Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

Abstract

The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation). This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression. A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wildtype promoter and is unresponsive to various concentrations of intracellular cAMP in vivo. The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro. However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression. The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon. The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed.L-Arabinose utilization in the bacterium Escherichia coli B/r requires the activation of three unlinked genetic loci by a single regulatory gene, araC (1, 2). The araBAD operon encodes three enzymes that are responsible for the initial catabolism of L-arabinose; the araE and araF genes encode proteins that are responsible for the transport of L-arabinose into the bacterium. In the presence of L-arabinose, araC protein is an activator of transcription for the araBAD operon and the araE and araF genes (1, 2). In the presence or absence of L-arabinose, araC protein is a repressor of its own synthesis (3, 4). In addition to regulation by its own protein product, araC gene expression is under positive control of the cAMP receptor protein (CRP) (3).The regulatory region for the araC gene is adjacent to the regulatory region for the araBAD operon. Their respective promoters are transcribed in opposite directions (5) and their transcriptional initiation sites are separated by 147 base pairs (bp) (6). The nucleotide sequence of this region of DNA has been determined (7,8) and it will be referred to as the ara regulatory region. Binding sites for regulatory proteins have been identified between the two transcriptional initiation sites (refs. 4 and 9; see Fig. 1). The localization of the regulatory protein binding sites in the promoter region has been the basis for several proposed models of the regulation of the araBAD operon and the araC gene (4, 9, 10). The portions of these models that are relevant to this work are the following. The DNase I protection studies form the basis for the most recent models for araBAD and araC expression (4, 9). Although consistent with physiological data, these models have...

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter

“…These include the concentration of arabinose used in inducing conditions (the lower the better), the ability of the strain to degrade arabinose (ara ϩ strains are better), and the physiological state of the culture due to the type of medium and amount and type of carbon sources present in the medium. Regarding the latter, since the araC-P BAD promoter system is subject to catabolite repression (32), levels of cyclic AMP will have a marked influence on expression. This could be important for strains growing in minimal medium with mixtures of carbon sources.…”

“…The uninduced levels can be reduced even further by growth in the presence of glucose. Glucose reduces levels of 3Ј,5Ј-cyclic AMP, thus lowering expression of the catabolite-repressed P BAD promoter (32). The properties of the mechanism of expression and repression of P BAD by AraC have been studied extensively, and their interactions have been dissected at the molecular level (reviewed in reference 41).…”

Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r

“…The sequence of the araBAD promoter mutants 1016 and 1036 was determined by Miyada et al (24). The AACT deletion in the AraC activator-binding site and the ACRP deletion in the CRP-binding site have been described (25,26). The solid-line bar adjacent to a number or letters indicates a deletion.…”

“…(i) ,-Galactosidase assay. Cultures (5 ml) of strain LA920 containing the desired araC plasmid were grown in supplemented M9 salts medium containing tetracycline (15 ,ug (24)(25)(26). The effect of these ara regulatory site mutations on this novel activation of araC gene expression was measured.…”

“…(iii) Autoregulation of araC gene expression requires AraC protein bound at araO1, a site which overlaps the RNA polymerase-binding site of the araC promoter, preventing initiation of transcription (14). (iv) Positive regulation of the araC gene and the araBAD operon by CRP-cAMP requires the complex bound to a site located upstream from araOl and from the araC transcription initiation site (20,26) (Fig. 1).…”