Regulation of arabinose and xylose metabolism in Escherichia coli

Abstract

Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.

FIG. 1.

Regulation of the arabinose and xylose metabolic gene circuits. (A) The arabinose metabolic and transporter genes are regulated by AraC (35). When bound with arabinose, AraC activates the transcription of the araBAD, araE, and araFGH operons and represses transcription from the araC operon. Expression of the AraE and AraFGH transporters increases the rate of arabinose uptake, further enhancing activation. (B) The xylose metabolic genes and transporters are regulated by XylR, an AraC-type positive regulator, in a manner analogous to that of arabinose (38). Xylose-bound XylR is believed to activate the transcription of the xylFGH, xylR, xylAB, and xylE operons. As with arabinose, expression of the XylE and XylFGH transporters increases the rate of xylose uptake and further enhances activation.

FIG. 2.

Comparison of P_xylA (A) and P_araB (B) promoter activity dynamics in response to different sugars.

FIG. 3.

Effects of varying arabinose and xylose concentrations on P_xylA (left) and P_araB (right) promoter activities. These experiments were performed using two transcriptional reporters: a GFP fusion to the P_araB promoter and an mCherry fusion to the P_xylA promoter. Expression is represented as relative fluorescence. Results are values obtained after 10 h of growth in 96-well microplates and are averages for three independent experiments. For all results, the standard deviations were less than 15 percent of the mean (data not shown).

FIG. 4.

Comparison of xylose and arabinose utilization. Symbols represent the concentration of arabinose when it is the sole sugar (▴) or when an equimolar mixture of arabinose and xylose is used (▪) and the concentration of xylose when it is the sole sugar (⋄) or when an equimolar mixture of arabinose and xylose is used (×).

FIG. 5.

Effects of varying arabinose and xylose concentrations on P_xylA promoter activity in a ΔaraC ΔaraBAD mutant. These experiments were performed using two transcriptional reporters: a GFP fusion to the P_araB promoter and an mCherry fusion to the P_xylA promoter. The results for the P_araB promoter are not shown, because the promoter is inactive and unresponsive to arabinose in the ΔaraC ΔaraBAD mutant. Results are values obtained after 10 h of growth in 96-well microplates and are averages for three independent experiments. For all results, the standard deviations were less than 15% of the mean for the concentrations tested (data not shown).

FIG. 6.

Comparison of P_xylA promoter activity in different mutants deficient in arabinose metabolism. Results are values obtained after 10 h of growth in 96-well microplates and are averages for three independent experiments.

FIG. 7.

Comparison of P_xylA promoter activities in strains with different arabinose transporter deletions. (A) Wild type; (B) ΔaraFGH; (C) ΔaraE; (D) ΔaraE ΔaraFGH. Results are values obtained after 10 h of growth in 96-well microplates and are averages for three independent experiments.

FIG. 8.

Comparison of xylose (A) and arabinose (B) utilization in a ΔaraE mutant. Symbols represent the concentration of arabinose when it is the sole sugar (▴) or when an equimolar mixture of arabinose and xylose is used (▪) and the concentration of xylose when it is the sole sugar (⋄) and when an equimolar mixture of arabinose and xylose is used (×).

FIG. 9.

Repression of the P_xylA promoter by constitutively active variants of AraC in the absence of arabinose. These experiments were performed using two transcriptional reporters: a YFP fusion to the P_xylA promoter and an mCherry fusion to the P_araB promoter. To facilitate comparison, the fluorescence values were normalized by their maximal value (raw data are given in the text). Results are values obtained after 5 h of growth in test tubes and are averages for three independent experiments.

FIG. 10.

DNA mobility shift assay demonstrating the binding of AraC to the P_xylA promoter using whole-cell extracts. The binding reaction mixture contained 0.1 ng of P³²-labeled DNA encompassing the P_xylA promoter. Lanes: A, no lysate; B, wild type; C, ΔaraC pProtet.E (empty plasmid); D, ΔaraC pAraC (plasmid expressing AraC); E, wild type plus 10 ng of the unlabeled I1-I1 AraC binding site; F, ΔaraC pAraC plus 10 ng of the unlabeled I1-I1 AraC binding site.

FIG. 11.

Schematic of the P_xylA, P_araB, and P_araE promoters. The CRP binding sites are shown in boldface, the XylR operator sites with straight underlines, and the AraC operator sites with wavy underlines. Annotations are based on RegulonDB (14). The putative AraC binding site, as determined through sequence analysis, is boxed.