Comparative analysis of Bacillus weihenstephanensis KBAB4 spores obtained at different temperatures

The impact of Bacillus weihenstephanensis KBAB4 sporulation temperature history was assessed on spore heat resistance, germination and outgrowth capacity at a temperature range from 7 to 30 degrees C. Sporulation rate and efficiency decreased at low temperature, as cells sporulated at 12, 20 and 30 degrees C with approximately 99% efficiency, whereas at 7 degrees C and 10 degrees C, a maximum 15% of sporulation was reached. Spores formed at 30 degrees C showed the highest wet heat resistance at 95 degrees C, with spores formed at 7 and 10 degrees C displaying only survival of 15min exposure at 70 degrees C, indicating their low level heat resistance. RT-PCR analysis revealed expression of sporulation sigma factor sigG, and germinant receptor operons gerI, gerK, gerL, gerR, gerS, and (plasmid-located) gerS2 to be activated in all sporulation conditions tested. Subsequent germination assays revealed a combination of inosine and L-Alanine to be very efficient, triggering over 99% of the spores to germinate, with spores obtained at 30 degrees C showing the highest germination rates (99%). Notably, spores obtained at 12, 20 and 30 degrees C, germinated at all tested temperatures, showing >70% spore germination even at temperatures as low as 5 degrees C. Less than 5% of spores obtained at 7 and 10 degrees C showed a germination response. Furthermore, spores produced at 12, 20 and 30 degrees C showed similar outgrowth effiency at these temperatures, indicating that low temperature sporulation history does not improve low temperature outgrowth performance. Insights obtained in sporulation and germination behaviour of B. weihenstephanensis KBAB4, in combination with the availability of its genome sequence, may contribute to our understanding of the behaviour of psychrotolerant spoilage and pathogenic Bacilli.