Identification of cyclic AMP-CRP binding sites in the intercistronic regulatory uxaCA-exuT region of Escherichia coli

Abstract

In Escherichia coli K-12 the two adjacent operons exuT and uxaCA are divergently transcribed and each possesses its own control region. We show that in vivo the expression of these two operons seems to be sensitive to catabolite repression and to be cAMP-dependent. The nucleotide sequence of 318 nucleotides, including the entire control region of uxaCA and the exuT operator, was determined and two cAMP-CRP binding sites were identified. INTRODUCTIONIn E. coil K-12, galacturonate is degraded according to the Ashwell pathway [1]. In the catabolism of galacturonate, the first steps involve, in order, the structural genes exuT, uxaC, uxaB and uxaA. The adjacent operons exuT and uxaCA, which are divergently transcribed, possess separate control regions and the precise location of the operator sites relative to endonuclease restriction sites was previously determined (see Fig. 1). The entire uxaCA control region (uxaCo and uxaCp) lies to the left of PstI whereas the exuTo operaior is located on the PstI-SalI fragment and the exuTp promoter lies to the right of Sail [2,3].To obtain a better picture of the genetic organization and of the control mechanisms that operate in the intercistronic uxaCA-exuT region, we have determined the nucleotide sequence of part of this region and we report here the catabolite receptor protein (CRP) binding sites inside this regulatory region. MATERIALS AND METHODSThe bacterial strains were E. coil K-12 derivatives: MC4100, araD139 AiacU169 rpsL thi [4]; 71/18, A(lac-pro) F'iacI q lacZAM15 pro + supE [51.Plasmids pREG10 and pREGll [21 derive from the lac fusion plasmid pMC874 [41. Plasrrtids pEMBL8 and pEMBL9 [5] were used for cloning and sequencing exu fragments. Procedures for isolation and analysis of plasmid DNA have been described [3]. DNase I footprinting was performed as described [6].