No apparent increase in Eltanexor in vivo number of phase dark spores was observed for spores of the deletion mutant (NVH-1307) supplemented with L-alanine, or the negative controls. Together with the absorbance measurements, this shows that the introduced disruption of the gerAA gene abolishes

the ability of B. licheniformis MW3 to use L-alanine as a germinant. The fact that the NVH-1311 complementation mutant showed a similar L-alanine triggered germination phenotype as the wild type spores, supports the hypothesis that an undisrupted copy of the gerAA, gerAB and gerAC genes, with flanking elements, are required for normal germination of B. licheniformis MW3 at these conditions. These findings were also supported by experiments performed with an alternative germination buffer; 50 mM Tris HCl pH 7.4 10 mM KCl (E. Klufterud, C. From; see more unpublished results). Figure 1 Germination of B. licheniformis with L-alanine. Germination is followed as a change in initial absorbance at 600 nm (A600) of phase bright spores in K-phosphate buffer

pH 7.2 at 30 °C after addition of 100 mM L-alanine. Complete germination (>99% phase Quisinostat ic50 dark spores as observed by phase contrast microscopy) was observed at ~40% of initial A600. The results shown are representative of experiments performed in duplicate on two individual spore batches repeated at least twice. Figure 2 Phase contrast images of B. licheniformis spores following L-alanine germination. Phase contrast images (100 x) showing B. licheniformis spores after 3 hours germination at 30 °C with 100 mM L-alanine or negative control (MQ) in K-phosphatebuffer pH 7.2. The displayed images are representative of experiments performed in duplicate on two individual spore batches repeated at least twice. An earlier study where germination in seven strains of B. licheniformis was investigated, showed that out of 24 amino acids tested, only L-alanine, L-cysteine and L-valine markedly stimulated germination [46].

In general, a greater germination response with L-alanine than with L-cysteine and L-valine was observed [46]. To assay the germination response of MW3, NVH-1307 click here and NVH-1311 to several amino acids, casein hydrolysate was used. Casein hydrolysate consists of a mixture of amino acids made from acid hydrolyzation of the milk protein casein and has been used as a germinant for Clostridium bifermentans and B. cereus in earlier studies [61–63]. In our study, casein hydrolysate proved to be a potent germinant for B. licheniformis, giving a rapid germination response (~70% phase dark spores as visualised by phase contrast microscopy) both for the wild type MW3 and the complementation mutant NVH-1311.