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.     

Characterization of Constitutive Galactose Permease Mutants in Salmonella typhimurium

Salmonella typhimurium strains, lacking both enzyme I and the phosphocarrier protein, HPr, of the phosphoenolpyruvate-sugar phosphotransferase system, cannot transport or metabolize glucose and other sugar substrates of this enzyme system. Mutants which regain the ability to specifically utilize glucose were found to constitutively synthesize a galactose permease by virtue of a mutation in the galR gene. This permease, shown to be an active transport system, does not require HPr or enzyme I for activity.     

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.     

Galactose metabolism in gal mutants of Salmonella typhimurium and Escherichia coli

Abstract We report a new pathway for galactose metabolism in Escherichia coli and Salmonella typhimurium . Growth of gal mutants on galactose is restored by the addition of pyrrolo-quinoline quinone (PQQ) to the medium. In such strains galactose is oxidized to galactonate by a PQQ-dependent, membrane-bound dehydrogenase. A pathway for galactonate metabolism in these organisms has already been described.     

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.     

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.     

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.     

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.     

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.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Aerotaxis in Salmonella typhimurium: role of electron transport

Sensory transduction in aerotaxis required electron transport, in contrast to chemotaxis, which is independent of electron transport. Assays for aerotaxis were developed by employing spatial and temporal oxygen gradients imposed independently of respiration. By varying the step increase in oxygen concentration in the temporal assay, the dose-response relationship was obtained for aerotaxis in Salmonella typhimurium. A half-maximal response at 0.4 microM oxygen and inhibition by 5 mM KCN suggested that the "receptor" for aerotaxis is cytochrome o. The response was independent of adenosine triphosphate formation via oxidative phosphorylation but did correlate with changes in membrane potential monitored with the fluorescent cyanine dye diS-C3-(5). Nitrate and fumarate, which are alternative electron acceptors for the respiratory chain in S. typhimurium, inhibited aerotaxis when nitrate reductase and fumarate reductase were induced. These results support the hypothesis that taxis to oxygen, nitrate, and fumarate is mediated by the electron transport system and by changes in the proton motive force. Aerotaxis was normal in Escherichia coli mutants that were defective in the tsr, tar, or trg genes; in S. typhimurium, oxygen did not stimulate methylation of the products of these genes. A cheC mutant which shows an inverse response to chemoattractants also gave an inverse response to oxygen. Therefore, aerotaxis is transduced by a distinct and unidentified signally protein but is focused into the common chemosensory pathway before the step involving the cheC product. When S. typhimurium became anaerobic, the decreased proton motive force from glycolysis supported slow swimming but not tumbling, indicating that a minimum proton motive force was required for tumbling. The bacteria rapidly adapted to the anaerobic condition and resumed tumbling after about 3 min. The adaptation period was much shorter when the bacteria had been previously grown anaerobically.     

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.     

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-.