cereus. Our current findings suggest that the protein is part Selleck SCH 900776 of an outer spore structure, most likely the exosporium or the interspace between the exosporium and the coat. The bacterial strains used in this study were the B. cereus type strain ATCC 14579 (Frankland & Frankland, 1887; Ivanova et al., 2003) and B. subtilis B252 (From et al., 2005). To create a bc1245 deletion mutant in B. cereus ATCC 14579, a shuttle vector modified from pMAD (Arnaud

et al., 2004) with a spectinomycin-resistant cassette in the restriction site SalI (Fagerlund, 2007) was used. Sequence information was obtained from the NCBI bacterial genome database (http://www.ncbi.nlm.nih.gov/guide) or the ergo database (Overbeek et al., 2003). Comparative genomic analyses of bc1245 were performed on selected members of the B. cereus group [B. cereus ATCC 14579 (GenBank: NC004722), B. cereus ATCC 10987 (GenBank: NC003909), Akt inhibitor B. cereus AH187 (GenBank: CP001177), Bacillus thuringiensis YBT-020 (GenBank: CP002508), B. anthracis str. Ames (GenBank: AE016879), Bacillus weihenstephanensis KBAB4 (GenBank: NC010184), B. mycoides DSM 2048 (GenBank: CM000742) and B. pseudomycoidesDSM12442 (GenBank: CM000745)] to investigate whether bc1245 is conserved. Putative σ-binding sites for the bc1245 promotor

were predicted by analyzing the 500-bp upstream region of bc1245 with DBTBS release 5 (Sierro et al., 2008). 4-Aminobutyrate aminotransferase To search for functional motifs, the amino acid sequence of BC1245 was submitted to ScanProSite, (http://www.expasy.ch/prosite; Bairoch et al., 1997). Quantitative PCR experiments were performed as described previously (van der Voort et al., 2010), and primers were designed by use of Primer 3 (Rozen & Skaletsky, 2000) for sigH, sigE, sigF, sigG, sigK, bc1245 and zcDNA (Table 1) using the chromosomal DNA sequence of B. cereus ATCC 14579 as a template.

PCR on genomic DNA was used to check primer efficiency (results not shown). RNA was isolated from two independent cultures withdrawn at different stages of sporulation of B. cereus ATCC 14579 grown in maltose sporulation medium (MSM) as described earlier (van der Voort et al., 2010). cDNA synthesis was performed with ~ 500 ng of total RNA and a mix of relevant reverse primers as described previously (van Schaik et al., 2007). Quantitative PCR was performed with 5 μM of each of the primer pairs listed in Table 1 using an ABI Prism 7700 with SYBR green technology (PE Applied Biosystems, Nieuwekerk a/d Ijssel, the Netherlands) as described previously (van Schaik et al., 2005). By comparing expression of the chosen genes with that of the reference 16S rRNA gene (zcDNA) levels, relative expression values were obtained with the REST-MCS program using the Pair Wise Fixed Reallocation Randomization Test (Pfaffl et al., 2002).