Genes Involved in the Uptake and Catabolism of Gluconate by Escherichia coli

Bächi, Brigitte; Kornberg, H. L.

doi:10.1099/00221287-90-2-321

Cited by 38 publications

(35 citation statements)

References 16 publications

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“…GntII, the subsidiary system, contains gntW and gntV, which are believed to encode another high-affinity gluconate transporter (41) and a thermosensitive gluconokinase (17,42), respectively. It is not known how GntII is regulated, although it has been reported that pyruvate might act as a metabolic repressor and that the gntS gene product might positively control expression of gntV (1,17). A fourth high-affinity gluconate transporter, gntP, was recently identified and, interestingly, was found not to be inducible by gluconate (18).…”

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confidence: 99%

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Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism

et al. 1997

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The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent K m of 6 M for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have ؊10 and ؊35 sequence elements typical of 70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 M gluconate, modestly induced by very low levels of gluconate (4 M), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

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“…coli possesses two systems for gluconate transport and phosphorylation, specified by two distinctly regulated sets of genes which are located in different regions of the genome (1,17,44). GntI, the main system, contains gntT, gntU, and gntK which code for high-and low-affinity gluconate transporters and a thermoresistant gluconokinase, respectively.…”

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Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism

et al. 1997

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“…For three decades, there was thought to be two systems for D-gluconate catabolism, GntI and GntII (1). The GntI system consists of gntT, gntU, and gntK, which encode high-and lowaffinity D-gluconate transporters and a thermoresistant gluconate kinase, respectively (19)(20)(21)29).…”

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Transcriptional Organization and Regulation of the l -Idonic Acid Pathway (GntII System) in Escherichia coli

2004

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The genetic organization of the idn genes that encode the pathway for L-idonate catabolism was characterized. The monocistronic idnK gene is transcribed divergently from the idnDOTR genes, which were shown to form an operon. The 215-bp regulatory region between the idnK and idnD genes contains promoters in opposite orientation with transcription start sites that mapped to positions ؊26 and ؊29 with respect to the start codons. The regulatory region also contains a single putative IdnR/GntR binding site centered between the two promoters, a CRP binding site upstream of idnD, and an UP element upstream of idnK. The genes of the L-idonate pathway were shown to be under catabolite repression control. Analysis of idnD-and idnK-lacZ fusions in a nonpolar idnD mutant that is unable to interconvert L-idonate and 5-ketogluconate indicated that either compound could induce the pathway. The L-idonate pathway was first characterized as a subsidiary pathway for D-gluconate catabolism (GntII), which is induced by D-gluconate in a GntI (primary gluconate system) mutant. Here we showed that the idnK and idnD operons are induced by D-gluconate in a GntI system mutant, presumably by endogenous formation of 5-ketogluconate from D-gluconate. Thus, the regulation of the GntII system is appropriate for this pathway, which is primarily involved in L-idonate catabolism; the GntII system can be induced by D-gluconate under conditions that block the GntI system.For three decades, there was thought to be two systems for D-gluconate catabolism, GntI and GntII (1). The GntI system consists of gntT, gntU, and gntK, which encode high-and lowaffinity D-gluconate transporters and a thermoresistant gluconate kinase, respectively (19)(20)(21)29). GntR negatively controls the GntI genes, as well as edd and eda of the EntnerDoudoroff pathway. The GntII system is composed of a thermosensitive gluconate kinase and a gluconate transporter, which provide for subsidiary catabolism of gluconate in GntI mutants (3,16). Recently, we discovered that the GntII system is, in fact, a pathway for catabolism of L-idonate, which proceeds via a D-gluconate intermediate (3). The discovery of this novel pathway solved the longstanding question of why there are two pathways for gluconate; GntI is primarily involved in gluconate catabolism, and GntII is responsible for idonate catabolism.The catabolic sequence for L-idonate is as follows: L-idonate is transported by the L-idonate transporter, IdnT; L-idonate is oxidized to 5-keto-gluconate (5KG) by L-idonate 5-dehydrogenase, IdnD; 5KG is reduced to D-gluconate by 5-keto-D-gluconate 5-reductase, IdnO; and D-gluconate is phosphorylated by a thermosensitive gluconate kinase, IdnK, to make 6-phosphogluconate (6PG), which is further catabolized via the Entner-Doudoroff pathway. Thus, IdnD and IdnO allow for the redox-coupled interconversion of L-idonate to D-gluconate via 5KG. The L-idonate catabolic pathway overlaps D-gluconate catabolism through the common intermediates D-gluconate and 6PG.While the biochemistry of the L...

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“…E. coli M6 is unable to grow on gluconate minimal medium because the mutations in gntT and gntU, the two transporters of the GntI system, eliminate its ability to transport gluconate in batch cultures with gluconate as the sole carbon and energy source. The gluconate transporter(s) of the GntII system (subsidiary system) are not induced under these growth conditions (1,15). Likewise, the gluconate transporter encoded by the gntP gene described in this report is subject to strong catabolite repression and is repressed when growing on gluconate or other sugars.…”

Section: Methodsmentioning

confidence: 83%

“…The GntII system, a subsidiary system for gluconate transport and phosphorylation, located in the 96-min region, includes the gntS gene, encoding another high-affinity transport system, and the gntV gene, encoding a thermosensitive glucokinase (15). Various observations have suggested that the genes of the GntI and the GntII systems are differentially regulated (1,5,19). The GntI system is specifically induced by gluconate and is regulated by the gntR gene (also located in the 75-min region) product, a repressor of the genes of the GntI system and the Entner-Doudoroff pathway but not of the genes of the GntII system (9, 19, 32).…”

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The gntP gene of Escherichia coli involved in gluconate uptake

et al. 1996

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The gntP gene, located between the fim and uxu loci in Escherichia coli K-12, has been cloned and characterized. Nucleotide sequencing of a region encompassing the gntP gene revealed an open reading frame of 447 codons with significant homology to the Bacillus subtilis gluconate permease. Northern (RNA) blotting indicated that the gntP gene was monocistronic and was transcribed as an mRNA with an apparent molecular size of 1.54 kb. The transcriptional start point was determined by primer extension analysis. The gntP gene was found to be under catabolite repression and was not induced by gluconate. Also, expression seemed to be stringently controlled. Several observations indicated that the GntP protein is an inner membrane protein; it contains characteristic membrane-spanning regions and was isolated predominantly from the inner-membrane fraction of fractionated host cells. A topology analysis predicted a protein with 14 membrane-spanning segments. The inability of a mutant strain to grow on gluconate minimal medium could be relieved by introduction of a plasmid encoding the gntP gene. Finally, the kinetics of GntP-mediated gluconate uptake were investigated, indicating an apparent K m for gluconate of 25 M.Gluconate is an important food source of Escherichia coli in its natural environment, the gut (26). In order to shunt gluconate into the main metabolic routes, the bacterium must carry out two initial operations. In the first, extracellular gluconate is imported into the cytoplasm, and in the second, the gluconate is phosphorylated to 6-phosphogluconate by a kinase. 6-Phosphogluconate can enter two central metabolic pathways; it is catabolized primarily through the Entner-Doudoroff pathway but also through the pentose phosphate pathway. Therefore, only two enzyme functions, a permease and a kinase, seem to be specifically required for gluconate catabolism. However, both of these functions are carried out by more than one enzyme. In fact, no less than three gluconate permeases and two kinases have been described hitherto. The 75-min region of E. coli K-12 contains the genes of the GntI system, i.e., gntT and gntU, encoding high-and low-affinity transport systems, respectively, and gntK, which encodes a gluconokinase. The GntII system, a subsidiary system for gluconate transport and phosphorylation, located in the 96-min region, includes the gntS gene, encoding another high-affinity transport system, and the gntV gene, encoding a thermosensitive glucokinase (15). Various observations have suggested that the genes of the GntI and the GntII systems are differentially regulated (1,5,19). The GntI system is specifically induced by gluconate and is regulated by the gntR gene (also located in the 75-min region) product, a repressor of the genes of the GntI system and the Entner-Doudoroff pathway but not of the genes of the GntII system (9, 19, 32). It is not known how the expression of the GntII system is controlled (15).At present it is not clear why this multitude of genes with seemingly redundant functions exists ...

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Genes Involved in the Uptake and Catabolism of Gluconate by Escherichia coli

Cited by 38 publications

References 16 publications

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism

Transcriptional Organization and Regulation of the l -Idonic Acid Pathway (GntII System) in Escherichia coli

The gntP gene of Escherichia coli involved in gluconate uptake

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