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, ␣-D-galactose and ␤-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 ␣-D-galactose and ␤-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 ␣ configuration inactivate the induction capacity of the sugar, the effect of substitutions in the ␤ configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.D-Galactose, also known as cerebrose, is a natural aldohexose which is ubiquitous in bacteria, plants, and animals, including human brains (28). It occurs also as part of more complex carbohydrates, e.g., oligo-and polysaccharides, glycoproteins, and glycolipids, and as components of bacterial cell walls. DGalactose has two anomeric forms, ␣-D-galactopyranose and ␤-D-galactopyranose ( Fig. 1) (36). Although the anomers spontaneously interconvert slowly in aqueous solution, an enzyme (mutarotase or aldose-1-epimerase) is responsible for interconverting the two optical varieties in monosaccharide forms in vivo (6,21). Mutarotases are present throughout the prokaryotic and eukaryotic phyla (6,31,34,37). Mutarotase syntheses are also induced in response to specific signals. For example, galactose mutarotase synthesis is induced by retinoic acid in human myeloid cells (30). In the bacterium Escherichia coli, enzymes of D-galactose metabolism (15,20), including the mutarotase (6), are encoded in an operon which is induced by D-galactose. It has been suggested previously that only ␤-Dgalactose is the inducer of the gal operon (7). Since D-galactose moieties in complex carbohydrates are anomerically specific, ␣ or ␤, we hypothesize that the mutarotase enzyme, which interconverts the ␣ and ␤ anomers, must be induced for the interconversion when either one of the two anomers is present in the cell, say, by hydrolysis of D-lactose, which generates ␤-Dgalactose, or of D-melibiose, which generates ␣-D-galactose. In this study, we showed by both in vivo and in vitr...

Solid-state NMR spectroscopy reveals anomer specific transport of galactose in the milk yeast Kluyveromyces lactis

“…We therefore conclude that b-anomer of galactose is capable of inducing the GAL/LAC regulon. A similar conclusion was also drawn in the E. coli gal operon by Lee et al (2008), although the molecular mechanism for the galactose induction is fundamentally different in these organisms.…”

“…A similar conclusion was also drawn in the E. coli gal operon by Lee et al . (), although the molecular mechanism for the galactose induction is fundamentally different in these organisms.…”

Cellular stress created by intermediary metabolite imbalances

“…After the cells were grown to OD600 of 0.5 in M63 minimal medium containing 0.3% D-fructose and 0.1% casamino acids, 0.3% D-galactose was added and cells were further grown for 1.5 h. The activity of ␤-galactosidase synthesis in cells was measured as described previously (30). Gus assay of galP1 and -P2 was carried out as previously described (20).…”

“…Does accumulation of UDP-galactose or any other intermediate cause induction of gal transcription in the galE Ϫ mutant cells? In vitro transcription assays showed that only D-galactose and not any of the metabolic intermediates, galactose-1-phosphate, UDPgalactose, or UDP-glucose, acts as an inducer of P1 or P2 transcription (20). Thus the accumulated UDP-galactose is unlikely to modulate gal transcription directly in vivo.…”

“…If the presence of D-galactose makes galE Ϫ mutant UTP deficient, as shown above, then we hypothesize that the P2 promoter in gal would be further derepressed by eliminating stuttering. We fused a reporter gene, gus, encoding ␤-glucuronidase, individually to the P1 and P2 promoter at the gus locus in the E. coli chromosome (20). The fusions of P1 and P2 were introduced into wild-type gal ϩ and galE Ϫ mutant cells and ␤-glucuronidase activities were measured in the presence and absence of D-galactose.…”