The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pacR, which is transcribed in the opposite direction. Site-directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature-dependent expression character of pac is preserved in the pacR translation-initiation mutant and the optimum temperature of PGA production was 22 degrees C in both wild type and mutant. At a higher temperature of 37 degrees C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pacR-mediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post-translational processing of the PGA precursor polypeptide.