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At least 127 records · Page 7

Computer-aided design of a proton pump

The use of transmembrane proton gradients in energy transduction is an almost universal feature of life on earth. These proton gradients are established and maintained by specialized assemblies of proteins which actively pump protons across membranes. One broad class of proton pumps uses captured light energy to drive the proton pumping. Our goal is to elucidate the minimum structural requirements of a light-driven proton-pump. There are two basic components to a simple light-driven proton pump: a source of photo-generated protons and a "gate-keeper" which prevents these protons from reattaching themselves to their source. A wide variety of molecules in the membrane, even as simple as polycyclic aromatic hydrocarbons, are capable of releasing protons when illuminated. Our work is therefore focused on the design of the "gate-keeper." Our initial model involves a pair of proton acceptors, coupled to each other by a transient water bridge, and supported in the membrane by a small bundle of peptide helices. Upon illumination, the proton source transfers its proton to the:- first acceptor of the gate-keeper. While the reverse reaction is highly probable, all that is needed to ensure irreversibility is a nonvanishing probability that the proton will be transferred to the second acceptor across a transient water bridge. Back transfer of the proton to the first acceptor, and thence to the proton source, is impeded by the free energy required to move the proton uphill towards the. proton source and by the disruption of the transient water bridge. As a prototypical water-bridged proton transfer system, we are studying the transfer of a proton across a water bridge from a formic acid to a formate anion. With a pK(sub alpha), of 3.7. formic acid is a good model for the acidic amino acids glutamate and aspartate which are good candidates for gate-keeper proton acceptors. Simulations of proton transfer reactions in a membrane are complicated by the quantum mechanical nature of the process breaking and forming chemical bonds.

New, Michael H.↗

Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.

NASA Discipline Neuroscience↗

Gating effects in Halobacterium halobium membrane transport

The transport of Na(+) via an H(+)/Na(+) antiporter and of aspartate and serine via Na(+)/amino acid symport systems was studied in Halobacterium halobium cell envelope vesicles. Gradients for H(+) were produced by illuminating the bacteriorhodopsin-containing vesicles at different light intensities, and the rate and extent of Na(+) transport were followed as functions of the electrochemical potential difference for protons. The coupling of Na(+) and H(+) gradients suggested a translocation stoichiometry of 2H(+)/Na(+) for the antiporter. The rate of Na(+) transport increases steeply above a critical transmembrane electrochemical proton gradient, and since the electrical and the chemical potentials of H(+) at this threshold point vary with the experimental conditions, while the sum of these potentials is constant, it was concluded that the gating of the Na(+) transport is caused by the total electrochemical gradient.

Lanyi, J. K.↗

Functional support of glutamate as a vestibular hair cell transmitter in an amniote

Although hair cells in the cochlea and in the vestibular endorgans of anamniotes are thought to release glutamate or a similar compound as their transmitter, there is little evidence in amniotes (which, unlike anamniotes, possess both type I and II hair cells) as to the nature of the hair cell transmitters in the vestibular labyrinth. We have recorded extracellularly from single semicircular canal afferents in the turtle labyrinth maintained in vitro and have bath-applied a number of transmitter agonists and antagonists to relate the effects of these substances to the actions of the endogenous transmitter substances. Both glutamate and aspartate strongly excite the afferents while GABA and carbachol have negligible or weak effects. In contrast to its lack of effect on afferent activity in some anamniotes, N-methyl-D-aspartate (NMDA) was also found to excite these afferents. Kynurenic acid reversibly reduced the resting firing rates of the afferents and the increases in firing due to the application of glutamate and aspartate. These findings provide preliminary support for the hypothesis that glutamate (or a related compound) is also a vestibular hair cell transmitter in amniotes.

NASA Discipline Neuroscience↗

AV3V lesions attenuate the cardiovascular responses produced by blood-borne excitatory amino acid analogs

Systemic injections of the excitatory amino acid (EAA) analogs, kainic acid (KA) and N-methyl-D-aspartate (NMDA), produce a pressor response in conscious rats that is caused by a centrally mediated activation of sympathetic drive and the release of arginine vasopressin (AVP). This study tested the hypothesis that the tissue surrounding the anteroventral part of the third ventricle (AV3V) plays a role in the expression of the pressor responses produced by systemically injected EAA analogs. Specifically, we examined whether prior electrolytic ablation of the AV3V region would affect the pressor responses to KA and NMDA (1 mg/kg iv) in conscious rats. The KA-induced pressor response was smaller in AV3V-lesioned than in sham-lesioned rats (11 +/- 2 vs. 29 +/- 2 mmHg; P < 0.05). After ganglion blockade, KA produced a pressor response in sham-lesioned but not AV3V-lesioned rats (+27 +/- 3 vs. +1 +/- 2 mmHg; P < 0.05). The KA-induced pressor response in ganglion-blocked sham-lesioned rats was abolished by a vasopressin V1-receptor antagonist. Similar results were obtained with NMDA. The pressor response to AVP (10 ng/kg iv) was slightly smaller in AV3V-lesioned than in sham-lesioned ganglion-blocked rats (45 +/- 3 vs. 57 +/- 4 mmHg; P < 0.05). This study demonstrates that the pressor responses to systemically injected EAA analogs are smaller in AV3V-lesioned rats. The EAA analogs may produce pressor responses by stimulation of EAA receptors in the AV3V region, or the AV3V region may play an important role in the expression of these responses.

Non-NASA Center↗

Analysis of the cyanobacterial amino acid metabolism with a precise genome-scale metabolic reconstruction of Anabaena sp. UTEX 2576

Filamentous cyanobacteria such as Anabaena sp. UTEX 2576 (a.k.a., Nostoc sp. PCC 7120) are a sustainable platform for commodity and specialty chemical production. Anabaena is a model cyanobacterium adopted to study production of nitrogen-containing metabolites useful for chemical, cosmetic, and pharmaceutical industries. Therefore, a precise description of the Anabaena metabolic network is desired to analyze their amino-acid metabolism and elucidate potential applications of these cyanobacteria as host organisms for biotechnological production. Secondary metabolite production depends on the metabolic availability of amino acids. We provide new insight on the biotechnological utilization of Anabaena after predicting phycocyanobilin and amino-acid production rates, using a genome-scale metabolic model (iDN1004). This metabolic reconstruction is a highly comprehensive representation of the global metabolism of Anabaena, which also contains experimental biomass equations and constraints under photoautotrophic and photodiazotrophic growth. Modelling results ranked proteinogenic amino acids based on predicted metabolic fluxes through amino-acid producing reactions. From these, l-aspartate, glycine, l-serine, l-valine, l-alanine, l-threonine, and l-leucine were selected as the best branching points to conduct metabolic engineering, where l-aspartate, l-serine, l-valine and glycine serve as potential precursors of the secondary metabolites Schizokinen (a siderophore), Sphingosine (a ceramide), Lyngbyatoxin A (an alkaloid), and Shinorine (a sunscreen).

59 BASIC BIOLOGICAL SCIENCES↗

Data for High Yield Production of 3-Hydroxypropionic Acid Using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Bioproducts↗

Light-activated amino acid transport in Halobacterium halobium envelope vesicles

Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

Macdonald, R. E.↗

Large-scale Genetic Characterization of a Model Sulfate-Reducing Bacterium

ABSTRACTSulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources, and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

Trotter, Valentine V↗

Large-scale genetic characterization of the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Treatment of cholinergic‐induced status epilepticus with polytherapy targeting GABA and glutamate receptors

Abstract Despite new antiseizure medications, the development of cholinergic‐induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia . 2005;46:142 demonstrated that the initiation and maintenance of cholinergic‐induced RSE are associated with trafficking and inactivation of gamma‐aminobutyric acid A receptors (GABA A R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N‐methyl‐ d ‐aspartate receptors (NMDAR) and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation ( Neurobiol Dis . 2013;54:225; Epilepsia . 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic‐induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic‐induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic‐induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine‐induced seizure in rats, organophosphorus nerve agent (OPNA)‐induced seizure in rats, and OPNA‐induced seizure in two mouse models: (1) carboxylesterase knockout (Es1 −/− ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock‐in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic‐induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic‐induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

59 BASIC BIOLOGICAL SCIENCES↗

Integration of proteomics and metabolomics into the Design, Build, Test, Learn cycle to improve 3-hydroxypropionic acid production in Aspergillus pseudoterreus

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4× after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

Pomraning, Kyle R.↗

Cyanobacterial carboxysome mutant analysis reveals the influence of enzyme compartmentalization on cellular metabolism and metabolic network rigidity

Cyanobacterial carboxysomes encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Genetic deletion of the major structural proteins encoded within the ccm operon in Synechococcus sp. PCC 7002 ( ΔccmKLMN ) disrupts carboxysome formation and significantly affects cellular physiology. In this study, we employed both metabolite pool size analysis and isotopically nonstationary metabolic flux analysis (INST-MFA) to examine metabolic regulation in cells lacking carboxysomes. Under high CO 2 environments (1%), the ΔccmKLMN mutant could recover growth and had a similar central flux distribution as the control strain, with the exceptions of moderately decreased photosynthesis and elevated biomass protein content and photorespiration activity. Metabolite analyses indicated that the ΔccmKLMN strain had significantly larger pool sizes of pyruvate (>18 folds), UDPG (uridine diphosphate glucose), and aspartate as well as higher levels of secreted organic acids (e.g., malate and succinate). These results suggest that the ΔccmKLMN mutant is able to largely maintain a fluxome similar to the control strain by changing in intracellular metabolite concentrations and metabolite overflows under optimal growth conditions. When CO 2 was insufficient (0.2%), provision of acetate moderately promoted mutant growth. Interestingly, the removal of microcompartments may loosen the flux network and promote RuBisCO side-reactions, facilitating redirection of central metabolites to competing pathways (i.e., pyruvate to heterologous lactate production). This study provides important insights into metabolic regulation via enzyme compartmentation and cyanobacterial compensatory responses.

09 BIOMASS FUELS↗

High yield production of 3-hydroxypropionic acid using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Biotechnology↗

EDTA-insoluble, calcium-binding proteoglycan in bovine bone

A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.

NASA Program Space Physiology and Countermeasures↗

Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System: The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation - Part 2

Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

Lowry, O. H.↗

Response of amino acids in hindlimb muscles to recovery from hypogravity and unloading by tail-cast suspension

Concentrations of glutamine, glutamate, aspartate (+ asparagine) and alanine were compared in hindlimb muscles of SL-3 and ground control rats. Alanine was lower in the soleus of flown rats but not of suspended animals, with no response in other muscles except a slight increase in the unloaded plantaris. With recovery, alanine in the soleus was elevated. Since no differences in alanine metabolism were found by isolated muscle, changes in muscle alanine are probably due to altered body use of this amino acid leading to varied plasma levels.

Tischler, M. E.↗

Regulation of L - and D -Aspartate Transport and Metabolism in Acinetobacter baylyi ADP1

Here, the regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N7-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways.

59 BASIC BIOLOGICAL SCIENCES↗