An appropriate evolutionary adaptation of germinant receptor expression/regulation is thus crucial to allow the cyclic transition between sporulation and germination upon environmental changes. In the ABT-263 in vivo construction of the complementation mutants in our study, certain precautions were therefore taken to avoid extensive over-expression of the complemented germinant receptor genes. By including some of the flanking regions of the gerAA, gerAB and gerAC fragment in the complementation plasmid, we wanted to maintain the native regulatory elements
of this locus. In addition, a shuttle-see more vector with an expected low or moderate copy number was sought as a basis for the complementation plasmid. To our knowledge, there is no shuttle-vector available for B. licheniformis where the copy number is demonstrated to be low or moderate. However, Arantes and Lereclus
 have constructed the pHT315 E. coli/B. thuringiensis shuttle-vector, with a copy number of ~ 15 per equivalent B. thuringiensis chromosome. This vector Selleckchem Defactinib has successfully been used in germinant receptor complementation studies in B. megaterium , and was thus considered as a reasonable choice for B. licheniformis. Despite that this vector has shown to be stably maintained in B. thuringiensis and B. megaterium without a selective pressure [52, 54], the antibiotic erythromycin had to be included to ensure persistence of the complementation plasmid during sporulation of the B. licheniformis complementation mutant NVH-1311. This could be due to a different segregation stability of the vector in B. licheniformis. Another possibility is that there is a potential Sulfite dehydrogenase elevated risk of plasmid curing due to sporulation at a high temperature. Sporulation of B. licheniformis MW3, NVH-1307 and NVH-1311 were performed at 50 °C since a pilot study showed that sporulation at this temperature
was faster, yielded more stable spores (less spontaneous germination) and a higher percentage of phase bright spores (results not shown). Disruption of gerAA abolish L-alanine and casein hydrolysate induced germination Decrease in absorbance at ~ 600 nm (A600) is used as a convenient method to monitor and compare germination of different spore populations [55, 56]. A fall in absorbance reflects a change in the refractive index (light scattering) of the multiple individual spores in a suspension, associated with germination events such as the excretion of spore’s depot of Ca2+-DPA, followed by water influx, cortex degradation and core swelling [51, 56–59]. Figure 1 shows a representative experiment where different strains of heat activated (65 °C 20 min) spores (in Phosphate buffer) are supplemented with the germinant L-alanine. At these conditions, a clear change in absorbance was observed for spores of wild type (MW3) and wild type complementation mutant (NVH-1311) supplemented with L-alanine. Less than a 5%/h decrease in absorbance was observed for spores of the disruption mutant (NVH-1307).