Induction of the galactose enzymes in Escherichia coli is independent of the C-1-hydroxyl optical configuration of the inducer D-galactose

Abstract

The two optical forms of aldohexose galactose differing at the C-1 position, alpha-D-galactose and beta-D-galactose, are widespread in nature. The two anomers also occur in di- and polysaccharides, as well as in glycoconjugates. The anomeric form of D-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other D-galactose-metabolizing enzymes are coded by genes that constitute an operon in Escherichia coli. The operon is repressed by the repressor GalR and induced by D-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of D-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the gal operon, specifically the mutarotase, be achievable by either anomer of D-galactose. Here we report in vivo and in vitro experiments showing that both alpha-D-galactose and beta-D-galactose are capable of inducing transcription of the gal operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the alpha configuration inactivate the induction capacity of the sugar, the effect of substitutions in the beta configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.

FIG. 1.

Generation of α and β anomers of d-galactose in cellular metabolism.

FIG. 2.

(A) Sequence of the translational fusion of gal promoters (P2⁺ P1⁺, P2⁺ P1⁻, and P2⁻ P1⁺) to the gusA reporter gene. (B) Genotype of E. coli SL3304 showing the relevant chromosomal markers, gal promoters fused to gusA at the gus locus, gal promoters directly fused to the galM gene but with the galETK region deleted, lacI::Tn10, and the mel locus. (C) Plasmid pJW-4* having the melR(Con) mutation.

FIG. 3.

In vitro transcription assays of gal (P2⁻ P1⁺) template DNA in the presence of GalR and various forms of d-galactose. α-MG, methyl-α-d-galactopyranoside; β-MG, methyl-β-d-galactopyranoside; α-PG, phenyl-α-d-galactopyranoside; β-PG, phenyl-β-d-galactopyranoside; 2DG, 2-deoxy-d-galactose.

FIG. 4.

In vitro derepression of the gal P1 promoter by α- and β-d-galactose.

FIG. 5.

Kinetics of dissociation of GalR from O_I by α- and β-d-galactose.

FIG. 6.

¹H NMR spectra of α- and β-d-galactose.