The Copy Number of the spoVA2mob Operon Determines Pressure Resistance of Bacillus Endospores

The spoVA(2mob) operon confers heat resistance to Bacillus spp., and the resistance correlates to the copy number of the operon. Bacillus endospores also exhibit a strong variation in resistance to pressure, but the underlying mechanisms of endospore resistance to pressure are not fully understood. We determined the effects of multiple spoVA(2mob) operons on high-pressure resistance in Bacillus endospores. The copy numbers of the spoVA(2mob) operon in 17 strains of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus velezensis, and Bacillus pumilus were determined via droplet digital PCR (ddPCR) and genome sequencing. These strains contained between 0 and 3 copies of the spoVA(2mob) operon; the quantification of the gene copy number by ddPCR was as accurate as whole-genome sequencing. We further tested the pressure resistance of 17 Bacillus endospores at 600 MPa and 80 degrees C. Strains with one or no spoVA(2mob) operon had significantly lower pressure resistance than strains with two or three copies of the operons (P < 0.001), indicating that redundant spoVA(2mob)operons in Bacillus contributed to higher pressure resistance of endospores. The copy number of the spoVA(2mob) operon was not related to the dipicolinic acid (DPA) content of endospores. Overall, the copy number of the spoVA(2mob) operon contributes to pressure resistance of Bacillus endospores. This improves our understanding of the pressure resistance mechanisms in Bacillus spp. and may inform the development of high-pressure sterilization in food processing. IMPORTANCE Bacillus spp. are considered pressure-resistant microorganisms, but the resistance mechanisms remain unknown. The spoVA(2mob) operon is a mobile genetic element, and it can transfer to pathogenic or spoilage organisms by horizontal gene transfer. Results in this study indicate that multiple copies of the spoVA(2mob) operon mediate high-pressure resistance of Bacillus endospores, and it might contribute to the identification of the source of pressure-resistant pathogens and spoilage organisms that may contaminate the food supply. The droplet digital PCR (ddPCR) system is well suited for analysis in some human diseases due to its high efficiency and capability to provide high precision; however, no relevant studies in food microbiology have been reported so far. This study demonstrates a novel application of ddPCR in food microbiology.