Anion channel forming activity from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

Abstract

The plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense secretes an anion channel forming activity (CFA) into the culture field. The CFA inserts spontaneously into planar lipid membranes when culture fluid of this species is added to the aqueous phase of the bilayer chamber. The channels formed are highly anion selective. The conductance decreases for larger anions (Cl- greater than SCN- greater than SO2-(4] and is practically zero for gluconate. The channels show a unique voltage dependence: (i) The single-channel conductance increases linearly with voltage up to 200 mV saturating at 250 mV with 25 +/- 1 pS (300 mM KCl). The channel is closed at negative voltage relative to the side of insertion (diode-type I-V curve). (ii) The average number of open channels also increases with voltage. The Poisson distribution of channel numbers indicates independent opening of the channels. Channel activity can be abolished by protease treatment of the planar bilayer. The channels can be blocked by indanyloxyacetic acid (IAA-94) and by pH greater than 10. The CFA was purified yielding one major band on the SDS gel with a relative molecular mass of 65,000. The putative involvement of the CFA in the toxicity of this plant pathogen is discussed and compared to other toxins like colicins and to the diptheria toxin group.