Effects of Global and Specific DNA-Binding Proteins on Transcriptional Regulation of the E. coli bgl Operon

Abstract

Using reporter gene (lacZ) transcriptional fusions, we examined the transcriptional dependencies of the bgl promoter (Pbgl) and the entire operon regulatory region (Pbgl-bglG) on eight transcription factors as well as the inducer, salicin, and an IS5 insertion upstream of Pbgl. Crp-cAMP is the primary activator of both Pbgl and the bgl operon, while H-NS is a strong dominant operon repressor but only a weak repressor of Pbgl. H-NS may exert its repressive effect by looping the DNA at two binding sites. StpA is a relatively weak repressor in the absence of H-NS, while Fis also has a weak repressive effect. Salicin has no effect on Pbgl activity but causes a 30-fold induction of bgl operon expression. Induction depends on the activity of the BglF transporter/kinase. IS5 insertion has only a moderate effect on Pbgl but causes a much greater activation of the bgl operon expression by preventing the full repressive effects of H-NS and StpA. While several other transcription factors (BglJ, RcsB, and LeuO) have been reported to influence bgl operon transcription when overexpressed, they had little or no effect when present at wild type levels. These results indicate the important transcriptional regulatory mechanisms operative on the bgl operon in E. coli.

Histone-like nucleoid structuring (H-NS) protein silences the beta-glucoside (bgl) utilization operon in Escherichia coli by forming a DNA loop

“…Our previous results have shown that H-NS and H-NSL30P are able to bind to the bgl promoter region in the absence of bglG , while H-NSL30P even showed a higher binding affinity [39] . Here, we further demonstrated H-NS binding to its downstream site within bglG , using the lacIQ promoter driving expression of the bglG - lacZ transcriptional fusion at the lac locus (Iq-G_Z).…”

“…In an earlier paper, we proposed that H-NS represses the bgl operon by forming a DNA loop between its two binding sites [39] . Here, we investigated this proposed DNA-looping mechanism in-depth.…”

“…We first determined the effect by preventing the oligomerization of H-NS using the hns L30P mutant (a missense mutation in the hns gene leading to a change of leucine to proline at the 30th codon) ( Fig. 2 A) [29] , [39] . This derivative of H-NS is still capable of dimerizing, thereby binding to the target DNA, but it has lost the ability to oligomerize [34] .…”

“…Likely, a similar mechanism applies to the repression of the bgl operon by H-NS, but this has not been demonstrated directly. Our prior studies have suggested that H-NS binding to its binding site(s) in the bgl promoter region is not sufficient to repress the operon, so we proposed that H-NS represses the bgl operon by the formation of a DNA loop between its two binding sites [39] . This proposed mechanism of bgl operon repression was investigated in-depth in this study.…”

“…Also, when H-NS is truncated at the N-terminus with loss of its DNA binding ability, another repressor of the bgl operon, StpA can function as a DNA-binding adapter of H-NS to repress the operon [37] , [38] . However, StpA is negatively regulated by H-NS and can only regulate the bgl operon when H-NS is absent [39] , [40] . Therefore H-NS is the major repressor of the bgl operon in wild-type E. coli cells.…”