New insights into the regulation of the pac gene from Escherichia coli W ATCC 11105

Abstract

The regulation of the pac gene encoding the penicillin G acylase of Escherichia coli W ATCC 11015 has been investigated by a molecular approach using lacZ as a reporter gene. This analysis revealed that a region of 170 bp located upstream of the pac structural gene contains the regulatory sequences that control its expression. The cAMP receptor protein is involved not only in the catabolite repression of penicillin G acylase production caused by glucose but also in the induction of pac gene expression by phenylacetic acid. Primer extension analyses have demonstrated that the transcription of the pac gene can be initiated from at least three different promoters. Although all these promoters are functional, their relative activity depends on the transcribed gene, the P1 and P3 promoters being more active in the presence of the pac gene, whereas the P2 promoter was stronger when the upstream region of the pac gene was fused to the lacZ reporter. A deletion of the region surrounding the 310 box of the P3 promoter produced a constitutive expression of the fused gene indicating that this sequence is required for phenylacetic acid induction and suggesting that the expression of the pac gene is regulated by a repression mechanism. This work reveals that the regulation of the pac gene is more complex than previously envisioned and provides new clues to investigate further this interesting regulatory system. ß

Biodegradation of Aromatic Compounds by Escherichia coli

“…The PacR repressor mediates the PA-dependent induction of the pac gene (60). However, induction of pac expression by PA has also been observed using P pac ::lacZ fusions in the absence of the pac gene (196,263). Involvement of IHF as an additional regulatory protein in pac transcription was also reported (300).…”

“…The pac gene of E. coli W is located in the vicinity of the hpa cluster (250) (Fig. 1A) and is transcribed as a monocistronic unit using different alternative promoters (190,263). The expression of the pac gene is induced by PA and phenoxyacetic acid and repressed by metabolic carbohydrates and polyalcohols (glucose, fructose, and glycerol) (309).…”

“…The expression of the pac gene is induced by PA and phenoxyacetic acid and repressed by metabolic carbohydrates and polyalcohols (glucose, fructose, and glycerol) (309). Although the repression by glucose appears to be a typical CRP-dependent process that is partially overcome by the addition of cAMP (106,196,263,300), the regulatory proteins involved in PA induction remain unknown. In this context, a pacR regulatory gene located inside the pac structural gene and transcribed in the opposite direction to pac has been identified (144).…”

Regulation of Penicillin G Acylase Gene Expression in Escherichia coli by Repressor PaaX and the cAMP-cAMP Receptor Protein Complex

“…10). Thus, although the pacR gene is located inside the pga structural gene (49), translational fusions (P pga ::lacZ and P pga ::phoA) that lack the pga structural gene continue to be inducible by PAA (20,50). Moreover, when we measured PGA activity in E. coli W cells transformed with plasmids expressing the pacR gene, we found normal levels of PGA activity rather than reduced levels as expected if PacR is a repressor (data not shown).…”

The PaaX Repressor, a Link between Penicillin G Acylase and the Phenylacetyl-Coenzyme A Catabolon of Escherichia coli W

“…1) (16,18,21,22,27,29,31). Despite the fact that this system was deeply studied, the regulatory proteins involved in PA induction still remained unclear.…”