The nptA gene of Vibrio cholerae encodes a functional sodium-dependent phosphate cotransporter homologous to the type II cotransporters of eukaryotes

Abstract

The nptA gene of Vibrio cholerae has significant protein sequence homology with type II sodium-dependent phosphate (P(i)) cotransporters found in animals but not previously identified in prokaryotes. The phylogeny of known type II cotransporter sequences indicates that nptA may be either an ancestral gene or a gene acquired from a higher eukaryotic source. The gene was cloned into an expression vector under the control of an inducible promoter and expressed in Escherichia coli. The results demonstrate that nptA encodes a functional protein with activity similar to that of the animal enzyme, catalyzing high-affinity, sodium-dependent P(i) uptake with comparable affinities for both sodium and phosphate ions. Furthermore, the activity of NptA is influenced by pH, again in a manner similar to that of the NaPi-2a subtype of the animal enzyme, although it lacks the corresponding REK motif thought to be responsible for this phenomenon. P(i) uptake activity, a component of which appeared to be sodium dependent, was increased in V. cholerae by phosphate starvation. However, it appears from the use of a reporter gene expressed from the nptA promoter that none of this activity is attributable to the induction of expression from nptA. It is thus proposed that the physiological function of NptA protein may be the rapid uptake of P(i) in preparation for rapid growth in nutrient-rich environments and that it may therefore play a role in establishing infection.

FIG. 1.

Physical map of the nptA locus on the V. cholerae chromosome, showing the relative positions of lgt-thyA and nhaR. Restriction sites used in the cloning and mapping procedures: P, PstI; E, EcoRI; Bg, BglII; A, AatII; H, HindIII; and X, XbaI. The short lines below the main restriction map indicate the DNA amplified from the V. cholerae chromosome for insertion into the expression vector pML-tac1. The EcoRI site at the 5′ end of nptA occurs naturally immediately upstream of the ribosome binding site; the SpeI site at the 3′ end was generated by the PCR to allow directional insertion into the vector. The dmpB reporter gene was inserted into the unique XbaI site within the nptA structural gene in a fragment carrying the entire nptA promoter region and part of nhaR. This was inserted into a p15A-based plasmid for analysis of expression of nptA in V. cholerae (see text).

FIG. 2.

Comparison between the sequence of the human NaPi-3a protein (the human homologue of NaPi-2a; GenBank accession no. AAA36354) and the deduced sequence of the nptA gene product (GenBank accession no. CAA09443). (A) Alignment of deduced amino acid sequences. ∗, identity; +, conservative substitution; -, deletion or nonconservative substitution. Underlined sequences I to VIII represent predicted transmembrane helices in the eight-helix model proposed by Murer et al. (25), and HI to HIV represent additional predicted hydrophobic membrane-associated hinge regions (38). Corresponding predicted transmembrane helix regions are underlined in each sequence. (B) Schematic diagram of proposed NaPi-3a and NptA structures based on the transmembrane helix predictions shown in panel A (based on the model in reference 38). Note the deletion in the large extracellular loop and the absence of transmembrane helix VIII in NptA.

FIG. 2.

Comparison between the sequence of the human NaPi-3a protein (the human homologue of NaPi-2a; GenBank accession no. AAA36354) and the deduced sequence of the nptA gene product (GenBank accession no. CAA09443). (A) Alignment of deduced amino acid sequences. ∗, identity; +, conservative substitution; -, deletion or nonconservative substitution. Underlined sequences I to VIII represent predicted transmembrane helices in the eight-helix model proposed by Murer et al. (25), and HI to HIV represent additional predicted hydrophobic membrane-associated hinge regions (38). Corresponding predicted transmembrane helix regions are underlined in each sequence. (B) Schematic diagram of proposed NaPi-3a and NptA structures based on the transmembrane helix predictions shown in panel A (based on the model in reference 38). Note the deletion in the large extracellular loop and the absence of transmembrane helix VIII in NptA.

FIG. 3.

Phylogenetic tree of representative NaPi-II cotransporter sequences. The sequences used were V. cholerae NptA (accession no. CAA09443), Caenorhabditis elegans NaPi cotransporter (AAA81148), human NaPi-IIa and NaPi-IIb (AAA36354 and AAC98695, respectively), mouse NaPi-IIa and NaPi-IIb (AAC42026 and AAC80007, respectively), rat NaPi-IIa (AAC37608), rabbit NaPi-IIa (I46534), sheep NaPi-IIa (CAA04715), opossum NaPi-IIa (AAA30978), bovine NaPi-IIb (CAA57345), Xenopus laevis NaPi-IIb (AAF21134), zebrafish NaPi-IIb2 (AF297180), flounder NaPi-IIb (AAB16821), trout NaPi-IIb2 (AF297186), and shark NaPi-IIb2 (AF297182).

FIG. 4.

Phosphate uptake in V. cholerae cells. Phosphate uptake was measured in 1 mM P_i with 10 μCi of ³²P_i per ml and 100 mM NaCl (or 100 mM choline chloride in the absence of Na⁺), pH 7.5. Means ± SEMs are shown (n = 4). (A) ▪, P_i uptake in the presence of sodium; ▴, P_i uptake in the absence of sodium; ▾, P_i uptake in the presence of sodium after phosphate starvation; •, P_i uptake in the absence of sodium after phosphate starvation. (B) Sodium-dependent component of the V. cholerae phosphate uptake shown in panel A (P_i uptake in the presence of sodium − P_i uptake in the absence of sodium). ▪, sodium-dependent P_i uptake of V. cholerae cells grown in minimal medium; ▴, sodium-dependent P_i uptake of V. cholerae cells after phosphate starvation. Sodium-dependent P_i uptake is enhanced 2.5 times after P_i starvation.

FIG. 5.

Phosphate uptake in E. coli cells transformed with pML-NPTtac. Phosphate uptake was measured in 1 mM P_i with 10 μCi of ³²P_i per ml and 100 mM NaCl (or 100 mM choline chloride in the absence of Na⁺), pH 7.5. Means ± SEMs are shown (n = 4). (A) P_i uptake without induction of NptA. ▪, P_i uptake in the presence of sodium; ▴, P_i uptake in the absence of sodium. (B) P_i uptake after induction of NptA expression. ▪, P_i uptake in the presence of sodium; ▴, P_i uptake in the absence of sodium; ▾, P_i uptake in the presence of sodium and 10 μM monensin. (C) Sodium-dependent components of the reactions in shown in panels A and B (uptake in the presence of sodium − uptake in the absence of sodium). ▪, sodium-dependent P_i uptake in noninduced cells; ▴, sodium-dependent P_i uptake after induction of NptA expression.

FIG. 6.

Effects of extracellular P_i, Na⁺, pH, and monensin on phosphate uptake in E. coli cells transformed with pML-NPTtac after induction. Phosphate uptake was measured in 1 mM P_i (varied in panel A) with 10 μCi of ³²P_i per ml and 100 mM NaCl (in panel B NaCl was replaced with choline chloride to preserve osmolarity) at pH 7.5 (varied in panel C). Means ± SEMs are shown (n = 4). (A) Effect of P_i on sodium-dependent phosphate uptake. (B) Effect of Na⁺ on sodium-dependent phosphate uptake. (C) Effect of pH on sodium-dependent phosphate uptake. (D) Effect of monensin on sodium-dependent phosphate uptake. As sodium-independent uptake amounted to less than 8% of the measured uptake in noninduced cells at the selected time point (4 min), sodium-dependent uptake was taken as being equal to uptake in the presence of sodium in these experiments.