PROMOTERS FROM CORYNEBACTERIUM-GLUTAMICUM - CLONING, MOLECULAR ANALYSIS AND SEARCH FOR A CONSENSUS MOTIF

Relatively limited information about promoter structures in Corynebact erium glutamicum has been available until now. With the aim of isolati ng and characterizing such transcription initiation signals, random Sa u3A fragments of C. glutamicum chromosomal DNA and of the corynebacter ial phage phi GA1 were cloned into the promoter probe vector pEKplCm a nd selected for promoter activity by chloramphenicol resistance of tra nsformed C. glutamicum cells, The nucleotide sequence of ten chromosom al and three phage fragments was determined and the transcriptional st art (TS) sites were localized by primer extension analyses. Additional ly, the promoters of five previously isolated C. glutamicum genes were cloned and mapped, All of the isolated promoters were also functional in the heterologous host Escherichia coli. A comparative analysis of the newly characterized promoter sequences together with published pro moters from C. glutamicum revealed conserved sequences centred about 3 5 bp (ttGcca) and 10 bp (TA.aaT) upstream of the TS site, The position of these motifs and the motifs themselves are comparable to the -35 a nd -10 promoter consensus sequences of other Gram-positive and Gramneg ative bacteria, indicating that they represent transcription initiatio n signals in C. glutamicum, However, the C. glutamicum consensus hexam er of the -35 region is much less conserved than in E. coli, Bacillus, Lactobacillus and Streptococcus.