Genetic requirements for growth of Escherichia coli K12 on methyl-alpha-D-glucopyranoside and the five alpha-D-glucosyl-D-fructose isomers of sucrose.
Strains of Escherichia coli K12, including MG-1655, accumulate methyl-alpha-D-glucopyranoside via the phosphoenolpyruvate-dependent glucose:phosphotransferase system (IICB(Glc)/IIA(Glc)). High concentrations of intracellular methyl-alpha-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-alpha-glucosidase that promotes growth of MG-1655 (pAP1) by catalyzing the in vivo hydrolysis of methyl-alpha-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 alpha-glucoside-specific transporter AglA (IICB(Agl))), strain MG-1655 (pAP2) metabolized a variety of other alpha-linked glucosides, including maltitol, isomaltose, and the following five isomers of sucrose: trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5), and palatinose alpha(1-->6). Remarkably, MG-1655 (pAP2) failed to metabolize sucrose alpha(1-->2). The E. coli K12 strain ZSC112L (ptsG::cat manXYZ nagE glk lac) can neither grow on glucose nor transport methyl-alpha-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-alpha-D-glucopyranoside, only AglA accepted other alpha-D-glucosides as substrates. Complementation experiments also revealed that IIA(Glc) was required for functional activity of both PtsG and AglA transporters. We conclude that AglA, AglB, and IIA(Glc) are necessary and sufficient for growth of E. coli K12 on methyl-alpha-D-glucoside and related alpha-D-glucopyranosides.
Pikis
Andreas
A
Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research/NIH, Bldg. 30, Convent Drive, Bethesda, MD 20892, USA.
Hess
Sonja
S
Arnold
Ingrid
I
Erni
Bernhard
B
Thompson
John
J
D000595:Amino Acid Sequence; D048168:Escherichia coli K12 / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005632:Fructose / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D007536:Isomerism; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D008757:Methylglucosides / Q000378:metabolism*; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism*; D014170:Transformation, Genetic; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D008757:Methylglucosides; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D005632:Fructose; C027020:methylglucoside; D013395:Sucrose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022359:phosphoenolypyruvate-alpha-methyl-D-glucoside phosphotransferase system; D000520:alpha-Glucosidases