Dicarboxylate Transport System Research Articles

Calcium receptor signaling and citrate transport.

The calcium sensing receptor (CaSR) in the distal nephron decreases the propensity for calcium stones. Here we investigate if the apical CaSR in the proximal tubule also prevents stone formation acting via regulation of apical dicarboxylate and citrate transport. Urinary citrate, partially reabsorbed as a dicarboxylate in the proximal tubule lumen, inhibits stone formation by complexing calcium. We previously demonstrated a novel apical calcium-sensitive dicarboxylate transport system in OK proximal tubule cells. This calcium-sensitive process has the potential to modulate the amount of citrate available to complex increased urinary calcium. Using isotope labeled succinate uptake in OK cells along with various pharmacologic tools we examined whether the CaSR alters apical dicarboxylate transport and through which signal transduction pathways this occurs. Our results indicate that in the proximal tubule CaSR adjusts apical dicarboxylate transport, and does so via a CaSR→Gq→PKC signaling pathway. Thus, the CaSR may decrease the propensity for stone formation via actions in both proximal and distal nephron segments.

Role of antioxidant phenolic compounds in plant responses to environmental stress

The betaproteobacterium Variovorax paradoxus strain TBEA6 is capable of using 3,3′-thiodipropionic acid (TDP) as sole carbon and energy source for growth. This thioether is employed for several industrial applications. It can be applied as precursor for the biotechnical production of polythioesters (PTE), which represent persistent bioplastics. Consequently, the genome of V. paradoxus strain TBEA6 was sequenced. The draft genome sequence comprises approximately 7.2 Mbp and 6852 predicted open reading frames. Furthermore, transposon mutagenesis to unravel the catabolism of TDP in strain TBEA6 was performed. Screening of 20,000 mutants mapped the insertions of Tn5::mob in 32 mutants, which all showed no growth with TDP as sole carbon source. Based on the annotated genome sequence together with transposon-induced mutagenesis, defined gene deletions, in silico analyses and comparative genomics, a comprehensive pathway for the catabolism of TDP is proposed: TDP is imported via the tripartite tricarboxcylate transport system and/or the TRAP-type dicarboxylate transport system. The initial cleavage of TDP into 3-hydroxypropionic acid (3HP) and 3-mercaptopropionic acid (3MP), which serves as precursor substrate for PTE synthesis, is most probably performed by the FAD-dependent oxidoreductase Fox. 3HP is presumably catabolized via malonate semialdehyde, whereas 3MP is oxygenated by the 3MP-dioxygenase Mdo yielding 3-sulfinopropionic acid (3SP). Afterwards, 3SP is linked to coenzyme A. The next step is the abstraction of sulfite by a desulfinase, and the resulting propionyl-CoA enters the central metabolism. Sulfite is oxidized to sulfate by the sulfite-oxidizing enzyme SoeABC and is subsequently excreted by the cells by the sulfate exporter Pse.

The genome of Variovorax paradoxus strain TBEA6 provides new understandings for the catabolism of 3,3′-thiodipropionic acid and hence the production of polythioesters

The betaproteobacterium Variovorax paradoxus strain TBEA6 is capable of using 3,3′-thiodipropionic acid (TDP) as sole carbon and energy source for growth. This thioether is employed for several industrial applications. It can be applied as precursor for the biotechnical production of polythioesters (PTE), which represent persistent bioplastics. Consequently, the genome of V. paradoxus strain TBEA6 was sequenced. The draft genome sequence comprises approximately 7.2Mbp and 6852 predicted open reading frames. Furthermore, transposon mutagenesis to unravel the catabolism of TDP in strain TBEA6 was performed. Screening of 20,000 mutants mapped the insertions of Tn5::mob in 32 mutants, which all showed no growth with TDP as sole carbon source. Based on the annotated genome sequence together with transposon-induced mutagenesis, defined gene deletions, in silico analyses and comparative genomics, a comprehensive pathway for the catabolism of TDP is proposed: TDP is imported via the tripartite tricarboxcylate transport system and/or the TRAP-type dicarboxylate transport system. The initial cleavage of TDP into 3-hydroxypropionic acid (3HP) and 3-mercaptopropionic acid (3MP), which serves as precursor substrate for PTE synthesis, is most probably performed by the FAD-dependent oxidoreductase Fox. 3HP is presumably catabolized via malonate semialdehyde, whereas 3MP is oxygenated by the 3MP-dioxygenase Mdo yielding 3-sulfinopropionic acid (3SP). Afterwards, 3SP is linked to coenzyme A. The next step is the abstraction of sulfite by a desulfinase, and the resulting propionyl-CoA enters the central metabolism. Sulfite is oxidized to sulfate by the sulfite-oxidizing enzyme SoeABC and is subsequently excreted by the cells by the sulfate exporter Pse.

Role of NaDC1 in Control of Urinary Excretion of α‐Ketoglutarate

Recent studies have suggested that urinary α‐Ketoglutarate (α‐KG) increases with alkali loads and decreases with acid loads. Also luminal α‐KG stimulates distal bicarbonate secretion via the Oxgr1 (GPR99) receptor.To investigate the role of sodium dependent dicarboxylate transporter, NaDC1, in regulation of α‐KG excretion, we studied NaDC1 knockout (KO) and Heterozygous (Het) mice on normal diet and with 72 hrs acid loading. Plasma and urine samples were analyzed by LC/Mass Spec.Also 14C‐α‐KG uptake was studied using HRPE cells overexpressing hNaDC1 (CUBS) and the proximal tubule (PT) OK cell line. OK cells express a novel calcium (Ca)‐sensitive dicarboxylate transport system.In mice, fractional excretion (FE) of α‐KG was dramatically increased in the KO and Het mice (WT 0.38+/‐0.05, Het 2.8+/‐1.3, KO 4.7+/‐0.9; p<0.05). Acidosis lowered FE significantly in all mice.In CUBS α‐KG uptake decreased when competed with the dicarboxylate succinate (Suc 2 mM) in both normal and <60 µM Ca buffers (0.64±0.11 to 0.25±0.10 and 0.63±0.15 to 0.29±0.11, p<0.01).In OK cells, α‐KG uptake increased when extracellular Ca was lowered to <60 µM (1.7±0.49 vs 2.5±0.6, p<0.01). Transport decreased when competed with Suc in both Ca buffers (1.3±0.39 vs 1.6±0.34, p<0.01).NaDC1 transports α‐KG and is the predominant, but not exclusive, mechanism of control of distal α‐KG delivery. Also α‐KG is transported via a Ca‐sensitive transporter in OK cells. Furthermore based on the FEs > 1.0, α‐KG is secreted into the PT simultaneously with reabsorption.Thus NaDC1 in the PT regulates acid‐base transport in the distal tubule via the control of α‐KG transport. NaDC1 represents a novel mechanism of PT–distal tubule communication in the kidney.NIH/NIDDK

Catabolite repression in Pseudomonas aeruginosa PAO1 regulates the uptake of C4‐dicarboxylates depending on succinate concentration

In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at μM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased.

Influence of C4-dicarboxylic acid transporters on succinate production

Current climate issues and the ongoing depletion of oil reserves have led to increased attention for biobased production processes. Not only has the production of bio-energy gained interest, but also the production of biochemicals. Succinate is one of those biochemicals. In the presented work, the dicarboxylic acid transport system of Escherichia coli was modified to enhance production of succinate, a highly attractive chemical building block. The engineering comprised the elimination of succinate uptake and the overexpression of succinate export. However, succinate export in Escherichia coli is normally only active under anaerobic conditions and import only under aerobic conditions. Therefore, the gene responsible for succinate import, dctA, was knocked out and the gene coding for succinate export, dcuC, was overexpressed with a constitutive artificial promoter. In the applied batch cultivations, these modifications increased succinate yield and specific production rate more than 50% in a ΔsdhAΔsdhB background (0.16 C-mole/C-mole glucose and 0.17 C-mole/C-mole biomass/h, respectively), but also revealed alternative succinate import proteins, YdjN and YbhI. Mutations in the genes coding for these proteins led to increased growth rates and specific production rates, however, they did not increase succinate yield (e.g. the deletion of ybhI resulted in a growth rate of 0.54 h−1 and a specific production rate of 0.23 C-mole/C-mole biomass/h).

Regulation of Uptake Hydrogenase and Effects of Hydrogen Utilization on Gene Expression in Rhodopseudomonas palustris

Rhodopseudomonas palustris is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The R. palustris genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the R. palustris regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in R. palustris cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in R. palustris.

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

New substrates for the dicarboxylate transport system of Sinorhizobium meliloti.

The dicarboxylate transport (Dct) system of Sinorhizobium meliloti, which is essential for a functional nitrogen-fixing symbiosis, has been thought to transport only dicarboxylic acids. We show here that the permease component of the Dct system, DctA, can transport orotate, a monocarboxylic acid, with an apparent K(m) of 1.7 mM and a V(max) of 163 nmol min(-1) per mg of protein in induced cells. DctA was not induced by the presence of orotate. The transport of orotate was inhibited by several compounds, including succinamic acid and succinamide, which are not dicarboxylic acids. The dicarboxylic acid maleate (cis-butenedioic acid) was not an inhibitor of orotate transport, which suggests that it was not recognized by DctA. However, maleate was an excellent inducer of DctA expression. Our evaluation of 17 compounds as inducers and inhibitors of transport suggests that substrates recognized by S. meliloti DctA must have appropriately spaced carbonyl groups and an extended conformation, while good inducers are more likely to have a curved conformation.

Carbon and nitrogen metabolism in Rhizobium

One of the paradigms of symbiotic nitrogen fixation has been that bacteroids reduce N2 to ammonium and secrete it without assimilation into amino acids. This has recently been challenged by work with soybeans showing that only alanine is excreted in 15N2 labelling experiments. Work with peas shows that the bacteroid nitrogen secretion products during in vitro experiments depend on the experimental conditions. There is a mixed secretion of both ammonium and alanine depending critically on the concentration of bacteroids and ammonium concentration. The pathway of alanine synthesis has been shown to be via alanine dehydrogenase, and mutation of this enzyme indicates that in planta there is likely to be mixed secretion of ammonium and alanine. Alanine synthesis directly links carbon catabolism and nitrogen assimilation in the bacteroid. There is now overwhelming evidence that the principal carbon sources of bacteroids are the C4-dicarboxylic acids. This is based on labelling and bacteroid respiration data, and mutation of both the dicarboxylic acid transport system (dct) and malic enzyme. L-malate is at a key bifurcation point in bacteroid metabolism, being oxidized to oxaloacetate and oxidatively decarboxylated to pyruvate. Pyruvate can be aminated to alanine or converted to acetyl-CoA where it either enters the TCA cycle by condensation with oxaloacetate or forms polyhydroxybutyrate (PHB). Thus regulation of carbon and nitrogen metabolism are strongly connected. Efficient catabolism of C4-dicarboxylates requires the balanced input and removal of intermediates from the TCA cycle. The TCA cycle in bacteroids may be limited by the redox state of NADH/NAD+ at the 2-ketoglutarate dehydrogenase complex, and a number of pathways may be involved in bypassing this block. These pathways include PHB synthesis, glutamate synthesis, glycogen synthesis, GABA shunt and glutamine cycling. Their operation may be critical in maintaining the optimum redox poise and carbon balance of the TCA cycle. They can also be considered to be overflow pathways since they act to remove or add electrons and carbon into the TCA cycle. Optimum operation of the TCA cycle has a major impact on nitrogen fixation.

Roles of DctA and DctB in signal detection by the dicarboxylic acid transport system of Rhizobium leguminosarum.

The dctA gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for dctB and dctD. The dctA promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader. dctAp also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in the dctA-dctB intergenic region (dctA101) which locks transcription of dctA at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix-) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation of dctB or dctD in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.

Identification of a Novel Gene Encoding the Yeast Mitochondrial Dicarboxylate Transport Protein via Overexpression, Purification, and Characterization of Its Protein Product

A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism. Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products. The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions. The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies. Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E. coli culture at a purity of 75%. The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e. Km = 1. 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined. The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported [14C]malonate uptake, whereas other anions tested did not. External substrate competition studies revealed a similar specificity profile. Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters. In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system. In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter. Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family. Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials.

Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons

Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate. The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.

Signal transduction in the Rhizobium meliloti dicarboxylic acid transport system

The gene products of the Rhizobium meliloti dctB and dctD genes, which control the expression of the C 4-dicarboxylic acid transporter DctA, were overproduced in Escherichia coli and purified. The purified sensor protein, DctB, was shown to have autophosphorylation activity in vitro and could subsequently phosphorylate the transcriptional activator, DctD. The presence of C 4-dicarboxylic acids did not affect either reaction. In vitro experiments aimed at investigating ‘crosstalk’ between cognate components demonstrated that the phospho-transfer activity was specific between DctB and DctD. Studies on truncated versions of the DctB protein in vitro revealed that the cytoplasmic domain of DctB had strong autophosphorylation activity. Data from gel retardation experiments demonstrated that once the activator protein, DctD, was phosphorylated it had increased affinity for binding to the dctA promoter DNA.

Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct)

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv. viciae

On the basis of enzyme assays, myo-inositol appears to be catabolized via 2-keto-myo-inositol and D-2,3-diketo-4-deoxy epi-inositol in Rhizobium leguminosarum bv. viciae, as occurs in Klebsiella aerogenes. The first two enzymes of the pathway, myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase were increased 7- and 77-fold, respectively, after growth of R. leguminosarum on myo-inositol compared to glucose. Cells of R. leguminosarum grown on glucose as the carbon source and then resuspended in myo-inositol, increased myo-inositol-dependent O2 consumption by sixfold in 3 h, confirming this to be an inducible pathway. Succinate, malate and glucose exhibited strong catabolite repression of the myo-inositol catabolic pathway with myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase being repressed by at least 68% and 80%, respectively, in all cases. A dicarboxylate transport mutant (dctA) was unable to repress either enzyme when grown on myo-inositol and succinate. There was no repression of the first two enzymes of the myo-inositol catabolic pathway in the background of constitutive expression of the dicarboxylate transport system, indicating a dicarboxylate must be present to cause repression. The data imply that dicarboxylates are required intra-cellularly to repress these enzymes. Three transposon mutants were isolated that cannot grow on myo-inositol as the sole carbon source and were unable to induce either myo-inositol dehydrogenase or 2-keto-myo-inositol dehydratase. The mutants were unaffected in the ability to nodulate peas and vetch. Furthermore, vetch plants infected with one mutant (RU360) reduced acetylene at the same rate as plants infected with the wild type. Bacteroids did not oxidize myo-inositol, nor were the catabolic enzymes detected, confirming myo-inositol is not important as an energy source in bacteroids. The possible role of myo-inositol in the rhizosphere is considered.

Renal transport mechanisms for xenobiotics: chemicals and drugs.

Using the stopped flow tubular lumen or peritubular capillary microperfusion method, the apparent Ki values of a large number of organic anions and cations against the respective transport systems were evaluated. Thereby the luminal transport system for monocarboxylates (lactate), the contraluminal and luminal transport systems for dicarboxylates (succinate), sulfate, and hydrophobic organic cations (tetraethylammonium or N1-methyl-nicotinamide), as well as contraluminal transport system for hydrophobic organic anions (para-aminohippurate, PAH) were characterized and their specificity determined. There is a partially overlapping substrate specificity between the PAH, dicarboxylate, and sulfate transport systems but also between the PAH and organic cation transport system. Xenobiotics and their metabolites are transported mainly by the organic anion (PAH) and organic cation transport systems. To test the complicated interactions possible a shot injection/urinary excretion method with simultaneous measurement of the intracellular concentration was developed. With this approach it is possible to evaluate (a) whether a substrate is net secreted or net reabsorbed, (b) whether interference with other substrates occurs, (c) whether interference takes place at the luminal or contraluminal cell side, and (d) whether cis-inhibition or trans-stimulation is the predominant mode of interaction. Finally, it will be discussed which ability a substrate must have to penetrate the cell membrane via a transporter, through the lipid bilayer, or both.

Aspartate transport in Rhizobium meliloti

Aspartate transport in Rhizobium meliloti was found to be mediated by at least two transport systems. High rates of aspartate uptake, necessary for growth on aspartate as a carbon source, required the dicarboxylate transport (Dct) system, which also transports succinate, fumarate and malate. The apparent K m for aspartate transport by this system was about 10 mm, compared to 15 βm for succinate. This difference in affinity was also apparent in competitive inhibition studies, which showed that succinate effectively inhibits aspartate transport. Although aspartate was not a preferred substrate, it was a very efficient inducer of the Dct system. Both the Dct system and a second aspartate transport system were capable of supplying aspartate for use as a nitrogen source. The second system had a lower apparent K m for aspartate transport (1·5 mm), and was competitively inhibited by glutamate. This aspartate-glutamate system was regulated independently from the Dct system, since it functioned in mutants lacking the Dct system regulatory genes dctB and dctD, and its induction did not coactivate the Dct system. Uptake kinetics in cultures growing on aspartate as nitrogen source showed rapid substrate exchange between extracellular and internal aspartate. R. meliloti was shown to be able to selectively activate the two uptake systems, and also regulated its metabolism as required to utilize aspartate as either carbon or nitrogen source.

Relationships between C4 dicarboxylic acid transport and chemotaxis in Rhizobium meliloti

The relationship between chemotaxis and transport of C4 dicarboxylic acids was analyzed with Rhizobium meliloti dct mutants defective in one or all of the genes required for dicarboxylic acid transport. Succinate, malate, and fumarate were moderately potent chemoattractants for wild-type R. meliloti and appeared to share a common chemoreceptor. While dicarboxylate transport is inducible, taxis to succinate was shown to be constitutive. Mutations in the dctA and dctB genes both resulted in the reduction, but not elimination, of chemotactic responses to succinate, indicating that transport via DctA or chemosensing via DctB is not essential for C4 dicarboxylate taxis, although they appear to contribute to it. Mutations in dctD and rpoN genes did not affect taxis to succinate. Aspartate, which is also transported by the dicarboxylate transport system, elicited strong chemotactic responses via a chemoreceptor distinct from the succinate-malate-fumarate receptor. Taxis to aspartate was unaltered in dctA and dctB mutants but was considerably reduced in both dctD and rpoN mutants, indicating that aspartate taxis is strongly dependent on elements responsible for transcriptional activation of dctA. Methylation and methanol release experiments failed to show a significant increase in methyl esterification of R. meliloti proteins in response to any of the attractants tested.