Comparative analysis of Bacillus weihenstephanensis

22 The impact of Bacillus weihenstephanensis KBAB4 sporulation temperature 23 history was assessed on spore heat resistance, germination and outgrowth capacity at a 24 temperature range from 7 to 30°C. Sporulation rate and efficiency decreased at low 25 temperature, as cells sporulated at 12, 20 and 30°C with approximately 99% 26 efficiency, whereas at 7°C and 10°C, a maximum 15% of sporulation was reached. 27 Spores formed at 30°C showed the highest wet heat resistance at 95°C, with spores 28 formed at 7 and 10°C displaying only survival of 15 min exposure at 70oC, indicating 29 their low level heat resistance. RT-PCR analysis revealed expression of sporulation 30 sigma factor sigG, and germinant receptor operons gerI, gerK, gerL, gerR, gerS, and 31 (plasmid-located) gerS2 to be activated in all sporulation conditions tested. 32 Subsequent germination assays revealed a combination of inosine and L-Alanine to be 33 very efficient, triggering over 99% of the spores to germinate, with spores obtained at 34 30°C showing the highest germination rates (99%). Notably, spores obtained at 12, 20 35 and 30°C, germinated at all tested temperatures, showing >70% spore germination 36 even at temperatures as low as 5°C. Less than 5% of spores obtained at 7 and 10°C 37 showed a germination response. Furthermore, spores produced at 12, 20 and 30°C 38 showed similar outgrowth effiency at these temperatures, indicating that low 39 temperature sporulation history does not improve low temperature outgrowth 40 performance. Insights obtained in sporulation and germination behaviour of B. 41 weihenstephanensis KBAB4, in combination with the availability of its genome 42 sequence, may contribute to our understanding of the behaviour of psychrotolerant 43 spoilage and pathogenic Bacilli. 44 45