[Analysis of function of the 452 bp sequence proceeding the pgiB gene in Bacillus stearothermophilus]

By random mutagenesis with chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine, a shuttle promoter probe vetor mutant pPGV5 (tr65) was obtained. A single site mutation from G to T was discovered at nucleotide + 238 of kan gene encoding kanamycin nucleotidyltransferae by sequence analysis. Using the gene pheB that encodes thermostable catechol 2'3-dioxygenase from Bacillus stearothermophilus FDTP-3 as the reporter gene, a transcriptional fusion plasmid pPGVPB452 was constructed and electroportated into B. stearothermophilus. By analyzing the reporter protein, it proved that the 452 bp sequence proceeding the coding region of phosphoglucoisomerase pgiB gene from B. stearothermophilus T521, which contained a putative promoter by sequence analysis, but has no transcriptional activity in B. stearothermophilus.