Genetic Requirements for Growth of Escherichia coli K12 on Methyl-α-D-glucopyranoside and the Five α-D-Glucosyl-D-fructose Isomers of Sucrose

Abstract

Strains of Escherichia coli K12, including MG-1655, accumulate methyl-α-d-glucopyranoside via the phosphoenolpyruvate-dependent glucose:phosphotransferase system (IICBGlc/IIAGlc). High concentrations of intracellular methyl-α-d-glucopyranoside 6-phosphate are toxic, and cell growth is prevented. However, transformation of E. coli MG-1655 with a plasmid (pAP1) encoding the gene aglB from Klebsiella pneumoniae resulted in excellent growth of the transformant MG-1655 (pAP1) on the glucose analog. AglB is an unusual NAD+/Mn2+-dependent phospho-α-glucosidase that promotes growth of MG-1655 (pAP1) by catalyzing the in vivo hydrolysis of methyl-α-d-glucopyranoside 6-phosphate to yield glucose 6-phosphate and methanol. When transformed with plasmid pAP2 encoding the K. pneumoniae genes aglB and aglA (an α-glucoside-specific transporter AglA (IICBAgl)), strain MG-1655 (pAP2) metabolized a variety of other α-linked glucosides, including maltitol, isomaltose, and the following five isomers of sucrose: trehalulose α(1→1), turanose α(1→3), maltulose α(1→4), leucrose α(1→5), and palatinose α(1→6). Remarkably, MG-1655 (pAP2) failed to metabolize sucrose α(1→2). The E. coli K12 strain ZSC112L (ptsG::cat manXYZ nagE glk lac) can neither grow on glucose nor transport methyl-α-d-glucopyranoside. However, when transformed with pTSGH11 (encoding ptsG) or pAP2, this organism provided membranes that contained either the PtsG or AglA transporters, respectively. In vitro complementation of transporter-specific membranes with purified general phosphotransferase components showed that although PtsG and AglA recognized glucose and methyl-α-d-glucopyranoside, only AglA accepted other α-d-glucosides as substrates. Complementation experiments also revealed that IIAGlc was required for functional activity of both PtsG and AglA transporters. We conclude that AglA, AglB, and IIAGlc are necessary and sufficient for growth of E. coli K12 on methyl-α-d-glucoside and related α-d-glucopyranosides. Strains of Escherichia coli K12, including MG-1655, accumulate methyl-α-d-glucopyranoside via the phosphoenolpyruvate-dependent glucose:phosphotransferase system (IICBGlc/IIAGlc). High concentrations of intracellular methyl-α-d-glucopyranoside 6-phosphate are toxic, and cell growth is prevented. However, transformation of E. coli MG-1655 with a plasmid (pAP1) encoding the gene aglB from Klebsiella pneumoniae resulted in excellent growth of the transformant MG-1655 (pAP1) on the glucose analog. AglB is an unusual NAD+/Mn2+-dependent phospho-α-glucosidase that promotes growth of MG-1655 (pAP1) by catalyzing the in vivo hydrolysis of methyl-α-d-glucopyranoside 6-phosphate to yield glucose 6-phosphate and methanol. When transformed with plasmid pAP2 encoding the K. pneumoniae genes aglB and aglA (an α-glucoside-specific transporter AglA (IICBAgl)), strain MG-1655 (pAP2) metabolized a variety of other α-linked glucosides, including maltitol, isomaltose, and the following five isomers of sucrose: trehalulose α(1→1), turanose α(1→3), maltulose α(1→4), leucrose α(1→5), and palatinose α(1→6). Remarkably, MG-1655 (pAP2) failed to metabolize sucrose α(1→2). The E. coli K12 strain ZSC112L (ptsG::cat manXYZ nagE glk lac) can neither grow on glucose nor transport methyl-α-d-glucopyranoside. However, when transformed with pTSGH11 (encoding ptsG) or pAP2, this organism provided membranes that contained either the PtsG or AglA transporters, respectively. In vitro complementation of transporter-specific membranes with purified general phosphotransferase components showed that although PtsG and AglA recognized glucose and methyl-α-d-glucopyranoside, only AglA accepted other α-d-glucosides as substrates. Complementation experiments also revealed that IIAGlc was required for functional activity of both PtsG and AglA transporters. We conclude that AglA, AglB, and IIAGlc are necessary and sufficient for growth of E. coli K12 on methyl-α-d-glucoside and related α-d-glucopyranosides. For almost 5