The gntP gene of Escherichia coli involved in gluconate uptake

Abstract

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 ...

GntP Is the Escherichia coli Fructuronic Acid Transporter and Belongs to the UxuR Regulon

“…Sequence analysis indicated a CRP binding site in the gntP regulatory region. This location, at position Ϫ42 with respect to the previously mapped transcription start site (10), is indicative of class II activation (3). A gntP-lacZ operon fusion was repressed 7-and 3.5-fold in the presence of glucose or gluconate, respectively, by comparison to LB medium alone (Table 2).…”

“…In particular, we are interested in gntP, which was implicated as being important for colonization of the mouse intestine by a human commensal E. coli strain (30). gntP is one of four genes which encode gluconate transporters (20), but previous studies showed that it is not induced by gluconate (10).…”

“…The gntP gene is predicted to be induced by glucuronate (27), but this has not been established experimentally. However, GntP does not appear to be involved in glucuronate catabolism: GntP is not homologous to the known glucuronate transporters, ExuT and KdgT, and GntP does not appear to transport glucuronate (10). Since GntP does not transport glucuronate and is not induced by gluconate, we explored the possibility that GntP transports one of the other sugar acids that support growth of E. coli, i.e., fructuronate (17).…”

“…The gntP gene is adjacent to and divergently transcribed from uxuAB (2,10). The uxuA and uxuB genes are negatively regulated by the product of the downstream gene, uxuR, and are induced for growth on glucuronate (1,2,8,(23)(24)(25)(26)(27).…”

Purification and characterization of the Escherichia coli thermoresistant gluconokinase encoded by the gntK gene

“…The GntlI was demonstrated to be repressed by the presence of pyruvate [3,8]. Recently, a thermoresistant gluconokinase was purified from E. coli and characterized [10], and another permease encoded by the gntP gene with the high affinity for gluconate was uncovered in E. coli [11].…”