Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro

Abstract

Bacterial strains, phages, and plasmids. The bacterial strains, phages, and plasmids used in this investigation are listed in Table 1. The bacterial strains were E. coli K-12 derivatives.Culture media. The media used for growth were identical to those described by Miller (26). The synthetic medium used was M63 medium (pH 7.2) (44) or M9 medium (pH 7.2) (26) and contained either glucose (5 g/liter), glycerol (5 g/liter), glucuronate (2.5 g/liter), galacturonate (2.5 g/liter), tagaturonate (2.5 g/liter), or fructuronate (2.5 g/liter); aldohexuronate-containing MacConkey media (Difco Laboratories) contained 15 mg of glucuronate or galacturonate per ml. When needed, ampicillin, chloramphenicol, and kanamycin were used at final concentrations of 25 jig/ml, and 973 on August 5, 2020 by guest

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

“…The role that CRP plays in the uxaCA and the exuT regulation is not well understood. It has previously been observed that deleting the restriction fragment lying to the left of the Sail site on the chromosome does not prevent the exuTexpression [2]. Since this deletion removes the CRP-binding site, we should tend to conclude that CRP plays no direct role in the exuT regulation.…”

“…The 1.8-kb SalI-BamHl-I fragment contains the entire control region of uxaCA and the exuTo operator ( Fig. 1) [2]. This restriction fragment was cloned into pEMBL8.…”

“…The uxaA-lacZ (plasmid pREG10) and exuT-lacZ (plasmid pREG11) previously constructed on a /ac fusion plasmid vector [2] were used to study the regulation of the uxaCA and exuT operons. Strain MC4100 which contains pREGI0 or pREGll was grown in the presence of glycerol, glucose or glucose plus cyclic AMP.…”

“…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].…”

“…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.…”