Dicarboxylic Acid Transport Research Articles

RNA expression changes driven by altered epigenetics status related to NASH etiology

Non-alcoholic fatty liver disease (NAFLD) is a growing health problem due to the increased obesity rates, among other factors. In its more severe stage (NASH), inflammation, hepatocellular ballooning and fibrosis are present in the liver, which can further evolve to total liver dysfunction or even hepatocarcinoma. As a metabolic disease, is associated to environmental factors such as diet and lifestyle conditions, which in turn can influence the epigenetic landscape of the cells, affecting to the gene expression profile and chromatin organization. In this study we performed ATAC-sequencing and RNA-sequencing to interrogate the chromatin status of liver biopsies in subjects with and without NASH and its effects on RNA transcription and NASH etiology. NASH subjects showed transcriptional downregulation for lipid and glucose metabolic pathways (e.g., ABC transporters, AMPK, FoxO or insulin pathways). A total of 229 genes were differentially enriched (ATAC and mRNA) in NASH, which were mainly related to lipid transport activity, nuclear receptor-binding, dicarboxylic acid transporter, and PPARA lipid regulation. Interpolation of ATAC data with known liver enhancer regions showed differential openness at 8 enhancers, some linked to genes involved in lipid metabolism, (i.e., FASN) and glucose homeostasis (i.e., GCGR). In conclusion, the chromatin landscape is altered in NASH patients compared to patients without this liver condition. This alteration might cause mRNA changes explaining, at least partially, the etiology and pathophysiology of the disease.

Abstract PR008: The SLC1A1/EAAT3 dicarboxylic amino acid transporter: An actionable, HIF-independent, hub that links epigenetic dysregulation to nucleotide metabolism in kidney cancer

Abstract Significance: The clear cell renal cell carcinomas (ccRCCs), which harbor the hallmark inactivation of the von Hippel Lindau tumor suppressor protein (pVHL), represent the majority of adult kidney cancers. Although, early stage/localized ccRCC is effectively managed by surgery and surveillance, therapeutic options for advanced stage disease are limited, highlighting the unmet need for novel therapies. Chromatin dysregulation is routinely observed in ccRCC and our previous work identified a distinct histone modification signature associated with pVHL-deficient ccRCCs, versus the pVHL-proficient chromophobe and papillary sub-types, which included elevated levels of bulk Histone H3 K27 acetylation (H3K27ac). H3K27ac marks transcriptionally activated regions in the genome. We hypothesized that, as in other biological contexts, H3K27ac accumulation in pVHL-deficient ccRCCs drives the expression of key oncogenes. Here, we functionally probed this dysregulated epigenetic program to identify actionable targets in kidney cancer. Experimental Approach and Results: We identified genes marked by elevated H3K27ac (ChIP-Seq) and higher expression (RNA-Seq), specifically in pVHL-deficient cells and prioritized the top 100 genes that encoded druggable products. Using a library representing these ORFs, we did an in vivo ‘sufficiency’ screen, to identify candidates whose expression promoted tumor growth in otherwise poorly tumorigenic pVHL-proficient cells. Our screen (and downstream validation studies) identified SLC1A1, which encodes an Asp/Glu transporter named EAAT3, as an oncogenic driver in ccRCC. Results: SLC1A1 expression was elevated in pVHL-deficient cells – but in a HIF-independent manner. Inactivation of SLC1A1 (using CRISPR/Cas9) led to fitness defects in a panel of ccRCC cell lines, along with a notable depletion of nucleotide biosynthesis precursors, likely because of the importance of the Asp carbon skeleton in this pathway. Consistent with this, esterified-Asp rescued the fitness defects associated with SLC1A1 loss. Moreover, SLC1A1 upregulation (high Asp/Glu) diminished dependency on Glutaminase; whereas, SLC1A1 loss (low Asp/Glu) increased Glutaminase dependency and promoted sensitivity to Glutaminase blockade. Finally, using clinical cohorts and cell-based studies, we found that SLC1A1 can impact its own transcriptional expression, but also the expression of certain other metabolic transporters involved in Asp/Glu metabolism (e.g. SLC22A11) and nucleotide metabolism (e.g SLC28A1). We speculate, these transcriptional alterations are driven by the conversion of Glu into a-ketoglutarate, a key cofactor for KDMs. Our ongoing studies are addressing the mechanistic basis of this transcriptional regulation and exploring the feasibility of pharmacologically targeting SLC1A1 in ccRCC. Altogether, we show that ccRCCs hijack nutrient reabsorption mechanisms (e.g. SLC1A1) to drive oncogenic programs. SLC1A1 thus links epigenetic dysregulation to a HIF-independent metabolic program regulating nucleotide biosynthesis/uptake. Citation Format: Abhishek A. Chakraborty. The SLC1A1/EAAT3 dicarboxylic amino acid transporter: An actionable, HIF-independent, hub that links epigenetic dysregulation to nucleotide metabolism in kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR008.

Engineering Saccharomyces cerevisiae for Succinic Acid Production From Glycerol and Carbon Dioxide.

Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in S. cerevisiae by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol’s higher reducing power (compared to sugars) for the production of the platform chemical succinic acid (SA) via a reductive, carbon dioxide fixing and redox-neutral pathway in a production host robust for organic acid production. Expression cassettes for three enzymes converting oxaloacetate to SA in the cytosol (“SA module”) were integrated into the genome of UBR2CBS-DHA, an optimized CEN.PK derivative. Together with the additional expression of the heterologous dicarboxylic acid transporter DCT-02 from Aspergillus niger, a maximum SA titer of 10.7 g/L and a yield of 0.22 ± 0.01 g/g glycerol was achieved in shake flask (batch) cultures. Characterization of the constructed strain under controlled conditions in a bioreactor supplying additional carbon dioxide revealed that the carbon balance was closed to 96%. Interestingly, the results of the current study indicate that the artificial “SA module” and endogenous pathways contribute to the SA production in a highly synergistic manner.

Evaluation of pyruvate decarboxylase-negative Saccharomyces cerevisiae strains for the production of succinic acid.

Dicarboxylic acids are important bio‐based building blocks, and Saccharomyces cerevisiae is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase‐negative S. cerevisiae strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of GPD1 and FUM1 and multi‐copy integration of the native PYC2 gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade‐off between redox cofactor supply and energy availability in the form of ATP.

Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid

BackgroundMalate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements.ResultsWe found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export.ConclusionsSince more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.

Hyperpolarized [1,4-13C2]Fumarate Enables Magnetic Resonance-Based Imaging of Myocardial Necrosis

ObjectivesThe aim of this study was to determine if hyperpolarized [1,4–13C2]malate imaging could measure cardiomyocyte necrosis after myocardial infarction (MI). BackgroundMI is defined by an acute burst of cellular necrosis and the subsequent cascade of structural and functional adaptations. Quantifying necrosis in the clinic after MI remains challenging. Magnetic resonance-based detection of the conversion of hyperpolarized [1,4–13C2]fumarate to [1,4–13C2]malate, enabled by disrupted cell membrane integrity, has measured cellular necrosis in vivo in other tissue types. Our aim was to determine whether hyperpolarized [1,4–13C2]malate imaging could measure necrosis after MI. MethodsIsolated perfused hearts were given hyperpolarized [1,4–13C2]fumarate at baseline, immediately after 20 min of ischemia, and after 45 min of reperfusion. Magnetic resonance spectroscopy measured conversion into [1,4–13C2]malate. Left ventricular function and energetics were monitored throughout the protocol, buffer samples were collected and hearts were preserved for further analyses. For in vivo studies, magnetic resonance spectroscopy and a novel spatial-spectral magnetic resonance imaging sequence were implemented to assess cardiomyocyte necrosis in rats, 1 day and 1 week after cryo-induced MI. ResultsIn isolated hearts, [1,4–13C2]malate production became apparent after 45 min of reperfusion, and increased 2.7-fold compared with baseline. Expression of dicarboxylic acid transporter genes were negligible in healthy and reperfused hearts, and lactate dehydrogenase release and infarct size were significantly increased in reperfused hearts. Nonlinear regression revealed that [1,4–13C2]malate production was induced when adenosine triphosphate was depleted by >50%, below 5.3 mmol/l (R2 = 0.904). In vivo, the quantity of [1,4–13C2]malate visible increased 82-fold over controls 1 day after infarction, maintaining a 31-fold increase 7 days post-infarct. [1,4–13C2]Malate could be resolved using hyperpolarized magnetic resonance imaging in the infarct region one day after MI; [1,4–13C2]malate was not visible in control hearts. ConclusionsMalate production in the infarcted heart appears to provide a specific probe of necrosis acutely after MI, and for at least 1 week afterward. This technique could offer an alternative noninvasive method to measure cellular necrosis in heart disease, and warrants further investigation in patients.

Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01.

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose-limited chemostat culture.

Currently, research is being focused on the industrial-scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial-scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose-limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild-type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial-scale production of fumaric acid are discussed.

The evolution of Jen3 proteins and their role in dicarboxylic acid transport in Yarrowia.

Jen proteins in yeast are involved in the uptake of mono/dicarboxylic acids. The Jen1 subfamily transports lactate and pyruvate, while the Jen2 subfamily transports fumarate, malate, and succinate. Yarrowia lipolytica has six JEN genes: YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D20108g, YALI0D24607g, and YALI0E32901g. Through phylogenetic analyses, we found that these genes represent a new subfamily, Jen3 and that these three Jen subfamilies derivate from three putative ancestral genes. Reverse transcription-PCR. revealed that only four YLJEN genes are expressed and they are upregulated in the presence of lactate, pyruvate, fumarate, malate, and/or succinate, suggesting that they are able to transport these substrates. Analysis of deletion mutant strains revealed that Jen3 subfamily proteins transport fumarate, malate, and succinate. We found evidence that YALI0C15488 encodes the main transporter because its deletion was sufficient to strongly reduce or suppress growth in media containing fumarate, malate, or succinate. It appears that the other YLJEN genes play a minor role, with the exception of YALI0E32901g, which is important for malate uptake. However, the overexpression of each YLJEN gene in the sextuple-deletion mutant strain ΔYLjen1-6 revealed that all six genes are functional and have evolved to transport different substrates with varying degrees of efficacy. In addition, we found that YALI0E32901p transported succinate more efficiently in the presence of lactate or fumarate.

“Malonate Uptake and Metabolism in Saccharomyces cerevisiae”

Malonyl-CoA plays an important role in the synthesis and elongation of fatty acids in yeast Saccharomyces cerevisiae. Malonyl-CoA is at a low concentration inside the cell and is produced mainly from acetyl-CoA through the enzyme acetyl-CoA carboxylase. It would be beneficial to find an alternative source of malonyl-CoA to increase its intracellular concentration and overall synthesis of the fatty acids. MatB gene from the bacteria Rhizobium leguminosarium bv. trifolii encodes for a malonyl-CoA synthetase which catalyzes the formation of the malonyl-CoA directly from malonate and CoA. However, results from high-performance liquid chromatography (HPLC) proved that Saccharomyces cerevisiae itself does not contain enough cytoplasmic malonate within them and is unable to uptake exogenously supplied malonate in the form of malonic acid. A dicarboxylic acid plasma membrane transporter with the ability to uptake exogenous malonic acid was identified from another species of yeast known as Schizosaccharomyces pombe and the gene encoding this transporter is identified as the mae1 gene. From the experiments thus far, the mae1 gene had been successfully cloned and transformed into Saccharomyces cerevisiae. The expression and functional ability of the encoded plasma membrane dicarboxylic acid transporter were also demonstrated and verified using specialized technologies such as RT-PCR, yeast immunofluorescence, HPLC, and LC-MS.

Effect of Gene Amplifications in Porphyrin Pathway on Heme Biosynthesis in a Recombinant Escherichia coli

A recombinant E. coli co-expressing ALA synthase (hemA), NADP-dependent malic enzyme (maeB), and dicarboxylic acid transporter (dctA) was reported to synthesize porphyrin derivatives including iron-containing heme. To enhance the synthesis of bacterial heme, five genes of the porphyrin biosynthetic pathway [pantothenate kinase (coaA), ALA dehydratase (hemB), 1-hydroxymethylbilane synthase (hemC), uroporphyrinogen III synthase (hemD), and uroporphyrinogen III decarboxylase (hemE)] were amplified in the recombinant E. coli co-expressing hemA-maeB-dctA. Pantothenate kinase expression enabled the recombinant E. coli to accumulate intracellular CoA. Intracellular ALA was the most enhanced by uroporphyrinogen III synthase expression, porphobilinogen by ALA dehydratase expression, and uroporphyrin and coproporphyrin by 1- hydroxymethylbilane synthase expression. The strain coexpressing coaA, hemA, maeB, and dctA produced heme of 0.49 micromol/g-DCW, which was twice as much from the strain without coaA expression. Further strain improvement for the porphyrin derivatives is discussed based on the results.

The Escherichia coliSLC26 homologue YchM (DauA) is a C4‐dicarboxylic acid transporter

The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM-dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C(4) -dicarboxylic acids and that its substrate specificity differs from the well-characterized succinate transporter, DctA. Accordingly ychM was re-designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C(4) -dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.

Renal dysfunction in methylmalonic acidurias: review for the pediatric nephrologist

Methylmalonic acidurias are a heterogeneous group of inborn errors of branched-chain amino acid metabolism. Depending on the underlying etiology, acute or chronic renal disease constitutes major (long-term) complications. In recent decades, overall survival has improved due to optimized treatment strategies based on the use of standardized emergency protocols and dialysis techniques. The majority of these patients, especially those having mut°, cblB, and cblA deficiency, are at increased risk of developing chronic kidney disease secondary to tubulointerstitial nephritis to require hemo- or peritoneal dialysis. Kidney and/or liver transplantation, as organ replacement, or even gene therapy on a limited scale, are controversially discussed treatment options in methylmalonic acidurias. The pathophysiological basis of renal disease has not been clarified in detail until now, but a severe mitochondrial dysfunction and an impairment of tubular dicarboxylic acid transport due to accumulated toxic metabolic compounds has been recently proposed. Another severe renal complication of methylmalonic acidurias is the occurrence of cblC-associated infantile atypical hemolytic syndrome, which can result in acute kidney injury. Close collaboration between (pediatric) nephrologists and metabolic specialists is required for the long-term management of these patients.

Symbiotic activity of alfalfa rhizobia (Sinorhizobium meliloti) strains with genetically modified transport of dicarboxylic acids

Inactivation of the genes involved in dicarboxylic acid transport in the alfalfa rhizobia Sinorhizobium meliloti—the structural gene of succinate permease dctA and its transcriptional regulators dctBD, nifA, and ntrA—leads to complete or partial loss of N2-fixing activity, while amplification of these genes leads to an increase in this activity. This considerably elevates the accumulation of nitrogen and carbon in alfalfa; however, the plant weight increases to a lesser degree, which is associated with an incomplete translocation of nitrogen to the aboveground organs. Factor analysis has demonstrated that the amplification of dctABD genes significantly influences these characteristics of symbiotic efficiency in all the performed experiments, while the effects of dctA, nifA, and ntrA depend on the plant cultivar and vegetation conditions.

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).

Dicarboxylic acid transport through hematite-coated sand

To better understand the behavior of low molecular weight organic acids in subsurface environments, the transport of three dicarboxylic acids (phthalic, maleic, and fumaric acid) in water-saturated columns packed with a hematite-coated sand was investigated in single and binary organic acid systems. Experiments were conducted at a single ionic strength (0.1 M) and at two pH values (4.1 and 5.3). In single-acid systems, the order of breakthrough at both pH values was fumaric acid, followed by maleic acid, and then phthalic acid. The shape of the breakthrough curves for the acids at the two pH values were similar except at pH 5.3 phthalic acid showed two adsorption fronts. The initial front only partially broke through, whereas the second front proceeded to complete breakthrough. This behavior resulted from a marked pH increase during phthalic acid adsorption and suggests that the single-acid systems behaved as dual-component systems with the organic acid and hydrogen ion as variables. The breakthrough curves for the binary organic acid systems showed organic acids with a higher adsorption affinity (e.g., phthalic acid) competitively displace organic acids with a lower adsorption affinity (e.g., fumaric acid). The dual-component effect observed for phthalic acid in the single-acid systems was suppressed in the mixed acid systems, perhaps reflecting pH changes that accompanied the desorption of the weakly-binding acids. These results may provide an important step toward further elucidating the processes controlling organic acid fractionation in the subsurface.

Characteristics of dicarboxylic amino acid transport by rabbit intestinal brush-border membrane vesicles.

Journal Article Characteristics of Dicarboxylic Amino Acid Transport by Rabbit Intestinal Brush-Border Membrane Vesicles Get access Nutrition Reviews, Volume 43, Issue 1, January 1985, Pages 30–32, https://doi.org/10.1111/j.1753-4887.1985.tb02391.x Published: 01 January 1985

AgtA, the Dicarboxylic Amino Acid Transporter of Aspergillus nidulans , Is Concertedly Down-Regulated by Exquisite Sensitivity to Nitrogen Metabolite Repression and Ammonium-Elicited Endocytosis

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

Potential Application of the Recombinant Escherichia coli-Synthesized Heme as a Bioavailable Iron Source

To investigate the potential use of microbial heme as an iron source, recombinant Escherichia coli coexpressing ALA synthase (HemA) as well as the NADP-dependent malic enzyme (MaeB) and dicarboxylic acid transporter (DctA) were cultured. The typical red pigment extracted from the recombinant E. coli after 38 h showed highest absorbance at 407 nm, and the amount of iron in 38.4 mg of microbial heme extract derived from 6-l fermentation broth was 4.1 mg. To determine the commercial potential of the recombinant E.coli-synthesized iron-associated heme as an iron source, mice were fed the iron-free provender with the microbial heme extract. The average body weight reduction of mice fed non-iron provender was 2.3%, whereas no detectable weight loss was evident in mice fed microbial heme addition after 15 days. The heme content of the blood from microbial heme fed mice was 4.2 mg/ml whereas that of controls was 2.4 mg/ml, which implies that the microbial heme could be available for use as an animal iron source.

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

BackgroundThe genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.ResultsThe experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second putative succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes.ConclusionThe genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.