Genetic and functional analysis of the cytK family of genes in Bacillus cereus

Abstract

CytK is a pore-forming toxin ofBacillus cereusthat has been linked to a case of necrotic enteritis. PCR products of the expected size were generated withcytKprimers in 13 of 29 strains. Six strains were PCR-positive for the related genehly-II, which encodes haemolysin II, a protein that is 37 % identical to the original CytK. Five of the strains were positive for both genes. The DNA sequences of putativecytKgenes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK. The authors have designated this newcytKvariantcytK-2, and refer to the originalcytKascytK-1. The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing.blastanalysis using thecytK-2gene sequences revealed very high homology with twocytK-2sequences in the genomes ofB. cereusstrains ATCC 14579 and ATCC 10987. The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins. The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1. Both native and recombinant CytK-2 proteins fromB. cereus1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1. It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains ofB. cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.