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The enzyme penicillin acylase is synthesized by Escherichia coli only at growth temperatures below 30 degrees C. The biochemical basis of this strict temperature-dependent formation of an enzyme was investigated. When the gene (pac) was under the control of the lacUV5 promoter it showed the same temperature-dependent expression as the chromosomally encoded gene transcribed from its own promoter. This indicates that translation of the pac mRNA rather than transcription of the gene is temperature-dependent. This conclusion could be further confirmed by Northern hybridisation and by analysis of pac-lacZ transcriptional fusions. TnphoA insertion mutagenesis and experiments in which the promoter and 5' sequence encoding the signal peptide of the pac gene was exchanged with those of the cyclodextrin glycosyltransferase gene from Klebsiella oxytoca localised the region of pac mRNA responsible for the temperature-sensitive translation to the 5'-untranslated region and/or the signal peptide. Extension of the 5 nucleotide long spacer separating the Shine-Dalgarno motif from the AUG initiation codon by one or three nucleotides lead to partial or full synthesis of penicillin acylase precursor at 40 degrees C, respectively. The precursor of penicillin acylase formed at 40 degrees C by the mutant variants or when placed under the control of a heterologous upstream region was associated with the membrane but could not be translocated. Taken together these data suggest that transport and translation of the penicillin acylase precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature. Moreover, evidence was also provided which indicates a direct effect of temperature on the conformation of the precursor and it is proposed that the lack of translation at high temperatures has been selected to prevent the accumulation of transport-incompetent protein locked in the membrane.

Molecular Basis of the Exclusive Low-Temperature Synthesis of an Enzyme in E. coli: Penicillin Acylase