, 2003). It has been shown that a thyX knockout mutant of C. glutamicum was more sensitive to a DHFR inhibitor compared with a wild-type strain. This could be because both ThyA and ThyX contribute to the synthesis of the one-carbon unit for the

biosynthesis of thymidine in C. glutamicum. Moreover, because only the ΔthyX strain exhibited poor survival during the stationary growth phase, it has been 17-AAG cell line suggested that the expression levels of thyA and thyX differ in response to different growth conditions (Fivian-Hughes et al., 2012; Park et al., 2010). Sigma factors are components of RNA polymerases that bind to the core subunits of the enzyme and confer specificity to the process of transcription initiation by recognition of promoter sequences of genes and operons. The presence of seven putative sigma factors, including SigA and SigB, in C. glutamicum reflects the ability of the bacterium to adapt to various stress conditions (Kalinowski et al., 2003; Pátek & Nešvera, 2011). SigB is an alternative sigma factor that is not essential for exponential growth. Genome-wide transcription profiles of the wild-type and ΔsigB strain strains of C. glutamicum have shown that SigB is involved in amino Natural Product Library acid metabolism, carbon metabolism, stress defense, membrane processes,

and phosphorus metabolism (Ehira et al., 2008). Our primary interest in the present study was to measure the levels of ThyA and ThyX during growth of C. glutamicum. Western blot analysis with ThyA and ThyX antiserum suggested that both proteins were expressed, and that ThyX was maintained at the same level in both late-exponential and stationary phase cells. We also carried out Western blot analysis of total protein from the wild-type, ΔsigB, and sigB-complemented strains. Our results showed that SigB is responsible for the level of ThyX during transition into

the stationary growth phase. The bacterial strains used in this study are listed in Table 1. Escherichia Galactosylceramidase coli and C. glutamicum strains were cultured at 37 and 30 °C, respectively, in Luria–Bertani (LB) medium. Minimal medium used for C. glutamicum was mineral C. glutamicum citrate (MCGC) (Von der Osten et al., 1989) with glucose added to a final concentration of 1% (w/v). Ampicillin (100 μg mL−1), kanamycin (50 μg mL−1), and WR99210-HCl (3 μM) were added when needed. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal, 3 μg mL−1) was used to monitor β-galactosidase production on plates. PCR was used to amplify the coding sequences of the thyA and thyX genes from C. glutamicum ATCC 13032. The DNA fragment corresponding to the thyA gene was amplified using primers pQETHYA1 and pQETHYA2, and the thyX gene was amplified using primers pETTHYX1 and pETTHYX2. The PCR fragments of thyA were digested with SmaI and HindIII, and then cloned into pQE82L (Qiagen), which was also digested with SmaI and HindIII, to yield plasmid pQE82L-thyA.