The entomopathogenic Bacillus thuringiensis serovar israelensis displays peculiar conjugative transfer capabilities, accounted for by the large conjugative plasmid pXO16 (350 kb). The efficient and fast conjugative transfers are accompanied by a macroscopic aggregation of bacterial partners. Moreover, pXO16 has proven capable of effective mobilization and the retro-transfer of both mobilizable and 'non-mobilizable' plasmids. In this work, the aggregation phenomenon is shown to promote pXO16 transfer while not being mandatory for transfer. Transfer of pXO16 to B. thuringiensis recipient strains that do not display aggregation is observed as well, hence enlarging the previously defined host range. The use of variant calling analysis of transconjugants allowed for observation of up to 791 kb chromosomal regions mobilization. Previous analysis of pXO16 did not reveal any Type IV Secretion System (T4SS) homologs, which suggested the presence of an unusual conjugative system. A FtsK/SpOIIIE ATPase gene proved here to be necessary for conjugative transfer. Additionally, the analysis of natural restriction-modification systems in both conjugative partners gave credit to a ssDNA transfer mechanism. A 'transfer israelensis plasmid' (tip) region containing this ATPase gene was shown to code for other potential T4SS proteins, illustrating a conjugative system distantly related to the other known Gram-positive T4SSs.
The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, 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 B. cytotoxicus 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 B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 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 B. cytotoxicus.
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