Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake.     

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.     

Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.     

Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1

The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.     

l-Serine transport in growing and maturing mouse oocytes

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis–Menten constant (K_m) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus–oocyte complexes.     

Transport of Biosynthetic Intermediates: Homoserine and Threonine Uptake in Escherichia coli

Although amino acid transport has been extensively studied in bacteria during the past decade, little is known concerning the transport of those amino acids that are biosynthetic intermediates or have multiple fates within the cell. We have studied homoserine and threonine as examples of this phenomenon. Homoserine is transported by a single system which it shares with alanine, cysteine, isoleucine, leucine, phenylalanine, threonine, tyrosine, and valine. The evidence for this being the sole system for homoserine transport is (i) a linear double-reciprocal plot showing a homoserine K(m) of 9.6 x 10(-6) M, (ii) simultaneous reduction by 85% of homoserine and branched-chain amino acid uptake in a mutant selected for its inability to transport homoserine, and (iii) simultaneous reduction by 94% of the uptake of homoserine and the branched-chain amino acids by cells grown in millimolar leucine. Threonine, in addition to sharing the above system with homoserine, is transported by a second system shared with serine. The evidence for this second system consists of (i) incomplete inhibition of threonine uptake by any single amino acid, (ii) only 70% loss of threonine uptake in the mutant unable to transport homoserine, and (iii) only 40% reduction of threonine uptake when cells are grown in millimolar leucine. In this last case, the remaining threonine uptake can only be inhibited by serine and the inhibition is complete.     

Resistance to serine in Bacillus subtilis: identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism

The Gram-positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.     

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.     

Na+-Dependent Transport of Amino Acids and Its Significance for Growth of a Lysine-producing Bacterium

A lysine-producing mutant Brevibacterium flavum HUT 8052, a threonine plus methionine (or threonine plus homoserine) auxotroph, grew rapidly as nearly as the wild strain in a medium supplemented with NaCl (60 µg/ml), threonine (100 µg/ml), and methionine (33.3 µg/ml). With NaCl concentrations less than 20 µg/ml, the mutant grew little or very slowly, The peculiar growth behavior of the mutant including the above phenomenon could be reasonably explained by the finding of Na⁺-dependent amino acids transport and the feedback inhibition of homoserine dehydrogenase by threonine in the bacterium.

The threonine transport was stimulated by Na⁺ and Li⁺. though the latter being less effective. The transport of threonine was inhibited by serine. The uptake of serine, isoleucine, leucine and valine was also stimulated by Na⁺     

A novel Porphyromonas gingivalis FeoB plays a role in manganese accumulation

FeoB is an atypical transporter that has been shown to exclusively mediate ferrous ion transport in some bacteria. Unusually the genome of the periodontal pathogen Porphyromonas gingivalis has two genes (feoB1 and feoB2) encoding FeoB homologs, both of which are expressed in bicistronic operons. Kinetic analysis of ferrous ion transport by P. gingivalis W50 revealed the presence of a single, high affinity system with a K(t) of 0.31 microM. FeoB1 was found to be solely responsible for this transport as energized cells of the isogenic FeoB1 mutant (W50FB1) did not transport radiolabeled iron, while the isogenic FeoB2 mutant (W50FB2) transported radiolabeled iron at a rate similar to wild type. This was reflected in the iron content of W50FB1 grown in iron excess conditions which was approximately half that of the wild type and W50FB2. The W50FB1 mutant had increased sensitivity to both oxygen and hydrogen peroxide and was avirulent in an animal model of infection whereas W50FB2 exhibited the same virulence as the wild type. Analysis of manganous ion uptake using inductively coupled plasma-mass spectrometry revealed a greater than 3-fold decrease in intracellular manganese accumulation in W50FB2 which was also unable to grow in manganese-limited media. The protein co-expressed with FeoB2 appears to be a novel FeoA-MntR fusion protein that exhibits homology to a manganese-responsive, DNA-binding metalloregulatory protein. These results indicate that FeoB2 is not involved in iron transport but plays a novel role in manganese transport.     

Effects of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, on glucose transport and metabolism in skeletal muscle.

The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.     

Comparative growth of Porphyromonas gingivalis strains in a defined basal medium

Porphyromonas gingivalis is an asaccharolytic bacterium whose metabolism is dependent on the uptake of small peptides and amino acids. The aim of this work was to study the growth of P. gingivalis in a defined basal medium (DBM) supplemented with various sources of proteins. The strain 49417 as well as other virulent isolates could grow in DBM containing 1% bovine serum albumin (BSA). Cells cultivated under this condition showed a slightly modified protein profile, and expressed hemagglutinating as well as proteolytic activities. Other natural proteins under investigation could not support the growth in the DBM. On the other hand, the strain 33277 as well as other avirulent strains of P. gingivalis could not use BSA as a substrate. The ability of P. gingivalis to grow in DBM-BSA is not entirely dependent on its ability to degrade the protein substrate as strain 33277 was able to extensively hydrolyse the molecule. Differences in either metabolic enzymes or peptide transport mechanisms may explain the distinctive behavior between virulent and avirulent strains. Data from this work suggest a relationship between nutritional requirements and virulence of P. gingivalis in an animal model. The DBM-BSA may represent a more appropriate medium for studies on the physiology of P. gingivalis.     

Characterization of MbrC involved in bacitracin resistance in Streptococcus mutans

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease.     

Amino acid transport by<Emphasis Type="Italic"> Sphingomonas</Emphasis> sp. strain Ant 17 isolated from oil-contaminated Antarctic soil

The Antarctic bacterial isolate Sphingomonas sp. strain Ant 17 utilized a wide range of L-isomer amino acids as the sole carbon and energy source for growth. The pH and temperature optima for growth on amino acids were pH 7.0 and 15°C, respectively. Growth on serine and tryptophan was inhibited by uncouplers and inhibitors of oxidative phosphorylation, but not by monensin, a Na⁺/H⁺ antiporter, suggesting that sodium gradients were not specifically required for growth on these amino acids. Serine transport was via a high-affinity (apparent K_m of 8 M) permease specific for both the L- and D-isomer. Tryptophan transport exhibited biphasic kinetics with both high-affinity (apparent K_m of 2.5 M) and low-affinity (non-saturable) uptake systems detected. The high-affinity system was specific for L-tryptophan, L-tyrosine, and L-phenylalanine whereas the low-affinity permease was specific for L-tryptophan and L-phenylalanine, but not L-tyrosine. Neither orthovanadate nor sodium arsenate, inhibitors of ATP-dependent permeases, had any significant inhibitory effect on the rate of serine and tryptophan transport. The protonophore carbonyl cyanide m-chlorophenylhydrazone completely abolished serine and tryptophan transport; maximum rates of solute uptake were observed at acidic pH values (pH 4.0–5.0) for both amino acids. These results suggest that an electrochemical potential of protons is the driving force for serine and tryptophan transport by Ant 17. These high-affinity proton-driven permeases function over environmental extremes (e.g. broad temperature and pH range) that are likely to prevail in the natural habitat of this bacterium.     

Porphyromonas gingivalis DPP-7 represents a novel type of dipeptidylpeptidase

A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

Transposition of Tn4351 in Porphyromonas gingivalis.

Genetic analysis of Porphyromonas gingivalis, an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751::*omega 4 has previously been used as a suicide vector to demonstrate transposition of Tn4351 in B. uniformis. The erythromycin resistance gene on Tn4351 functions in Bacteroides and Porphyromonas. Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 x 10(-7) from matings between Escherichia coli HB101 containing R751::*omega 4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn4351 and half contained both Tn4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn4351-mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis-Tn4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis.     

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.     

A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium.

PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.