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.

Keywords: Crp; DNA loop; Fis; H-NS; StpA; bgl operon; insertion sequences (IS); β-glucosides.

Figure 1

The bgl promoter (Pbgl) activities in the wild type and various isogenic genetic backgrounds. Cells were grown in M63 minimal media with shaking at 37 °C. At least four samples were collected at OD₆₀₀ values of 0.2 to 1.0 during the exponential growth phase. Bacterial samples were subject to β-galactosidase assays as described in Section 4, and the enzyme activities were calculated using the equation [(OD₄₂₀−1.75 × OD₅₅₀)/(sample volume in mL × time in min)] × 1000. For a given test strain, the slope of OD₆₀₀ values versus β-galactosidase activities was referred to as the promoter activity. (A) Diagram showing the lacZ transcriptional reporter for the bgl promoter (Pbgl-lacZ). Pbgl (−205 to + 54 relative to the transcriptional start site) with no terminators was fused to the upstream region of the lacZ’s RBS (that is, to TTTCACACAGGAAACAGCT) at the lac locus, replacing lacI and the lacI/lacZ intergenic region. The native bgl operon remained intact. However, for strain Bgl⁺, there is an IS5 element oriented in the inverse direction and inserted at −207.5 upstream of the bglG translation start site. For both the promoter reporter and the native bgl operon, the blue bars represent the Crp binding sites (O_Crp) while the red bars represent the proposed H-NS binding sites (O_HNS). (B) Pbgl activities in cells grown with glycerol as the primary carbon source. (C) Pbgl activities in cells grown with glycerol and salicin as carbon sources.

Figure 2

The bgl operon activities in the wild type and its various genetic backgrounds. Culture preparation, sample collection, and β-galactosidase assays were carried out as in Figure 1 (see Section 4). (A) Diagram showing the lacZ transcriptional reporter for the entire regulatory region of the bgl operon (Pbgl-bglG-lacZ). The region carrying Pbgl and bglG, including both terminators (−205 to + 1127 relative to the bglG transcriptional start site), was fused upstream of the lacZ’s RBS (that is, TTTCACACAGGAAACAGCT) at the lac locus. Strain Bgl⁺ carries an IS5 element oriented in the inverse direction and inserted at −207.5 upstream of the bglG translation start site. For all other strains, the native bgl operon remains unchanged. For both the operon reporter and the native bgl operon, the blue bars represent the Crp binding sites (O_Crp) while the red bars represent the proposed H-NS binding sites (O_HNS). (B) bgl operon activities in cells grown in M63 with glycerol as the primary carbon source. (C) bgl operon activities in cells grown with glycerol and salicin as carbon sources.

Figure 3

Inhibitory effect of StpA on bgl operon expression in the absence of H-NS. Using the operon reporter, Pbgl-bglG-lacZ (at the lac locus), the bgl operon transcriptional activities were assayed, comparing the stpA single mutant (∆stpA), the hns single mutant (∆hns), and the hns/stpA double mutant (∆hns∆stpA). Cells were cultured in M63 with glycerol and salicin. Sample preparation and β-galactosidase assays were carried out as in Figure 1.

Figure 4

IS5 insertion stimulates both promoter and operon activities. (A) Diagram showing IS5 insertion at the Pbgl reporter (IS5Pbgl-lacZ). An IS5 element in the reverse direction is located at -207.5 upstream of the bglG translation start site. The native bgl operon is unchanged. (B) Diagram showing IS5 insertion at the bgl operon reporter (IS5Pbgl-bglG-lacZ). IS5 orientation and location are the same as in Figure 4A. In both (A) and (B), the blue bars represent the Crp binding sites (O_Crp) while the red bars represent the proposed H-NS binding sites (O_HNS). (C) Effects of IS5 insertion on Pbgl activities. (D) Effects of IS5 insertion on bgl operon activities. In both (C) and (D), test strains were cultured in M63 with glycerol and salicin as carbon sources. Sample collections and β-galactosidase assays were carried out as in Figure 1.

Figure 5

Fis represses bgl operon expression when it is activated by IS insertion. (A) Fis repression of bgl operon expression after activation by IS insertion. (B) Antagonism between Fis and Crp in regulating the bgl operon. In both (A) and (B), IS5 insertion is present both in the native operon and the operon lacZ reporter. Cells were cultured in M63 with glycerol and salicin as carbon sources. Sample collection and β-galactosidase assays were carried out as in Figure 1.

Figure 6

Possible requirement of DNA looping for bgl operon silencing. (A) Effect of H-NSL30P on bgl operon expression. H-NSL30P is an H-NS derivative that carries a proline residue instead of leucine at residue 30 in the protein. This derivative is thought to maintain its DNA-binding capability but is deficient in oligomerization [64,65], thereby failing to bridge two or more DNA loci together. Using the operon reporter, Pbgl-bglG-lacZ, the effect of this mutant H-NS on bgl operon expression was determined by comparing the wild type H-NS and the absence of H-NS. (B) Diagram of a truncated bgl operon reporter (tPbgl-bglG-lacZ). It is the same as the regular operon reporter Pbgl-bglG-lacZ except that the regulatory region upstream of the Crp operator (believed to carry an H-NS binding site) in Pbgl has been removed. The blue bars represent the Crp binding sites (O_Crp) while the red bars represent the proposed H-NS binding sites (O_HNS). (C) The bgl operon activity using a reporter lacking the proposed H-NS binding site in the upstream regulatory region. (D) Effect of H-NSL30P on Pbgl. In (A,C,D), test strains were cultured in M63 with glycerol and salicin as carbon sources at 37 °C. Sample collection and β-galactosidase assays were carried out as in Figure 1.