Vegetative Insecticidal Protein (Vip1Ac) of Bacillus thuringiensis HD201:  Evidence for Oligomer and Channel Formation

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.

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

“…It also shares 33 to 38% identity with the C. spiroforme toxin, the B. anthracis protective antigen, and toxin B of C. difficile at amino acids 142 to 569 (23,26,29). Vip2 shares Ͼ30% sequence identity with the clos-…”

Clostridium botulinum C2 Toxin

“…To localize the binding site of chloroquine, which is presumably the same as for C2I, sequence comparisons of binding components C2II, PA, iota b, and VIP1 (Ac, B. thuringiensis; Aa, B. cereus) of the binary toxins C2, anthrax, iota, and the vegetative insecticidal proteins, respectively, were performed. The sequences of these binding components are significantly homologous, but they differ in binding affinity for chloroquine and related compounds (12,29,40,41). Chloroquine binds to the PA channel with much higher affinity than to C2II, but it has a low affinity for binding to iota b and does not bind to VIP1.…”

“…Heptamerization and Pore Formation Are Influenced by Mutations E399A and D426A-After proteolytic activation, C2II, PA of anthrax toxin, iota b of iota toxin, and the VIPs form heptamers (12,33,47). These heptamers are essential for binding and component-induced channel formation in biological and artificial membranes (12,28,29,41,48).…”

“…These heptamers are essential for binding and component-induced channel formation in biological and artificial membranes (12,28,29,41,48). Here the influence of the mutation of several different amino acids on heptamerization as a prerequisite for channel formation was investigated.…”

“…There C2I exerts its catalytic activity (1)(2)(3). Other members of the family of binary toxins are the iota toxin of Clostridium perfringens (4,5), ADP-ribosyltransferase of Clostridium difficile (6 -8), Clostridium spiroforme toxin (9,10), the vegetative insecticidal proteins (VIPs) of Bacillus cereus and Bacillus thuringiensis (11,12), and the anthrax toxin of Bacillus anthracis (13,14).…”