In the presence of dethiobiotin, only 9 of the genes listed in Table 1 were differentially expressed, all showing an increased mRNA level similar to those under biotin limitation. The most strongly regulated see more genes were bioB, the gene encoding biotin synthase converting dethiobiotin to biotin (11.3 fold higher than with biotin), cg2884 (5.6 fold) and bioY (4.4 fold). Transcriptional organisation of the putative bioYMN operon As the chromosomal location of bioY, bioM and bioN and their biotin-dependent gene expression patterns indicated that these genes might form an operon, RT-PCR was applied to test this hypothesis (Figure 1). Total RNA

isolated from C. glutamicum ATCC 13032 was transcribed into cDNA by using random hexamer primers in a reverse transcriptase reaction. The resulting products were then used for PCR amplifications A to C (Figure 1 Doxorubicin upper panel). As shown in the middle panel of Figure 1, cDNA created with random hexamer primers allowed the amplification of a bioY fragment (reaction A) and a bioMN fragment (reaction C),

pointing to an co-transcription of the latter two genes. But further evidence was obtained that bioYMN are co-transcribed, since PCR amplification using primers annealing to bioY and to bioM yielded a PCR product covering the intergenic region and parts of both genes (reaction B). As an internal control in the RT-PCR assays, we used dnaE encoding a

subunit of DNA polymerase. Besides reactions A, B and C three additional control reactions (AN, BN, CN) were performed; these were identical to reactions A to C, respectively, except that reverse transcriptase was omitted from the initial reactions. The fact that no PCR products were obtained in these reactions confirmed that the RNA was not contaminated with chromosomal DNA. Figure 1 Transcriptional organization of the bioYMN locus in C. glutamicum. (upper panel) Scheme showing the bioYMN locus in C. glutamicum and the RT-PCR reactions used to determine co-transcription of bioY, bioM and bioN. RNA from C. glutamicum WT was transcribed into cDNA ever with random primers. Subsequently, cDNAs were used as templates for the PCR reactions labeled A-C. (middle panel) Results from the RT-PCR analyses described above. The lower DNA fragment visible lanes A-C represents dnaE, and RT-PCR of dnaE served as positive control in all reactions. The upper bands in lanes A, B and C correspond to the products of the PCR reactions A-C indicated in A. Reactions AN, BN and CN represent controls confirming the absence of DNA in the RNA preparation. The reactions were identical to the PCR reactions as shown in lanes A-C except that reverse transcriptase was omitted in the cDNA reactions. (lower panel) The bioYMN locus is shown schematically.