming proteins. Importantly, in most strains, the amount of cell wall-linked corynomycolates was not sufficient to cover the bacterial surface ; interestingly, the occurrence of a cell wall fracture plane correlated with the amount of non-covalently bound lipids of the strains. Furthermore, these lipids were shown to spontaneously form liposomes, indicating that they may participate in a bilayer structure. Altogether, the data suggested that the cell wall permeability barrier in corynebacteria involved both covalently linked corynomycolates and non-covalently bound lipids of their cell envelopes.

Protective antigen (PA) of the tripartite anthrax toxin binds to a cell surface receptor and mediates the transport of two enzymatic components, edema factor and lethal factor, into the cytosol of host cells. Here recombinant PA(63) from Bacillus anthracis was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels. The heptameric PA(63)-channel contains a binding site for 4-aminoquinolones, which block ion transport through PA in vitro. This result allowed a detailed investigation of ligand binding and the stability constants for the binding of chloroquine, fluphenazine, and quinacrine to the binding site inside the PA(63)-channel were determined using titration experiments. Open PA(63)-channels exhibit 1/f noise in the frequency range between 1 and 100 Hz, whereas the spectral density of the ligand-induced current noise was of Lorentzian type. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of ligand binding. The on-rate constants of ligand binding were between 10(6) and 10(8) M(-1) s(-1) and were dependent on the ionic strength of the aqueous phase, sidedness of ligand addition, as well as the orientation and intensity of the applied electric field. The off-rates varied between approximately 10 s(-1) and 2600 s(-1) and depended mainly on the structure of the ligand.

The anthrax toxin complex consists of three different molecules, protective antigen (PA), lethal factor (LF), and edema factor (EF). The activated form of PA, PA63, forms heptamers that insert at low pH in biological membranes forming ion channels and that are necessary to translocate EF and LF in the cell cytosol. LF and EF are intracellular active enzymes that inhibit the host immune system promoting bacterial outgrowth. Here, PA63 was reconstituted into artificial lipid bilayer membranes and formed ion-permeable channels. The heptameric PA63 channel contains a binding site for LF on the cis side of the channel. Full-size LF was found to block the PA63 channel in a dose- and ionic-strength-dependent way with half-saturation constants in the nanomolar concentration range. The binding curves suggest a 1:1 relationship between (PA63)7 and bound LF that blocks the channel. The presence of a His6 tag at the N-terminal end of LF strongly increases the affinity of LF toward the PA63 channel, indicating that the interaction between LF and the PA63 channel occurs at the N terminus of the enzyme. The LF-mediated block of the PA63-induced membrane conductance is highly asymmetric with respect to the sign of the applied transmembrane potential. The result suggested that the PA63 heptamers contain a high-affinity binding site for LF inside domain 1 or the channel vestibule and that the binding is ionic-strength-dependent.

No abstract

The binding component (Vip1Ac) of the ADP-ribosylating vegetative insecticidal protein (Vip) of Bacillus thuringiensis HD201 was isolated from the supernatant of cell cultures. Vip1Ac protein solubilized at room temperature ran as oligomers on SDS-PAGE. These oligomers were not resistant to heating. Mass spectroscopic analysis of this high molecular mass band identified it as Vip1Ac. The protein formed in artificial lipid bilayer membranes channels with two conductance states of about 350 and 700 pS in 1 M KCl. The channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero-current membrane potential measurements showed that the Vip1Ac channel has a slightly higher permeability for chloride than for potassium ions. Asymmetric addition of Vip1Ac to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. The functional role of Vip1Ac and its relationship to other ADP-ribosylating toxins are discussed.