Proton efflux associated with melibiose permease activity in Salmonella typhimurium.

Transport of thiomethyl-β-D-galactoside (TMG) via the melibiose permease system (TMG permease II) in $\text{Salmonella typhimurium}$ is known to be a sodium-dependent co-transport system. We have shown that this co-transport of sodium and TMG is associated with extrusion of protons from the cells. The rate and extent of proton extrusion during TMG uptake were measured in wild-type cells and mutants containing internal and extended deletions in the $\text{pts}$ locus. No differences between these various strains were noted.     

Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ～1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ～1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ～500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.     

Citrate transport in Salmonella typhimurium.

Citrate was rapidly metabolized in wild-type cells of Salmonella typhimurium but actively accumulated in both aconitase mutants and fluorocitrate-poisoned cells. In aconitase mutants citrate was transported by a single high affinity system (K_m 23 μm, V_max 27.2 nmol min^?1 mg^?1), characterized by a single pH optimum of 7.0 and a Q₁₀ of 3.0, and was stimulated by Na⁺. cis-Aconitate, tricarballylate, trans-aconitate, and dl-fluorocitrate were weak competitive inhibitors of citrate transport whereas various other tricarboxylic acid cycle intermediates and carboxylates were ineffective. Spontaneous citrate transport mutants were unable to oxidize citrate, cis-aconitate, or tricarballylate. Such mutants were specific for citrate and transported dicarboxylates normally whereas dicarboxylate transport mutants transported and oxidized citrate normally. In whole cells of an aconitase mutant citrate transport was strongly dependent on an energy source. d(?)-Lactate dehydrogenase mutants were singularly defective in energization by d(?)-lactate. Membrane vesicles of wild-type cells were capable of energized transport by d(?)-lactate or ascorbate-phenyl-methyl sulfonate. Citrate transport in whole cells was primarily energized aerobically, and ATPase deficient mutants were still able to transport citrate in whole cells.     

Galactose transport in Salmonella typhimurium.

We have studied the various systems by which galactose can be transported in Salmonella typhimurium, in particular the specific galactose permease (GP). Mutants that contain GP as the sole galactose transport system have been isolated, and starting from these mutants we have been able to select point mutants that lack GP. The galP mutation maps close to another mutation, which results in the constitutive synthesis of GP, but is not linked to galR. Growth of wild-type strains on glaactose induces GP but not the beta-methylgalactoside permease (MGP). Strains lacking GP are able to grow slowly on galactose, and MGP is induced; however, D-fucose is a much better inducer of MGP. Induction of GP or MGP is not prevented by a pts mutation, although this mutation changes the apparent Km of MGP for galactose. pts mutations have no effect on GP. GP has a rather broad specificity: galactose, glucose, mannose, fucose, 2-deoxygalactose, and 2-deoxyglucose are substrates, but only galactose and fucose can induce this transport system.     

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

The melibiose permease of Salmonella typhimurium (MelB_St) catalyzes the stoichiometric symport of galactopyranoside with a cation (H⁺, Li⁺, or Na⁺) and is a prototype for Na⁺-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelB_St have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelB_St, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na⁺-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelB_St is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na⁺-coupled MFS transporters.     

Regulation of L-cystine transport in Salmonella typhimurium.

A kinetic analysis of L-cystine uptake in wild-type Salmonella typhimurium indicates the presence of at least two, and possibly three, separate transport systems. CTS-1 accounts for the majority of uptake at 20 muM L-cystine, with a Vmax of 9.5 nmol/min per mg and a Km of 2.0 muM; CTS-2 is a low-capacity, higher-affinity system with a Vmax of 0.22 nmol/min per mg and a Km of 0.05 muM; a third, nonsaturable process has been designated CTS-3. We find that wild-type CTS-1 levels are at least 11 times higher in sulfur-limited cells than in L-cystine-grown cells. Pleiotropic cysteine auxotrophs of the types cysE (lacking serine transacetylase) and cysB- (lacking a regulatory element of positive control) have very low levels of CTS-1 even when grown under conditions of sulfur limitation, which response is analogous to that previously observed for cysteine biosynthetic enzymes (N . M. Kredich, J. Biol. Chem. 246:3474-3484, 1971). CTS-1 is induced in cysE mutants by growth in the presence of O-acetyl-L-serine (the product of serine transacetylase), again paralleling the behavior of the cysteine biosynthetic pathway. Strain DW25, a prototrophic cysBc mutant, which is constitutive for cysteine biosynthesis, is also derepressed for CTS-1 when grown on L-cystine. Since CTS-1 is regulated by sulfur limitation, O-acetyl-L-serine, and the cysB gene product, the same three conditions controlling cysteine biosynthesis, we propose that this transport system is a part of the cysteine regulon.     

Proton movements coupled to sugar transport via the galactose transport system in Salmonella typhimurium.

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium. The addition of galactose to lightly buffered suspensions of anaerobic, non-metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellularpH as galactose and protons enter the cell together. Other substrates for this transport system, D-fucose, 2-deoxygalactose, glucose and 2-deoxyglucose similarly cause an influx of protons when transported. In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx. Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non-energized conditions. At pH 7.2 the permeant anion SCN- increases both the rate and extent of galactose-induced proton influx; at pH 6 the rate, but not the extent is increased by SCN-.     

sn-Glycerol-3-phosphate transport in Salmonella typhimurium

Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased.     

Na+-dependent methyl beta-thiogalactoside transport in Salmonella typhimurium.

We have studied the role of sodium ions in methyl beta-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in Salmonella typhimurium. TMG uptake via TMG II in anaerobic, straved and metabolically poisoned cells is dependent on an inward-directed Na+ gradient. Cells which have been partially depleted of endogenous substrates show H+ extrusion upon sodium-stimulated TMG influx. Measurements of the electrochemical H+ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plug Na+ uptake.     

Biochemical studies of melibiose metabolism in wild type and mel mutant strains of Salmonella typhimurium

I identified two enzyme activities, alpha-galactosidase and a galactoside permease, required for melibiose metabolism by Salmonella typhimurium. These activities are very low under normal growth conditions, but their production can be induced by melibiose and gratuitously by melibiitol. Melibiose induction is severely inhibited by glucose, but the glucose effect can be countered by 3', 5' cyclic adenosine monophosphate. I isolated two phenotypic classes of mutants not able to utilize melibiose as a carbon source. One class, Car(-), is deficient in the phosphotransferase system. The other, Mel, lacks either alpha-galactosidase, galactoside permease, or both functions.     

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.     

Fine-structure map of the histidine transport genes in Salmonella typhimurium.

Afine-structure genetic map of the histidine transport region of the Salmonella typhimurium chromosome was constructed. Twenty-five deletion mutants were isolated and used for dividing the hisJ and hisP genes into 8 and 13 regions respectively. A total of 308 mutations, spontaneous and mutagen induced, have been placed in these regions by deletion mapping. The histidine transport operon is presumed to be constituted of genes dhuA, hisJ, and hisP, and the regulation of the hosP and hisJ genes by dhuA is discussed. The orientation of this operon relative to purF has been established by three-point crosses as being: purF duhA hisJ hisP.     

Role of Gly117 in the cation/melibiose symport of MelB of Salmonella typhimurium

The melibiose permease of Salmonella typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+), and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na(+)-binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding of cosubstrates depended on the physical-chemical properties of the side chain. Compared with WT MelB(St), the Gly117 → Ala mutant exhibited little difference in either cosubstrate binding or stimulation of melibiose transport by Na(+) or Li(+), but all other mutations reduced melibiose active transport and efflux, and decreased the apparent affinity for Na(+). The bulky Trp at position 117 caused the greatest inhibition of melibiose binding, and Gly117 → Arg yielded less than a 4-fold decrease in the apparent affinity for melibiose at saturating Na(+) or Li(+) concentration. Remarkably, the mutant Gly117 → Arg catalyzed melibiose exchange in the presence of Na(+) or Li(+), but did not catalyze melibiose translocation involving net flux of the coupling cation, indicating that sugar is released prior to release of the coupling cation. Taken together, the findings are consistent with the notion that Gly117 plays an important role in cation binding and translocation.     

Genetics of sulfate transport by Salmonella typhimurium

Sixty-four mutants were isolated from the LT-2 wild-type strain of Salmonella typhimurium by selecting for chromate resistance. The majority of lesions were shown to lie in the cysA gene. (i) The mutants cannot take up sulfate, a finding which verifies the role of cysA in sulfate transport. In addition, 52 sulfate-transport mutants isolated without chromate selection were defective in the cysA gene. (ii) Most had less than 25% of the binding activity of the wild-type strain. (iii) Most had normal sulfite reductase (H(2)S-nicotinamide adenine dinucleotide phosphate oxidoreductase, EC 1.8.1.2) activity. (iv) Their sulfate-binding protein (binder) appears electrophoretically and immunologically normal. (v) Amber cysA mutants also make apparently normal binder in small amounts. (vi) All classical cysA mutants tested, including two with long deletions, had normal binding activity. From these observations, it is suggested that the cysA gene does not code for the binder. But many mutations in this gene reduce the binding activity in some unknown way. Other mutants, identified as cysB mutants, had neither binding nor uptake activities and their sulfite reductase activities were similarly reduced, thus confirming the regulatory role of the cysB gene. When binder was detectable, it had wild-type properties. No mutations in the binder gene were found among more than 100 mutants examined. Thus, sulfate binding has not been established as a part of sulfate transport. However, the production of binder is intimately connected with cysA, the established sulfate transport gene, and is regulated by the same mechanism that regulates both transport and the rest of the cysteine biosynthetic pathway.