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 For almost 50 years, methyl-␣-D-glucopyranoside (␣-MGlc)3 has served as a model substrate for probing the mechanism(s) of sugar transport in bacteria. Fortuitously (but importantly), ␣-MGlc is not metabolized by Escherichia coli K12, and transport data were interpretable without the ambiguities caused by further catabolism of the accumulated sugar. Early studies of ␣-MGlc uptake by cells of E. coli introduced the "permease" concept to bacterial physiologists, and "active transport" was incorporated in the lexicon of microbial energetics (1-5). The toxicity of ␣-MGlc toward E. coli was soon recognized, and Rogers and Yu (2) unexpectedly found that the glucose analog was recoverable from cells in both free and phosphorylated form. An understanding of these observations awaited the serendipitous discovery, and subsequent biochemical dissection, of the bacterial phosphoenolpyruvate-dependent sugar:phosphotransferase system (PEP-PTS; EC 2.7.1.69) by Roseman and co-workers (6, 7). The multicomponent PEP-PTS includes membrane-localized, sugar-specific transporters (enzymes IICB) that are fused or associated with a third domain (IIA) and two general cytoplasmic proteins (enzyme I and HPr). Collectively, these interactive components constitute a phospho-relay that (in five sequential stages) transfers the high energy phosphoryl moiety from PEP to catalyze the simultaneous phosphorylation and translocation of sugars through the cytoplasmic membrane (for reviews of PTS functions and nomenclature see . Completion of the chromosomal DNA sequence of E. coli K12 strain MG-1655 in 1997 (15), confirmed that ptsG encodes one (IICB Glc /PtsG) of two glucose-specific PTS transporters, whereas the genes for the general proteins and IIA Glc reside within a separate ptsHIcrr operon. After the discovery of other PEP-PT systems, it became apparent that ␣-MGlc was a reasonably specific substrate for the PtsG transporter in E. coli. , it was established that ␣-MGlc6P was accumulated via PtsG to toxic concentrations and that intracellular ␣-MGlc was formed via dephosphorylation of ␣-MGlc6P (Fig. 1, left).Our interest in the transport and bactericidal effects of ␣-MGlc(6P) in E. coli K12 stems from the fact that Klebsiella pneumoniae ATCC 23357 (a taxonomically close relative) readily ferments ␣-MGlc as an energy source for growth. Furthermore, and again in contrast with E. coli K12 strains, K. pneumoniae also metabolizes the following five O-␣-linked isomers of sucrose: trehalulose, turanose, maltulose, leucrose, and palatinose (20, 21). We presented evidence (21) for a tricistronic ␣-glucoside (Agl) operon comprising genes aglR, aglA, and aglB. We suggested that the three genes encode (in order), a transcriptional regulatory protein (AglR), an ␣-glucoside PTS transporter AglA (IICB Agl Swiss-Prot accession number Q9AGA7), and an Mn

Engineering of Escherichia coli to facilitate efficient utilization of isomaltose and panose in industrial glucose feedstock

“…3). Our results showed that the recombinant could assimilate many types of sugars and sugar alcohols, including α-methyl-glucoside, isomaltose, turanose, maltulose, and isomaltulose, which had been reported to be assimilated by AglA and AglB (Pikis et al 2006). In contrast, galactinol, maltotriitol, and erlose could not be assimilated by the recombinant.…”

“…Evaluation of the substrate specificity of GlvA and GlvC in E. coli Pikis et al reported that that expression of aglA and aglB from K. pneumoniae in E. coli allowed the cells to assimilate α-methyl-glucoside, isomaltose, trehalulose, turanose, maltulose, leucrose, and isomaltulose (Pikis et al 2006). To investigate whether the heterologous expression of glvA and glvC enabled E. coli to assimilate other types of carbon sources, particularly panose, we performed growth tests using M9 solid medium supplemented with various types of sugars and sugar alcohols as a sole carbon source.…”

“…The expression of either glvA or glvC alone is not sufficient to confer upon the recombinant the ability to assimilate any of the tested sugars and sugar alcohols except α-methyl-glucoside. It is known that α-methyl-glucoside is transported by PtsG of E. coli with concomitant phosphorylation (Pikis et al 2006). GlvA can hydrolyze the phosphorylated α-methyl-glucoside, conferring the ability to grow on the medium containing α-methyl-glucoside.…”

“…Maltose, isomaltose, and panose were measured using an ICS-3000 Ion Chromatography System with a CarboPac PA1 column (Dionex, CA, USA). Data of MG1655 (pAP2) which expresses aglA and aglB from K. pneumoniae are described in the reference (Pikis et al 2006) and listed in this table for comparison to show what kinds of sugars and sugar alcohols were newly assimilated by heterologous expression of glvA and glvC DP degree of polymerization, NDG no detectable growth, − minimal growth, + slight growth, ++ clear growth Fig. 3 Growth of E. coli recombinants on M9 solid medium containing panose as a sole carbon source.…”

“…There are 21 different EII complexes encoded in the E. coli chromosome; these complexes are involved in the transport of approximately 20 different carbohydrates (Escalante et al 2012). Pikis et al reported that the heterologous expression of Klebsiella pneumoniae aglA (a singlechain polypeptide of EIIC and EIIB that mediates the transport and phosphorylation of sucrose and various other α-linked glucosides) and aglB (a phospho-α-glucosidase) confers upon E. coli cells the ability to utilize isomaltose (Pikis et al 2006;Thompson et al 2001). Although E. coli K-12 strains have homologs of aglA and aglB , these seemed to be cryptic or nonfunctional truncated proteins (Reizer 1994;Thompson et al 1995).…”