Complementation

of the sigB mutation in the KH4 strain restored resistance to the level of the wild-type strain (Fig. 5), indicating that the resistance of wild-type and sigB-complemented KH5 to WR99210-HCl was increased compared to KH4. This is presumably due to sufficient amounts of thymidylate and tetrahydrofolate being available through ThyX activity, along with an alternative folate reductase that is resistant to WR99210-HCl. We previously showed that the thyX gene is located on an operon with dapB and dapA, and that these genes are transcribed in a single transcriptional unit as dapB-thyX-dapA (Pátek et al., 1997; Park et al., 2010). Two putative −35 and −10 promoter regions of dapB (p1-dapB1 and p2-dapB2) IWR 1 have been identified. Sequences of TAGACT for the −10 region and CAGCAC for the −35 region were found in p1-dapB1, while CATAAT for the −10 region and TCGCCC for the −35 region were found for p2-dapB2 (Pátek et al., 1996). One or both of these promoter regions appeared to contain the promoter sequences recognized by SigB of C. glutamicum, tAnAAT for the −10 region and cgGCaa for the −35 region, as the fourth and fifth adenines of the −10 region of the SigB-recognized genes are

highly conserved (Larisch et al., 2007). The observation that a ΔsigB mutation resulted in a considerable decrease in the level of ThyX is an indication that the SigB protein acts as a regulator to induce expression O-methylated flavonoid of thyX. However,

the ΔsigB AUY-922 molecular weight mutation did not completely eliminate the synthesis of ThyX, suggesting that SigA is also able to direct transcription from the promoters and cooperates with SigB in transcription of thyX, as these promoter regions were similar to the promoter sequences suggested to be recognized by SigA, TA(c/t)aaT for the −10 region and ttGcca for the −35 region (Pátek et al., 1996). In this case, SigA would preferentially recognize the promoter sequences of thyA, as the ΔsigB mutation exhibited no effect on the expression of thyA. Thus, it is likely that the expression of thyA and thyX differ in response to different growth conditions of C. glutamicum. The C. glutamicum ΔsigB strain failed to grow in medium containing 3 μM WR99210-HCl, suggesting a role of SigB in the regulation of thymidine synthesis which is independent of the coupling activity of DHFR and ThyA. The fact that SigB can confer resistance to WR99210-HCl suggests that SigB regulates the expression of alternative folate reductase(s), which could help to compensate for the loss of DHFR activity for growth. Experiments thus far have indicated that the level of ThyX appears to be regulated by two sigma factors, SigA and SigB, in C. glutamicum.