The construction of a more easily transformable mutant, B. licheniformis MW3, has largely overcome this challenge [50]. In order to facilitate the understanding of germinant/receptor interactions in B. licheniformis, we have constructed disruption and complementation mutants of the gerAA locus in B. licheniformis MW3. Spores of these mutants have been studied in germination assays with L-alanine, casein hydrolysate and the non-nutrient germinant Ca2+Dipicolinic acid (Ca2+DPA).

These studies reveal that gerA is AZD1480 datasheet a main germinant receptor complex of B. licheniformis recognising amino acid(s), and supports the view that L-alanine is an important nutrient-germinant for this species. Results and Discussion Construction of the disruption and complementation mutants To elucidate the role of the hypothetical GerA proteins during spore germination, a disruption mutant of the gerAA locus in B. licheniformis MW3 was constructed. B. licheniformis MW3 was used as target strain due to its superior transformability compared to its fully sequenced parent strain DSM 13 [50]. The gerAA Momelotinib cost mutant, NVH-1307, was constructed

so that a part of the gerAA gene was substituted with a spectinomycin resistance cassette. This will cause the mutant to acquire spectinomycin resistance, and in addition, affect a potential phenotype related to the disrupted gene. If the target gene is part of an operon, which is the case of gerAA, downstream transcripted genes will also be affected, and the receptor non functional. Sequence analysis showed that in addition to harbouring the spectinomycin cassette in the gerAA locus, NVH-1307 also harboured two

additional mutations (one base substitution and one base deletion) in the gerAA locus. These mutations were most likely acquired during PCR amplification of the fragments used to construct the disruption vector (pMAD_SpRΔgerAA). These mutations were “”accepted”" (not corrected) due to their location in the gene targeted for disruption. However, in construction of the plasmid used for gerAA complementation, a polymerase with a higher expected fidelity was applied to limit the Amino acid risk of such mutations. Sequence analysis of the complementation plasmid pHT315_MW3gerA revealed no mutations in the amplified gerA operon when compared to the sequence of Veith et al.[48]. Genetic modification studies have shown that the germination rates could be significantly increased when specific germinant receptors are over-expressed in B. subtilis [51]. Thus, expression of germinant receptors is apparently not optimised for maximal spore germination, forwarded as a possible evolutionary strategy to prevent premature germination at nutrient conditions inadequate for sustained vegetative growth [3]. Very high levels of receptor expression could on the other hand have a Fedratinib research buy negative effect on the sporulation process [51].