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

Abstract

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.

Microinjection and growth of bacteria in the cytosol of mammalian host cells

“…PrfA is known to be active particularly when the bacteria grow in the cytosol of host cells (8,15), and the induced expression of this sugar uptake system by the host cell cytosol environment may enable L. monocytogenes to utilize glucose-1-phosphate (24), which is possibly derived from glycogen of the host cell. Similar uptake systems for phosphorylated sugars are also present in E. coli (27) and possibly other bacteria, but their expression in the cytosolic milieu of host cells may be less efficient than the PrfA-regulated uptake system of L. monocytogenes. Further experiments will show whether this PrfA-controlled uptake of glucose-1-phosphate is the only specific metabolic requirement for cytosolic growth of L. monocytogenes.…”

Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation inListeria

“…The OPA family also includes the extensively characterized HP transporter UhpT from E. coli (18,35,36). We in fact identified the listerial hpt gene by the structural similarities of its encoded product with the enterobacterial UhpT permease.…”

“…1C), plus four new putative members of the PrfA regulon. One gene encoded a protein similar to UhpT, a hexose phosphate permease found in enteric bacteria (18) (Fig. 1 A).…”

Topology of OxlT, the Oxalate Transporter of Oxalobacter formigenes , Determined by Site-Directed Fluorescence Labeling

“…Because of its assignment to the MFS, one expects OxlT to show a topological organization that includes 12 transmembrane segments arranged in two groups of six segments each (24,25,30), and because circular dichroism spectroscopy (8) shows OxlT to be about 65% ␣-helix, one also expects these segments to be structured as transmembrane ␣-helices. A topological model of OxlT had provisionally identified the expected transmembrane segments, based on the interpretive tools noted above and also by comparisons with the experimentally determined topologies of other bacterial examples in the MFS, including LacY (5), UhpT and GlpT (12,15), and RhaT (34). In the latter cases, as in many others, topology had been extensively probed by use of fusions with a reporter protein, but it seemed possible that this approach could be less informative for OxlT, which has two (putative) transmembrane segments (TM2 and TM11) enriched for polar residues.…”