Na(+)/glutamine (asparagine) cotransport by Staphylococcus lugdunensis and Corynebacterium amycolatum.

Staphylococcus lugdunensis and Corynebacterium amycolatum each have a Na(+)/glutamine cotransporter that displays an ordered reaction sequence at the extracellular surface, with sodium binding (K(m) of 6.5 mM) before glutamine (K(m) of 50 microM). Asparagine is low-affinity substrate (K(m) approximately 1 mM) for each system.

Structure/function relationships in OxlT, the oxalate-formate transporter of oxalobacter formigenes. Assignment of transmembrane helix 11 to the translocation pathway.

OxlT, the oxalate:formate antiporter of Oxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 --> Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate.

Primary structure and properties of the Na+/glucose symporter (Sg1S) of Vibrio parahaemolyticus.

Previously, we cloned and sequenced a DNA fragment from Vibrio parahaemolyticus and found four open reading frames (ORFs). Here, we clearly demonstrate that one of the ORFs, ORF1, is the gene (sglS) encoding a Na+/glucose symporter (SglS). We characterize the Na+/glucose symporter produced in Escherichia coli mutant (JM1100) cells which lack original glucose transport activity and galactose transport activity. We also show that phlorizin, a potent inhibitor of the SGLT1 Na+/glucose symporter of animal cells, inhibited glucose transport, but not galactose transport, via the SglS system.

Sequence of a Na+/glucose symporter gene and its flanking regions of Vibrio parahaemolyticus.

The nucleotide sequence of an approximately 6 kbp segment of chromosomal DNA of Vibrio parahaemolyticus was determined. The nucleotide sequence revealed four open reading frames (ORFs) in this region. Hydropathy profiles of the deduced amino acid sequence of the ORFs indicate that ORF1 encodes a hydrophobic polypeptide with typical characteristics of a membrane transport protein. All other ORFs encode hydrophilic polypeptides. ORF1 showed significant amino acid sequence similarity to proteins of the SGLT (Na+/glucose symporter) family, and the amino acid sequence of ORF4 showed very high similarity to several bacterial transcriptional repressor proteins (GalR-LacI family). We observed elevated glucose transport activity in cells harboring a plasmid carrying the DNA region corresponding to ORF1, and the glucose transport was greatly stimulated by Na+. Thus, we believe that ORF1 encodes a Na+/glucose symporter.

Properties of a Na+/galactose (glucose) symport system in Vibrio parahaemolyticus.

We have investigated galactose transport in a mutant strain of Vibrio parahaemolyticus that lacks a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system) and a trehalose-PTS. Cells of the V. parahaemolyticus actively transported D-galactose and Na+ greatly stimulated the transport. Maximum stimulation of D-galactose transport activity was observed at 10mM NaCl, and Na+ could be replaced with Li+. Addition of galactose to the cell suspension under anaerobic conditions elicited Na+ uptake. Therefore, we conclude that this organism accomplishes galactose transport by a Na+/solute symport mechanism. Judging from inhibition results, D-galactose, D-glucose and to a lesser extent alpha-D-fucose are substrates of this transport system. The Na+/galactose symport system exhibited a high affinity for D-galactose (Km: 40 microM) and showed a relatively lower affinity for D-glucose (Km: 420 microM), but the maximum velocities for galactose and glucose transport were almost same (about 52 nmol/min per mg protein). The Na+/D-galactose symport system was induced by either D-galactose or alpha-D-fucose, and repressed by D-glucose.

Characterization of a glucose transport system in Vibrio parahaemolyticus.

Cells of a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system)-negative mutant of Vibrio parahaemolyticus transport D-glucose in the presence of Na+. Maximum stimulation of D-glucose transport was observed at 40 mM NaCl, and Na+ could be replaced partially with Li+. Addition of D-glucose to the cell suspension under anaerobic conditions elicited Na+ uptake. Thus, we conclude that glucose is transported by a Na+/glucose symport mechanism. Calculated Vmax and Km values for the Na(+)-dependent D-glucose transport were 15 nmol/min/mg of protein and 0.57 mM, respectively, when NaCl was added at 40 mM. Na+ lowered the Km value without affecting the Vmax value. D-Glucose was the best substrate for this transport system, followed by galactose, alpha-D-fucose, and methyl-alpha-glucoside, judging from the inhibition pattern of the glucose transport. D-Glucose itself partly repressed the transport system when cells were grown in its presence.