Two nucleotide transport proteins in Chlamydia trachomatis, one for net nucleoside triphosphate uptake and the other for transport of energy

Abstract

The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1Ct and Npt2Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His10-Npt1Ct catalyzed ATP and ADP transport in an exchange mode. The apparent Km values were 48 (ATP) and 39 (ADP) microM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His10-Npt2Ct transported all four ribonucleoside triphosphates with apparent Km values of 31 microM (GTP), 302 microM (UTP), 528 microM (CTP), and 1,158 microM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2Ct. This observation indicated that His10-Npt2Ct acts as a nucleosidetriphosphate/H+ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1Ct provides chlamydiae with energy whereas Npt2Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.

FIG. 1

Alignment of predicted amino acid sequences of Npt1_Ct, Npt2_Ct, and RpTLC. Numbers indicate amino acid positions; dots mark artificial sequence gaps introduced to improve similarity between the proteins.

FIG. 2

Hydropathy analysis of the predicted amino acid sequences of Npt1_Ct, Npt2_Ct, and RpTLC. Hydropathy analysis was carried out as described by von Heijne (28). The values of H, which are essentially identical, have been offset to separate the plots for clarity. The predicted transmembrane domains in RpTLC are shown at the top.

FIG. 3

Time dependency of [α-³²P]ATP (●) and [α-³²P]ADP (○) uptake into intact E. coli cells expressing npt1_Ct. IPTG-induced E. coli cells harboring plasmid pJT167 were incubated with 50 μM [α-³²P]ATP or [α-³²P]ADP for the indicated time periods. Control cells were not induced (−IPTG). Data are means of three independent experiments; standard deviations were less than 8% of the mean values.

FIG. 4

Exchange-mediated efflux of intracellular radioactivity. IPTG-induced E. coli cells expressing npt1_Ct were preloaded by incubation with [α-³²P]ATP at 25 μM. After removal of external radioactivity, cells were resuspended in phosphate buffer with or without 1 mM AMP, ADP, or ATP.

FIG. 5

Time dependency of [α-³²P]ATP, [α-³²P]ADP, [α-³²P]GTP, [α-³²P]UTP, and [α-³²P]CTP uptake into intact E. coli cells expressing npt2_Ct. (A) Time dependency of [α-³²P]ATP (0.8 mM; ●) or [α-³²P]ADP (1 mM; ○) uptake. (B) Time dependency of [α-³²P]GTP (0.05 mM), [α-³²P]UTP (0.5 mM), and [α-³²P]CTP (0.5 mM) uptake.