| Summary: | The thermotolerant representative of the <i>Bacillus cereus</i> group, <i>Bacillus cytotoxicus</i>, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of <i>B. cytotoxicus</i> has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two <i>B. cytotoxicus</i> strains, E8.1 and E28.3. The complementation of the <i>cytK-1</i> KO mutation was implemented in a mutant strain lacking in the <i>cytK-1</i> gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the <i>cytK-1</i> KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that <i>cytK-1</i> KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in <i>B. cytotoxicus.</i>
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