Participation of regulator AscG of the beta-glucoside utilization operon in regulation of the propionate catabolism operon

Abstract

The asc operon of Escherichia coli is one of the cryptic genetic systems for beta-D-galactoside utilization as a carbon source. The ascFB genes for beta-D-galactoside transport and catabolism are repressed by the AscG regulator. After genomic SELEX screening, AscG was found to recognize and bind the consensus palindromic sequence TGAAACC-GGTTTCA. AscG binding was detected at two sites upstream of the ascFB promoter and at three sites upstream of the prpBC operon for propionate catabolism. In an ascG-disrupted mutant, transcription of ascFB was enhanced, in agreement with the repressor model of AscG. This repression was indicated to be due to interference of binding of cyclic AMP-CRP to the CRP box, which overlaps with the AscG-binding site 1, as well as binding of RNA polymerase to the promoter. Under conditions of steady-state E. coli growth in a rich medium, the intracellular level of AscG stayed constant at a level supposedly leading to tight repression of the ascFB operon. The level of prpR, encoding the activator of prpBCDE, was also increased in the absence of AscG, indicating the involvement of AscG in repression of prpR. Taken together, these data suggest a metabolic link through interplay between the asc and prp operons.

FIG. 1.

Gel mobility shift assay of AscG-target DNA complex formation. Fluorescently labeled DNA probes (1.0 pmol each) containing a SELEX segment from the ascG-ascF spacer (A) or the prpR-prpB spacer (B) were incubated at 37°C for 30 min in the absence (lane 1) or presence of increasing concentrations of AscG (lane 2, 2.5 pmol; lane 3, 5.0 pmol; and lane 4, 10 pmol). The reaction mixtures were directly subjected to PAGE. P, probe DNA; C1 to C3, AscG-probe DNA complexes.

FIG. 2.

Identification of AscG box sequence. A fluorescently labeled SELEX segment (1.0 pmol) from the ascG-ascF spacer (A) or the prpR-prpB spacer (B) was incubated in the absence or presence of increasing concentrations of purified AscG (2.5, 5.0, 10, and 20 pmol, from right to left) or CRP (0.3, 0.6, 1.25, and 2.5 pmol, from right to left) and then subjected to DNase I footprinting assays. Lanes A, T, G, and C represent the respective sequence ladders.

FIG. 3.

Locations of promoters and AscG- and CRP-binding sites. (A) The transcription initiation sites of ascG and ascF and the binding sites for AscG and CRP are shown along the 256-bp-long ascG-ascF spacer. The transcription initiation sites were determined by primer extension assay (data not shown), while the binding sites for AscG and cAMP-CRP were determined by DNase I footprinting assay as described in the legend to Fig. 2. The numbers in parentheses represent distances from the transcription initiation site of ascF, which is under the control of AscG. Open boxes at the termini of the DNA bar represent the initiation codons of ascG and ascF. (B) The transcription initiation sites of prpR and prpB and the binding sites for AscG and CRP are shown along the 200-bp-long prpR-prpB spacer. The binding sites for AscG and cAMP-CRP were determined by DNase I footprinting assay as described in the legend to Fig. 2. The numbers in parentheses represent distances from the transcription initiation site of prpR, which is under the control of AscG. Boxes at the termini of the DNA bar represent the initiation codons of prpR and prpB.

FIG. 4.

Consensus sequence of AscG box and its location within the asc and prp operons. (A) The sequences represent those protected by AscG within the ascG-ascF and prpR-prpB spacer regions (see Fig. 2 and 3). The consensus sequence of the AscG box was identified after comparison of these AscG-binding sequences. The numbers in parentheses represent distances (bp) from the transcription initiation site of ascF or prpR. The underlined nucleotides represent those identical with the consensus AscG box. (B) The sequences represent those protected by cAMP-CRP within the ascG-ascF and prpR-prpB spacer regions (see Fig. 2 and 3). The numbers in parentheses represent distances (bp) from the transcription initiation site of ascF or prpR.

FIG. 5.

Assay of the acsF and prpR promoters in wild-type and ascG mutant E. coli. The ascF and prpR promoter fragments were inserted into the TFP promoter assay vector pGRP, and the resulting promoter plasmids, pGRPascF (A) and pGRPprpR (B), were transformed into wild-type KP7600 and its ascG-disrupted mutant, JD27135. The promoter activities were determined by measuring the GFP/RFP ratio. Closed bars represent the promoter activities in wild-type KP7600, while open bars represent the promoter activities in the ascG mutant JD27135.

FIG. 6.

Northern blot analysis of asc and prp operon RNAs. Overnight cultures of wild-type E. coli KP7600 (wt) and its ascG-disrupted mutant JD27135 (ΔascG) in either LB or M9-glucose medium were transferred into the respective fresh medium. After shaking culture at 37°C, cells were harvested in the middle of log phase (OD₆₀₀, 0.6). Total RNA was isolated as described in Materials and Methods and fractionated by PAGE in the presence of urea. After blotting of RNA onto filters, ascF, ascB, prpR, and prpB RNAs were detected with fluorescently labeled probes. The probes used were prepared by PCR, using the primers described in Table 1. The total amounts of RNA samples were adjusted based on the staining intensity of rRNA with ethidium bromide.

FIG. 7.

Determination of intracellular concentrations of AscG, CRP, and PrpR. Wild-type E. coli KP7600 was grown in LB medium at 37°C for various times. Cells were harvested at turbidities of 0.3, 0.6, 0.9, and 1.5, and whole-cell lysates were prepared as described in Materials and Methods. Quantitative Western blot analysis was performed by a standard procedure (11), using anti-AscG, anti-CRP, and anti-PrpR antibodies. The intensities of immunoblot bands were measured with an LAS-1000 Plus Lumino image analyzer and Image Gauge (Fuji Film).

FIG. 8.

Model of the regulatory roles of AscG. AscG was found to regulate not only the ascFB operon but also the prpR gene, which encodes a positive regulator of the prpBC operon for propionate catabolism. Two AscG-binding sites were identified in the ascF promoter, while three sites were identified in the prpR promoter.