Induction of an active transport system for glucose 6-phosphate in Escherichia coli

Pogell, Burton M.; Maity, Bijoy R.; Frumkin, Samuel; Shapiro, Susan

doi:10.1016/0003-9861(66)90047-6

Cited by 56 publications

(34 citation statements)

References 14 publications

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“…Both the Pit and Pst systems are highly specific for P i (30). Another two transporters accept P i as a low-affinity analogue for either glycerol-3-phosphate (glpT) (18) or glucose-6-phosphate (uhpT) (29,53), but in the absence of Pit and Pst activity, these latter two systems cannot support cell growth when supplied with P i (38).…”

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Characterization of PitA and PitB from Escherichia coli

Harris

Webb

Howitt³

et al. 2001

J Bacteriol

122

115

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Escherichia coli contains two major systems for transporting inorganic phosphate (P i ). The low-affinity P i transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the highaffinity Pst system (pstSCAB) is induced at low external P i concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P i transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P i transporter that may be repressed at low P i levels by the pho regulon. While a pitB ؉ cosmid clone allowed growth on medium containing 500 M P i , E. coli with wild-type genomic pitB (pitA ⌬pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB ⌬pstC345 triple mutant. The mutation ⌬pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB ؉ pitA ⌬pstC345 strain to utilize P i , with P i uptake rates significantly higher than background levels. In addition, the apparent K m of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.Escherichia coli contains at least two major systems for transporting inorganic phosphate (P i ). The low-affinity inorganic phosphate transporter (Pit) is dependent on the proton motive force for energy and is constitutively expressed (30,31,49). When P i is plentiful, this is the major uptake system for phosphate, with a reported apparent K m (K m app ) of 25 M (30) to 38 M (50) in whole cells and 11.9 M in membrane vesicles (43). If the external P i concentration is below the millimolar range, the high-affinity phosphate-specific transport (Pst) system is induced. This has a K m app of around 0.2 M (30, 50). The Pst system is a complex of four proteins, including a periplasmic binding protein, which is energized by ATP and belongs to the ABC transporter family (7,15,47). The pst operon contains five genes under pho regulon control (1,40,41), which induces a range of genes when the phosphate supply is limited. Both the Pit and Pst systems are highly specific for P i (30). Another two transporters accept P i as a low-affinity analogue for either glycerol-3-phosphate (glpT) (18) or glucose-6-phosphate (uhpT) (29, 53), but in the absence of Pit and Pst activity, these latter two systems cannot support cell growth when supplied with P i (38).Divalent cations, such as Mg 2ϩ or Ca 2ϩ

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confidence: 99%

Characterization of PitA and PitB from Escherichia coli

Harris

Webb

Howitt³

et al. 2001

J Bacteriol

122

115

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“…Cells of Escherichia coli possess an inducible active transport system which is required for growth on glucose 6-phosphate (Glu6P), fructose 6-phosphate (Fru6P), and several other phosphorylated sugars and related compounds (4,7,21). This transport system, encoded by the uhpT gene, consists of a single polypeptide chain of 463 amino acids.…”

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Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium

1992

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Expression of the Escherichia coli sugar phosphate transport system, encoded by the uhpT gene, is regulated by external glucose 6-phosphate through the action of three linked regulatory genes, uhpABC. The nucleotide sequence of the uhp region cloned from Sabnonella typhimurium was determined. The deduced Uhp polypeptide sequences from the two organisms are highly related. Comparison with the corrected sequence from E. coli revealed that the four uhp genes are closely spaced, with minimal intergenic distances, and that uhpC is nearly identical in length to uhpT, both of which have substantial sequence relatedness along their entire lengths. To facilitate analysis of uhp gene function, we isolated insertions of a kanamycin resistance (Km) cassette throughout the uhp region. In-frame deletions that removed almost the entire coding region of individual or multiple uhp genes were generated by use of restriction sites at the ends of the Km cassette. The phenotypes of the Km insertions and the in-frame deletions confirmed that all three regulatory genes are required for Uhp function. Whereas the deletion of uhpA completely abolished the expression of a uhpT-lacZ reporter gene, the deletion ofuhpB or uhpC resulted in a partially elevated basal level ofexpression that was not further inducible.These results indicated that UhpB and perhaps UhpC play both positive and negative roles in the control of uhpT transcription., Translational fusions of the uhpBCT genes to topological reporter gene phoA were generated by making use of restriction sites provided by the Km cassette or with transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with models predicting that UhpC and UhpT have identical transmembrane topologies, with 10 to 12 transmembrane segments, and that UhpB has 4 to 8 amino-terminal transmembrane segments that anchor the polar carboxyl-terminal half of the protein to the cytoplasmic side of the inner membrane.

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“…Although the UhpT transport system mediates the entry of a number of different sugar phosphates, the synthesis of the transporter is induced only by exogenous glucose 6-phosphate (Glu6P) or 2-deoxyglucose 6-phosphate (21,42). The large pool of Glu6P normally present inside a cell does not trigger induction.…”

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Promoter elements required for positive control of transcription of the Escherichia coli uhpT gene

et al. 1992

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The uhpABCT locus of Escherichia coli encodes the transport system which allows the cell to accumulate a variety of sugar phosphates in unaltered form. The expression of uhpT, the gene encoding the transport protein, is regulated by the uhpABC gene products. The UhpA protein is required for expression; its deduced amino acid sequence shows that it belongs to a subfamily of bacterial transcription regulators including NarL, DegU, and FixJ. Members of this subfamily have an amino-terminal phosphorylation domain characteristic of so-called two-component regulators, such as OmpR, CheY, PhoB, and NtrC, and a carboxyl-terminal domain conserved among many transcriptional activators, including LuxR and MalT. The major sequence elements in the uhpT promoter that are needed for uhpT expression were investigated. Northern (RNA) hybridization analysis showed that the uhpT transcript was only present in cells induced for UhpT transport activity. The start site of transcription was identified by primer extension. Comparison of the regions upstream of the uhpT transcription start site in E. coli and SalmoneUla typhimurium suggested the presence of four sequence elements that might be involved in promoter function: a typical -10 region, a short inverted repeat centered at -32, a long inverted repeat centered at -64, and a cyclic AMP receptor protein-binding sequence centered at -103. Deletion and linker substitution mutations in the promoter demonstrated that the presence of the cyclic AMP receptor protein-binding site resulted in about an eightfold increase in promoter activity and that the -64, -32, and -10 elements were essential for promoter function. In vivo titration of transcriptional activator UhpA by the intact or mutant promoters on multicopy plasmids identified the -64 element as the UhpA-binding site. The two halves of the -64 inverted repeat did not contribute equally to promoter function and did not have to be intact for UhpA titration. The sequence recognized by UhpA is predicted to be 5'-GGCAAAACNNNGAAA.

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Induction of an active transport system for glucose 6-phosphate in Escherichia coli

Cited by 56 publications

References 14 publications

Characterization of PitA and PitB from Escherichia coli

Characterization of PitA and PitB from Escherichia coli

Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium

Promoter elements required for positive control of transcription of the Escherichia coli uhpT gene

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