Corynebacterium glutamicum cells were routinely cultured at 30 °C in MB (Follettie et al., 1993) medium. MCGC minimal media for C. glutamicum were prepared as described previously (Park et al., 2008). Escherichia coli DH-10B (Invitrogen) was used for plasmid Selleckchem AZD4547 construction and propagation. Escherichia coli cells were cultured at 37 °C in LB (Sambrook & Russell, 2001). Antibiotics were added at the following concentrations (μg mL−1): 30 ampicillin, 20 chloramphenicol, and 30 kanamycin. The sensitivity of C. glutamicum cells to diamide was assessed on MB plates as described previously (Kim et al., 2005). We utilized standard molecular cloning, transformation, and electrophoresis protocols (Sambrook & Russell, 2001). Plasmids were introduced

into the C. glutamicum cells by electroporation. Restriction enzymes and DNA modifying enzymes were purchased from Takara Bio and used according to the manufacturer’s instructions. PCR was carried out as previously described (Kim et al., 2005). Total RNA was prepared using a NucleoSpin RNA II Kit (Macherey-Nagel). cDNA conversion was carried out using a DyNAmo™ cDNA Synthesis Kit (Finnzymes). RT-qPCR was performed as described previously (Lee et al., 2009). CFX96™ Real-Time PCR Detection System (Bio-Rad) was used for gene expression analysis.

Standard curve, ERK pathway inhibitor expression normalization, and standard error values were obtained with CFX Manager™ software ver. 1.5 (Bio-Rad), which employs the ΔΔCt method. Normalization was performed with 16S rRNA gene. Verification of RT-qPCR products was performed by melting curve and peak analysis. CYTH4 The following primers were used: NCgl0663, 5′-ACCCAACTTGGTGGTCAGATGGAA-3′ and 5′-TTGAGCAGCGGAACCATAGACCAT-3′; NCgl2984, 5′-ACGAGAAGATTCGCTTCGTCACCA-3′ and 5′-AATCTTCACCAGTGACGGTGTCGT-3′; NCgl0328, 5′-ATCGCCCTTGTTATTGCTACCGGA-3′ and 5′-AGTAGCTGTTGTCGATGCGCCTAT-3′; NCgl1022, 5′-GCCAACAATGAGGTGGGAACCATT-3′ and 5′-AACGCGTCAACTCCCAAGTCAAAG-3′; NCgl2053, 5′-ACTTCGACCAGACTTTGCAG-3′ and 5′-AAGAGGGTTTCCGAAGGTTG-3′; NCgl2971, 5′-TCAAGCACATCACCGTCAAG-3′ and 5′-TGGAATCAACTGGAAGGGTC-3′; whcA, 5′-AAATGGCGACCCAGATGCAT-3′ and

5′-TATCTAAGGCATCGGCGC-3′; 16S rRNA gene, 5′-ACCCTTGTCTTATGTTGCCAG-3′ and 5′-TGTACCGACCATTGTAGCATG-3′; spiA, 5′-ACATCTTTACGTAAGGTGGCGGGA-3′ and 5′-CTGCTTTGCACTACCCTTCGCAAT-3 The C. glutamicum ∆spiA mutant strain was constructed according to the method described by Schäfer et al. (1994). Briefly, a DNA fragment was prepared from the C. glutamicum genome by crossover PCR utilizing the primers F1 5′-GTTGCCCAGGCCCACGACCAGT-3′, R1 5′-TTTCAGGTCGCGCTTCTAGACAACAATCCCGCCAGCTCATCA-3′, F2 5′-GATTGTTGTCTAGAAGCGCGACCTGAAAACCCTCCTC-3′, and R2 5′-TTCCCTGCACTTCCCGCCACCTTA-3′. The amplified fragment was cloned into the pGEM-T-easy vector (Promega). The EcoRI fragment was then isolated and inserted into EcoRI-digested pK19mobsacB (Schäfer et al., 1994). The subsequent procedures were conducted as described previously (Hwang et al., 2002), and the chromosomal deletion of spiA in C.