Genetic and functional analysis of the cytK family of genes in Bacillus cereus.
CytK is a pore-forming toxin of Bacillus cereus that has been linked to a case of necrotic enteritis. PCR products of the expected size were generated with cytK primers in 13 of 29 strains. Six strains were PCR-positive for the related gene hly-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 putative cytK genes 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 new cytK variant cytK-2, and refer to the original cytK as cytK-1. The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing. blast analysis using the cytK-2 gene sequences revealed very high homology with two cytK-2 sequences in the genomes of B. cereus strains 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 from B. cereus 1230-88 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 of B. cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.
Fagerlund
Annette
A
Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway.
Ween
Ola
O
Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway.
Lund
Terje
T
Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway.
Hardy
Simon P
SP
School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK.
Granum
Per E
PE
Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway.
D000595:Amino Acid Sequence; D000818:Animals; D001409:Bacillus cereus / Q000235:genetics* / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity; D001483:Base Sequence; D018938:Caco-2 Cells; D002522:Chlorocebus aethiops; D003603:Cytotoxins / Q000235:genetics* / Q000633:toxicity; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells
D001427:Bacterial Toxins; D003603:Cytotoxins; D004269:DNA, Bacterial; D008051:Lipid Bilayers