Formation of ion channels by Colicin B in planar lipid bilayers

Abstract

The gene for the antibacterial peptide colicin B was cloned and transformed into a host background where it was constitutively overexpressed. The purified gene product was biologically active and formed voltage-dependent, ion-conducting channels in planar phospholipid bilayers composed of asolectin. Colicin B channels exhibited two distinct unitary conductance levels, and a slight preference for Na+ over Cl-. Kinetic analysis of the voltage-driven opening and closing of colicin channels revealed the existence of at least two conducting states and two nonconducting states of the protein. Both the ion selectivity and the kinetics of colicin B channels were highly dependent on pH. Excess colicin protein was readily removed from the system by perfusing the bilayer, but open channels could be washed out only after they were allowed to close. A monospecific polyclonal antiserum generated against electrophoretically purified colicin B eliminated both the biological and in vitro activity of the protein. Membrane-associated channels, whether open or closed, remained functionally unaffected by the presence of the antiserum. Taken together, our results suggest that the voltage-independent binding of colicin B to the membrane is the rate-limiting step for the formation of ion channels, and that this process is accompanied by a major conformational rearrangement of the protein.

Calorimetric Investigations of the Structural Stability and Interactions of Colicin B Domains in Aqueous Solution and in the Presence of Phospholipid Bilayers

“…The decreased thermal stability of ColB at lowered pH correlates with the previously observed requirement of acidic conditions for optimal channel and molten globule formation of ColB (30), increased membrane association of ColA (31,32), augmented membrane-insertion rate of ColIa (33,34), and pH-sensitive conformational changes of ColE1 in the presence of membranes (35). The unfolding profile of diphtheria toxin has been published previously (36).…”

The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin

“…Conjugal transfer of pCP13 and pRK415 derivatives to Bordetella strains was accomplished by triparental matings with E. coli DH5␣ as the plasmid donor strain and DH5␣ (pRK2013) as the source of mobilization functions. Suspensions of donor, helper, and recipient strains in SS plus 10 mM MgSO 4 were combined at an estimated 1:1:3 cell ratio, spotted onto Luria-Bertani or blood agar plates (for B. bronchiseptica recipients) or Bordet-Gengou agar plates (for B. pertussis recipients), and incubated at 37ЊC for 3 to 5 h. Bacteria were streaked from mating spots directly onto agar plates containing the appropriate selective antibiotics and crude colicin B (3,12). Plates were incubated at 37ЊC for 1 to 4 days, at which time transconjugant colonies were apparent.…”

Isolation and characterization of Bordetella bronchiseptica mutants deficient in siderophore activity

“…After 0.5 ml of the cell mixture was spread onto LB agar containing 20 mM MgSO4 (44), the cells were dried onto the surface of the medium and incubated for 6 h at 370C. The cells were harvested and plated onto LB agar containing kanamycin and nalidixic acid and also spread with 30 to 50 pl of a colicin B-enriched bacterial lysate (approximately 5 mg of total protein per ml) to additionally discourage growth of E. coli donors (9). Transconjugants were patched onto CAS agar for assessment of siderophore activity.…”