THE EXPRESSION OF THE PENICILLIN-G AMIDASE GENE OF ESCHERICHIA-COLI BY PRIMER EXTENSION ANALYSIS

Escherichia coli ATCC 11105 and JM109, transformed with a multicopy plasmid carrying the penicillin G amidase (PGA) gene, were grown at 26 degrees and 37 degrees C, in the presence or the absence of phenylacetic acid (PAA) or of glucose. A method based on primer extension was developed to quantify in vivo levels of PGA mRNAs. A unique transcription start site was found to be used in all the fermentation conditions tested. This site is located 28 nucleotides upstream of the initiation codon. Its utilization is subjected to catabolic repression and is induced by PAA. This site is used at 37 degrees C, but the PGA mRNA level in E. coli ATCC 11105 is lower at 37 degrees C than at 26 degrees C. Induction of the pga gene by PAA was found to be more efficient in the producer strain. Taking into account the amount of PGA mRNA present in the cells at 37 degrees C, one would expect the production of active PGA at this temperature. This is not the case. Thus, at 37 degrees C, expression is blocked at a step after transcription.