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Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli. [ABSTRACT FROM AUTHOR]

Microbial acetate metabolism is an important part of marine carbon cycling. We present a comprehensive study to constrain microbial acetate metabolism and its regulation in surface seawater of the northwest Pacific Ocean. We found that acetate oxidation (rate constant k: 0.016–0.506 day−1) accounted for 77.6%–99.4% of the total microbial acetate uptake, suggesting that acetate was predominantly used as a microbial energy source. Acetate also served as a significant biomass carbon source, as reflected by the elevated contribution of acetate assimilation to bacterial carbon production. Acetate turnover was largely influenced by water mass mixing and nutrient conditions. Atmospheric deposition was a source of acetate in surface water and this process can also impact the microbial acetate uptake. Microbial utilization of acetate could account for up to 25.9% of the bacterial carbon demand, suggesting the significant role of acetate metabolism in microbial carbon cycling in the open ocean. Plain Language Summary: Acetate is a key metabolic intermediate during the mineralization of organic matter and microbial acetate metabolism plays a crucial role in marine carbon cycling. However, the microbial metabolism of acetate and its environmental controls in pelagic waters remain poorly understood. We investigated biogeochemical cycling of acetate in the Kuroshio‐Oyashio extension region of northwest Pacific Ocean. Acetate concentrations in surface waters were generally low and atmospheric deposition was a source of acetate in the upper ocean. Acetate was used preferentially as a microbial energy source and the proportion of acetate devoted to bacterial growth or respiration largely depended on local nutrient conditions. Rapid biological turnover of acetate contributed significantly to bacterial carbon demand, exceeding the contributions of other compounds such as methanol. These results constrain the atmospheric sources and microbial sinks of marine acetate and reveal the ecological significance of microbial acetate metabolism in the open ocean. Key Points: Microbial uptake rates of acetate ranged from 1.7 to 495 nmol L−1 day−1 in surface waters of the northwest Pacific OceanAcetate is used preferentially as a microbial energy source rather than as a biomass carbon sourceMicrobial acetate metabolism is influenced by water mass mixing and atmospheric deposition [ABSTRACT FROM AUTHOR]

Background: Previous studies have confirmed the involvement of EmaSR (ethanol metabolism a sensor/regulator) in the regulation of Acinetobacter baumannii ATCC 19606 ethanol and acetate metabolism. RNA-seq analysis further revealed that DJ41_568-571, DJ41_2796, DJ41_3218, and DJ41_3568 regulatory gene clusters potentially participate in ethanol and acetate metabolism under the control of EmaSR. Methods: This study fused the EmaSR regulon promoter segments with reporter genes and used fluorescence expression levels to determine whether EmaSR influences regulon expression in ethanol or acetate salt environments. The enzymatic function and kinetics of significantly regulated regulons were also studied. Results: The EmaSR regulons P2796 and P3218 exhibited > 2-fold increase in fluorescence expression in wild type compared to mutant strains in both ethanol and acetate environments, and PemaR demonstrated a comparable trend. Moreover, increases in DJ41_2796 concentration enhanced the conversion of acetate and succinyl-CoA into acetyl-CoA and succinate, suggesting that DJ41_2796 possesses acetate: succinyl-CoA transferase (ASCT) activity. The kcat/KM values for DJ41_2796 with potassium acetate, sodium acetate, and succinyl-CoA were 0.2131, 0.4547, and 20.4623 mM−1s−1, respectively. Conclusions: In A. baumannii, EmaSR controls genes involved in ethanol and acetate metabolism, and the EmaSR regulon DJ41_2796 was found to possess ASCT activity. [ABSTRACT FROM AUTHOR]

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO2-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA- derived metabolites. [ABSTRACT FROM AUTHOR]

Pretreatment of lignocellulosic biomass produces growth inhibitory substances such as furfural which is toxic to microorganisms. Acinetobacter baylyi ADP1 cannot use furfural as a carbon source, instead it biotransforms this compound into difurfuryl ether using the reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydrogenases AreB and FrmA during aerobic acetate catabolism. However, NADH consumption for furfural biotransformation compromises aerobic growth of A. baylyi ADP1. Depending on the growth phase, several genes related to acetate catabolism and oxidative phosphorylation changed their expression indicating that central metabolic pathways were affected by the presence of furfural. During the exponential growth phase, reactions involved in the formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (icd gene) and NADH (sfcA gene) were preferred when furfural was present. Therefore a higher NADH and NADPH production might support furfural biotransformation and biomass production, respectively. In contrast, in the stationary growth phase genes of the glyoxylate shunt were overexpressed probably to save carbon compounds for biomass formation, and only NADH regeneration was appreciated. Finally, disruption of the frmA or areB gene in A. baylyi ADP1 led to a decrease in growth adaptation and in the capacity to biotransform furfural. The characterization of this physiological behavior clarifies the impact of furfural in Acinetobacter metabolism. [ABSTRACT FROM AUTHOR]

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 µmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax, acetate of 0.9 µmol mg-1 min-1 versus Vmax, acetyl-CoA of 0.3 µmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 µmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d. [ABSTRACT FROM AUTHOR]

Komagataella phaffii (a.k.a. Pichia pastoris) requires histidine for optimal growth when cultured in a medium containing yeast extract, peptone (YP), and acetate (YPA). We demonstrate that HIS4‐deficient, K. phaffii strain GS115 exhibits a growth defect on YP‐media containing acetate, but not on other carbon sources. K. phaffii X33, a prototroph, grows better than K. phaffii GS115 (his4), a histidine auxotroph in YPA. Normal growth of GS115 is restored either by the expression of HIS4 or by culturing in YPA containing ≥0.6 mM histidine. In the presence of histidine, expression of several genes is altered, including those encoding key subunits of mitochondrial ATP synthase, transporters of amino acids and nutrients, as well as biosynthetic enzymes. Thus, histidine should be included as an essential component for optimal growth of K. phaffii histidine auxotrophs cultured in YPA.

Background: Previous studies have confirmed the involvement of EmaSR (ethanol metabolism a sensor/regulator) in the regulation of Acinetobacter baumannii ATCC 19606 ethanol and acetate metabolism. RNA-seq analysis further revealed that DJ41_568-571, DJ41_2796, DJ41_3218, and DJ41_3568 regulatory gene clusters potentially participate in ethanol and acetate metabolism under the control of EmaSR. Methods: This study fused the EmaSR regulon promoter segments with reporter genes and used fluorescence expression levels to determine whether EmaSR influences regulon expression in ethanol or acetate salt environments. The enzymatic function and kinetics of significantly regulated regulons were also studied. Results: The EmaSR regulons P2796 and P3218 exhibited > 2-fold increase in fluorescence expression in wild type compared to mutant strains in both ethanol and acetate environments, and PemaR demonstrated a comparable trend. Moreover, increases in DJ41_2796 concentration enhanced the conversion of acetate and succinyl-CoA into acetyl-CoA and succinate, suggesting that DJ41_2796 possesses acetate: succinyl-CoA transferase (ASCT) activity. The kcat/KM values for DJ41_2796 with potassium acetate, sodium acetate, and succinyl-CoA were 0.2131, 0.4547, and 20.4623 mM−1s−1, respectively. Conclusions: In A. baumannii, EmaSR controls genes involved in ethanol and acetate metabolism, and the EmaSR regulon DJ41_2796 was found to possess ASCT activity.

Numerous technological advances have been made in the depolymerization and fermentation of lignocellulosic biomass. Nevertheless, economic feasibility is still a major concern for the bioconversion of lignocellulose into value-added products. Acetate—the most abundant carbon source after the sugars in cellulosic hydrolysates—has been considered a fermentation inhibitor, but it can also be a good substrate in industrial biotechnological processes. Co-consumption of acetate and cellulosic sugars by yeasts will improve the economic feasibility of any bioconversion processes using cellulosic hydrolysates. This study investigates state-of-the-art technologies for the utilization of the hemicellulose fractions of lignocellulosic biomass, rich in acetate and xylose. Furthermore, the potentials of acetate- and xylose-rich hydrolysates will be highlighted in diversifying the product profiles for lignocellulosic bioprocesses from bioethanol to drop-in fuels and other value-added chemicals. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a well-known nosocomial pathogen that can survive in different environments through the use of intricate networks to regulate gene expression. Two-component systems (TCS) form an important part of such regulatory networks, and in this study, we describe the identification and characterization of a novel EmaSR TCS in A. baumannii. We constructed a Tn5-tagged mutagenesis library, from which an emaS sensor kinase gene and emaR response regulator gene were identified. We found that emaS/emaR single-mutants and double-mutants were unable to replicate in M9 medium with 1% ethanol as the single carbon source. Motility and biofilm formation were negatively affected in double-mutants, and transcriptomic analysis showed that mutation of emaSR dysregulated genes required for carbon metabolism. In addition, emaS/emaR single-mutants and double-mutants were unable to survive in acetic acid- and sodium acetate-containing medium, indicating that the EmaSR TCS is also important for acetate metabolism. Furthermore, virulence against Galleria mellonella was diminished in emaS/emaR single- and double-mutants. Taken together, these results show that this novel EmaSR TCS is involved in the regulation of A. baumannii ethanol metabolism and acetate metabolism, with important implications on motility, biofilm formation, and virulence if mutated. Further research on the underlying mechanisms is warranted. [ABSTRACT FROM AUTHOR]

Komagataella phaffii (a.k.a. Pichia pastoris) requires histidine for optimal growth when cultured in a medium containing yeast extract, peptone (YP), and acetate (YPA). We demonstrate that HIS4-deficient, K. phaffii strain GS115 exhibits a growth defect on YP-media containing acetate, but not on other carbon sources. K. phaffii X33, a prototroph, grows better than K. phaffii GS115 (his4), a histidine auxotroph in YPA. Normal growth of GS115 is restored either by the expression of HIS4 or by culturing in YPA containing ≥0.6 mM histidine. In the presence of histidine, expression of several genes is altered, including those encoding key subunits of mitochondrial ATP synthase, transporters of amino acids and nutrients, as well as biosynthetic enzymes. Thus, histidine should be included as an essential component for optimal growth of K. phaffii histidine auxotrophs cultured in YPA. [ABSTRACT FROM AUTHOR]

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A system-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the major substrates available in various nutrient niches such as the mammalian gut and a keto diet. A substantial amount of acetate gets secreted in aerobic metabolism. Therefore, investigating the study on acetate metabolism is highly significant. It is known that the global regulators fis and arcA regulate acetate uptake genes in E. coli under glucose conditions. This study deciphered the growth and flux distribution of E. coli transcription regulatory knockouts Δfis, ΔarcA and double deletion mutant, ΔarcAΔfis under acetate using 13C-metabolic flux analysis (MFA), which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~ 1.2–1.6-fold) with a proportionate increase in acetate uptake rates compared to the wild type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes via the TCA cycle, anaplerotic pathway and gluconeogenesis, which conferred an advantage of a faster growth rate with better carbon usage in all the mutants. This resulted in higher metabolic fluxes through the TCA cycle (~ 18–90%), lower gluconeogenesis (~ 15–35%) and higher CO2 and ATP production with the proportional increase in growth rate. The study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate, thus acting as potential candidates for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products. Key Points • Mutants exhibited a better balance between energy and precursor synthesis than WT. • Leveraged in the unravelling of regulatory control under various nutrient shifts. • Metabolic readjustment resulted in optimal biomass requirement and faster growth. [ABSTRACT FROM AUTHOR]

Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function, and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic, but data on the use of other potential carbon substrates are lacking in humans. This prospective and blinded cohort study was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure then, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from 13C-labeled substrates resulting in an M + 2 isotopologue that contains 2 13C atoms. Comparing the fate of three major carbon sources, mean enrichment of M + 2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate in 278 CCs from 21 women ≤34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p = 0.0004, t-test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M + 2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: −6.02, CI: −1.74,-13.79, p = 0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility. Abbreviations: CCs: cumulus cells; COC: cumulus-oocyte complex; LC-MS: liquid chromatography-mass spectrometry; acetyl-CoA: acetyl-Coenzyme A; CoA: Coenzyme A; [ABSTRACT FROM AUTHOR]

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized “EDEMP cycle” (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed (“fluxed”) through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

• Acetate metabolic pathway via glyoxylate shunt in K. marxianus was revealed. • Enhanced glyoxylate shunt increased acetate metabolism to 122.7 % at 40 °C. • Overexpressing GPD2 and FDH1 further increased acetate uptake by 3.9 folds. • Toxic compounds detoxification enhanced lignocellulosic biomass conversion. • Foresight for the acetate utilization as an intermediate for reducing CO 2. Lignocellulosic biomass is a typical agriculture waste and a potential feedstock for the green biomanufacturing. However, insufficient utilization of carbon sources caused by inhibitors, especially acetic acid, hinders the economic bioconversion. Here, we demonstrated the acetate metabolic pathway via glyoxylate shunt in nonconventional yeast Kluyveromyces marxianus. Acetate metabolism increased to 122.7 % at 40 °C after overexpressing ICL1 and MLS1 , key genes of glyoxylate shunt revealed by transcriptomic data. Strengthening GPD2 and FDH1 further enhanced the robustness of the K. marxianus , acetate metabolism was significantly increased. Finally, the modified K. marxianus 1727–5-ICL1-MLS1-GPD2-FDH1 boosted the efficiency for xylose uptake up to 1.3 folds in non-detoxified lignocellulosic hydrolysate, benefiting from a 3.9-fold increase in acetate consumption. Our results propose a novel strategy for xylose assimilation by coupling acetate metabolism, contributing the bioconversion of sustainable lignocellulosic biomass. It also provides a new scheme for the total carbon utilization of lignocellulosic biomass, including carbon dioxide (CO 2) released from the biofuel production to achieve a circular economy. [ABSTRACT FROM AUTHOR]

Acetate is a characteristic by-product of Escherichia coli K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP. Mutants in the AckA-Pta pathway differ from each other in the potential to produce and accumulate acetyl-phosphate. In the publication at hand, we investigated different mutants in the acetate pathway, both under aerobic as well as anaerobic conditions. While under aerobic conditions only small changes in growth rate were observed, all acetate mutants showed severe reduction in growth rate and changes in the by-product pattern during anaerobic growth. The AckA– mutant showed the most severe growth defect. The glucose uptake rate and the ATP concentration were strongly reduced in this strain. This mutant exhibited also changes in gene expression. In this strain, the atoDAEB operon was significantly upregulated under anaerobic conditions hinting to the production of acetoacetate. During anaerobic growth, protein acetylation increased significantly in the ackA mutant. Acetylation of several enzymes of glycolysis and central metabolism, of aspartate carbamoyl transferase, methionine synthase, catalase and of proteins involved in translation was increased. Supplementation of methionine and uracil eliminated the additional growth defect of the ackA mutant. The data show that anaerobic, fermentative growth of mutants in the AckA-Pta pathway is reduced but still possible. Growth reduction can be explained by the lack of an important ATP generating pathway of mixed acid fermentation. An ackA deletion mutant is more severely impaired than pta or ackA-pta deletion mutants. This is most probably due to the production of acetyl-P in the ackA mutant, leading to increased protein acetylation.

Acetate is a characteristic by-product of Escherichia coli K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP. Mutants in the AckA-Pta pathway differ from each other in the potential to produce and accumulate acetyl-phosphate. In the publication at hand, we investigated different mutants in the acetate pathway, both under aerobic as well as anaerobic conditions. While under aerobic conditions only small changes in growth rate were observed, all acetate mutants showed severe reduction in growth rate and changes in the by-product pattern during anaerobic growth. The AckA– mutant showed the most severe growth defect. The glucose uptake rate and the ATP concentration were strongly reduced in this strain. This mutant exhibited also changes in gene expression. In this strain, the atoDAEB operon was significantly upregulated under anaerobic conditions hinting to the production of acetoacetate. During anaerobic growth, protein acetylation increased significantly in the ackA mutant. Acetylation of several enzymes of glycolysis and central metabolism, of aspartate carbamoyl transferase, methionine synthase, catalase and of proteins involved in translation was increased. Supplementation of methionine and uracil eliminated the additional growth defect of the ackA mutant. The data show that anaerobic, fermentative growth of mutants in the AckA-Pta pathway is reduced but still possible. Growth reduction can be explained by the lack of an important ATP generating pathway of mixed acid fermentation. An ackA deletion mutant is more severely impaired than pta or ackA-pta deletion mutants. This is most probably due to the production of acetyl-P in the ackA mutant, leading to increased protein acetylation. [ABSTRACT FROM AUTHOR]

Acetate and the related metabolism of acetyl-coenzyme A (acetyl-CoA) confer numerous metabolic functions, including energy production, lipid synthesis, and protein acetylation. Despite its importance as a nutrient for cellular metabolism, its source has been unclear. Recent studies have provided evidence to support the existence of a de novo pathway for acetate production derived from pyruvate, the end product of glycolysis. This mechanism of pyruvate-derived acetate generation could have far-reaching implications for the regulation of central carbon metabolism. In this Opinion, we discuss our current understanding of acetate metabolism in the context of cell-autonomous metabolic regulation, cell–cell interactions, and systemic physiology. Applications relevant to health and disease, particularly cancer, are emphasized. Our understanding of the origin of acetate in physiological and disease states is currently evolving and is not limited to exogenous acetate uptake. The regulation of the production of endogenous pyruvate-derived acetate remains largely unknown. Pathways of metabolism during periods of nutritional excess and limitation allow metabolic coupling to confer fitness advantages to proximal and systemic cellular partners. Preferential uptake of acetate by certain tissues is driven not only by the availability of transporters but by environmental pressures such as hypoxia and nutrient scarcity. [ABSTRACT FROM AUTHOR]

This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation. [ABSTRACT FROM AUTHOR]

This study demonstrated the degradation of unionized acetate in anaerobic digestion at 45°C through the identification of kinetics and inhibition parameters. The kinetic parameters, Ks and rmax, were determined using Monod-based model for three different conditions, namely uninhibited, inhibited, and systems with high substrate condition. The Ks values for uninhibited condition were in the range of 0.124 to 0.191 mg/L as unionized HAc. Ks value of inhibited condition were at 0.027 mg/L as unionized HAc. Ks values for systems with high substrate condition were found to be in the range of 0.237 to 0.279 mg/L as unionized HAc. As for rmax, a 35°C anaerobic digestion system showed the highest value at 0.166 mg/L/day - greater than the values of all 45°C systems under all experimental conditions. Additionally, the inhibition parameter KI was also determined using the Michaelis-Menten model. The parameter was determined for inhibitory conditions resulting from high free NH3 content. The inhibition type was uncompetitive with KI value of 0.072 mg/L as unionized HAc. The outcomes suggested that the methanogens responsible for the digestion process at 45°C were thermo-tolerant acetateutilizing methanogens of Methanosarcinaceae species, and the system will be totally inhibited with the presence of high free NH3 content. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major threat to global health, with the emergence of multi-drug and extensively drug-resistant strains posing a serious challenge. Thereby, understanding the molecular basis of MTB virulence and disease pathogenesis is critical for developing effective therapeutic strategies. Targeting proteins involved in central metabolism has been recognized as a promising therapeutic approach to combat MTB. In this regard, the enzyme AckA of the acetate metabolic pathway which produces acetate from acetyl phosphate, is an important drug target for various pathogenic organisms. Therefore, this study aimed to identify potential AckA inhibitors through in silico methods, including molecular modeling, molecular dynamics simulation (MDS), and high-throughput virtual screening (HTVS) followed by ADMETox, MMGBSA, Density Functional Theory (DFT) calculations. HTVS of one million compounds from the ZINC database against AckA resulted in the top five hits (ZINC82048449, ZINC1219737510, ZINC1771921358, ZINC119699567, and ZINC1427100376) with better binding affinity and optimal binding free energy. MDS studies on complexes revealed that key residues, Asn195, Asp266, Phe267, Gly314, and Asn318 played a significant role in stable interactions of the top-ranked compounds to AckA. These outcomes provide insights into the optimal binding of the leads to inhibit the acetate pathway and aid in the rational design of novel therapeutic agents. Thus, the identified leads may act as promising compounds for targeting AckA and may serve as a potential therapeutic modality for treating TB. Our findings offer valuable insights into the inhibition of the acetate pathway, while also serving as a blueprint for rational drug design. The identified leads hold promise as compelling compounds for targeting AckA, thereby offering a potential therapeutic avenue for tackling TB. Thus, our study uncovers a pathway toward promising TB therapeutics by elucidating AckA inhibitors. By leveraging in silico methodologies, potent compounds that hold the potential to thwart AckA's role in MTB's acetate pathway have been unveiled. This breakthrough fosters optimism in the quest for novel and effective TB treatments, addressing a global health challenge with renewed vigor. [Display omitted] • This study hold significant value due to the conserved binding site residues across different acetate kinases. • The conserved ATP binding site acts as a promising avenue for the development of ATP competitive inhibitors. • The compound ZINC1219737510 was identified as a potent acetate kinase inhibitor. • Extending the scope for probing chemical and structural diversity of compounds that have the potential to interact with acetate kinase. [ABSTRACT FROM AUTHOR]

In the past few years, the yeast Dekkera bruxellensis has gained much of attention among the so-called non-conventional yeasts for its potential in the biotechnological scenario, especially in fermentative processes. This yeast has been regarded as an important competitor to Saccharomyces cerevisiae in bioethanol production plants in Brazil and several studies have reported its capacity to produce ethanol. However, our current knowledge concerning D. bruxellensis is restricted to its aerobic metabolism, most likely because wine and beer strains cannot grow in full anaerobiosis. Hence, the present work aimed to fulfil a gap regarding the lack of information on the physiology of Dekkera bruxellensis growing in the complete absence of oxygen and the relationship with assimilation of nitrate as nitrogen source. The ethanol strain GDB 248 was fully capable of growing anaerobically and produces ethanol at the same level of S. cerevisiae. The presence of nitrate in the medium increased this capacity. Moreover, nitrate is consumed faster than ammonium and this increased rate coincided with a higher speed of glucose consumption. The profile of gene expression helped us to figure out that even in anaerobiosis, the presence of nitrate drives the yeast cells to an oxidative metabolism that ultimately incremented both biomass and ethanol production. These results finally provide the clues to explain most of the success of this yeast in industrial processes of ethanol production. [ABSTRACT FROM AUTHOR]

Acetate is a key intermediate of organic matter mineralization, but its metabolism remains largely unconstrained in the pelagic ocean. We conducted an integrated biogeochemical study to investigate microbial acetate cycling in the northern Gulf of Mexico with the goal of elucidating the importance of acetate as a carbon and energy source. Acetate was used primarily as an energy source, as evidenced by observed oxidation rates (rate constant k: 0.06–0.22 day−1) that varied between 42% and 96% of total biological acetate uptake (i.e., assimilation + oxidation; k: 0.06–0.34 day−1). The assimilation of acetate into biomass (k: 0.01–0.20 day−1) illustrated the potential significance of acetate as a biomass carbon source, particularly in nutrient‐rich coastal waters. No relationship between acetate assimilation or oxidation and environmental factors, such as chlorophyll and nutrients, was observed. However, elevated acetate uptake in reduced oxygen waters characterized by particulate organic carbon mineralization suggests that acetate metabolism may be a good proxy for particulate organic carbon breakdown. Molecular genetic analysis revealed that SAR11 Alphaproteobacteria were the most abundant heterotrophic bacteria and suggest that they may utilize acetate. At some sites, acetate carbon may have accounted for up to 50.4% of the bacterial carbon production. These results suggest that acetate may serve as an important carbon and energy source for heterotrophic bacteria thus revealing a potentially significant role of acetate for dissolved organic carbon cycling in the ocean. Key Points: Acetate was used primarily as an energy source but was also an important structural carbon source, especially in coastal watersAcetate metabolism was not correlated with chlorophyll or nutrients but tracked POC degradation and O2 consumption in the water columnThe carbon incorporated into biomass from acetate may support a substantial fraction of bacterial production [ABSTRACT FROM AUTHOR]

2017 marks the 60th anniversary of Krebs’ seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism. [ABSTRACT FROM AUTHOR]

[Display omitted] • Kinetic analysis showed antibiotic exposure stimulated methanogenesis. • Metagenome-assembled genomes (MAGs) discovered functional microbiomes. • Network analysis revealed targeted cooperation responding to antibiotic exposure. • Isotope tracing confirmed the dominance of acetoclastic methanogenesis. Understanding the source of methane (CH 4) is of great significance for improving the anaerobic fermentation efficiency in bioengineering, and for mitigating the emission potential of natural ecosystems. Microbes involved in the process named direct interspecies electron transfer coupling with CO 2 reduction, i.e., electrons released from electroactive bacteria to reduce CO 2 into CH 4 , have attracted considerable attention for wastewater treatment in the past decade. However, how the synergistic effect of microbiota contributes to this anaerobic carbon metabolism accompanied by CH 4 production still remains poorly understood, especial for wastewater with antibiotic exposure. Results show that enhancing lower-abundant acetoclastic methanogens and acetogenic bacteria, rather than electroactive bacteria, contributed to CH 4 production, based on a metagenome-assembled genomes network analysis. Natural and artificial isotope tracing of CH 4 further confirmed that CH 4 mainly originated from acetoclastic methanogenesis. These findings reveal the contribution of direct acetate cleavage (acetoclastic methanogenesis) and provide insightsfor further regulation of methanogenic strategies. [ABSTRACT FROM AUTHOR]

To investigate the underlying causes of instability, reactor overload was induced by implementing step-type disturbances in the organic load rate (OLR), followed by a comprehensive characterization of microbial communities (genes), process parameters, and kinetics parameters. The results revealed significant reductions in k acetate and k propionate , with decreases of 52.87 ± 0.14 % and 73.23 ± 0.94 %, respectively, compared to their maximum values (1.74 ± 0.09 gVS/(gVSS∙d) and 0.71 ± 0.05 gVS/(gVSS∙d)), when the OLR was set at 3 kgVS/(m3∙d) and the methane yield (MY) was stabilized at 0.52 ± 0.02 L/gVS. Similarly, at an OLR of 3.5 kgVS/(m3∙d), the significant deviations of k acetate and k propionate from their normal range (k acetate : 0.71 ± 0.02–1.74 ± 0.09 gVS/(gVSS∙d); k propionate : 0.39 ± 0.02–0.71 ± 0.05 gVS/(gVSS∙d)) were observed and a substantial decline in MY subsequently was observed. Notably, the continuous and significant decline in the relative abundance of SPOB, AMA, and their related metabolic genes (prpE , ACADM , paaF , PCCA , MCEE , sucD , cdhC , and metF) in response to the high OLR stress was observed, providing valuable insights into the reduction in k acetate and k propionate , ultimately leading to their accumulation. Moreover, intricate interactions between acetate and propionate metabolism under overload conditions were revealed, with propionate metabolism displaying the highest sensitivity. [Display omitted] • The acetate and propionate metabolism were blocked simultaneously. • Both acetate and propionate metabolism reciprocal influenced on each other. • Propionate metabolism displayed the highest sensitivity. • The regulation of k acetate and k propionate was influenced by some microbes (genes). [ABSTRACT FROM AUTHOR]

The CrbS/R system is a two-component signal transduction system that regulates acetate utilization in Vibrio cholerae, P. aeruginosa, and P. entomophila. CrbS is a hybrid histidine kinase that belongs to a recently identified family, in which the signaling domain is fused to an SLC5 solute symporter domain through aSTAC domain. Upon activation by CrbS, CrbR activates transcription of the acs gene, which encodes an acetyl-CoA synthase (ACS), and the actP gene, which encodes an acetate/solute symporter. In this work, we characterized the CrbS/R system in Pseudomonas fluorescens SBW25. Through the quantitative proteome analysis of different mutants, we were able to identify a new set of genes under its control, which play an important role during growth on acetate. These results led us to the identification of a conserved DNA motif in the putative promoter region of acetate-utilization genes in the Gammaproteobacteria that is essential for the CrbR-mediated transcriptional activation of genes under acetate-utilizing conditions. Finally, we took advantage of the existence of a second SLC5-containing two-component signal transduction system in P. fluorescens, CbrA/B, to demonstrate that the activation of the response regulator by the histidine kinase is not dependent on substrate transport through the SLC5 domain.

The regulation of metabolic networks has been shown to be distributed and shared through the action of metabolic cycles. Biochemical cycles play important roles in maintaining flux and substrate availability for multiple pathways to supply cellular energy and contribute to dynamic stability. By understanding the cyclic and acyclic flows of matter through a network, we are closer to understanding how complex dynamic systems distribute flux along interconnected pathways. In this work, we have applied a cycle decomposition algorithm to a genome-scale metabolic model of Chlamydomonas reinhardtii to analyse how acetate supply affects the distribution of fluxes that sustain cellular activity. We examined the role of metabolic cycles which explain the down regulation of photosynthesis that is observed when cells are grown in the presence of acetate. Our results suggest that acetate modulates changes in global metabolism, with the pentose phosphate pathway, the Calvin-Benson cycle and mitochondrial respiration activity being affected whilst reducing photosynthesis. These results show how the decomposition of metabolic flux into cyclic and acyclic components helps to understand the impact of metabolic cycling on organismal behaviour at the genome scale. [ABSTRACT FROM AUTHOR]

The CrbS/R system is a two-component signal transduction system that regulates acetate utilization in Vibrio cholerae, P. aeruginosa, and P. entomophila. CrbS is a hybrid histidine kinase that belongs to a recently identified family, in which the signaling domain is fused to an SLC5 solute symporter domain through aSTAC domain. Upon activation by CrbS, CrbR activates transcription of the acs gene, which encodes an acetyl-CoA synthase (ACS), and the actP gene, which encodes an acetate/solute symporter. In this work, we characterized the CrbS/R system in Pseudomonas fluorescens SBW25. Through the quantitative proteome analysis of different mutants, we were able to identify a new set of genes under its control, which play an important role during growth on acetate. These results led us to the identification of a conserved DNA motif in the putative promoter region of acetate-utilization genes in the Gammaproteobacteria that is essential for the CrbR-mediated transcriptional activation of genes under acetate-utilizing conditions. Finally, we took advantage of the existence of a second SLC5-containing two-component signal transduction system in P. fluorescens, CbrA/B, to demonstrate that the activation of the response regulator by the histidine kinase is not dependent on substrate transport through the SLC5 domain. [ABSTRACT FROM AUTHOR]

The ketone body, β-hydroxybutyrate (βOHB), is metabolised by the brain alongside the mandatory brain fuel glucose. To examine the extent and circumstances by which βOHB can supplement glucose metabolism, we studied guinea pig cortical brain slices using increasing concentrations of [U-C]D-βOHB in conjunction with [1-C] d-glucose under conditions of normo- and hypoglycaemia, as well as under high potassium (40 mmol/L K) depolarization in normo- and hypoglycaemic conditions. The contribution of βOHB to synthesis of GABA was also probed by inhibiting the synthesis of glutamine, a GABA precursor, with methionine sulfoximine (MSO). [U-C]D-βOHB at lower concentrations (0.25 and 1.25 mmol/L) stimulated mitochondrial metabolism, producing greater total incorporation of label into glutamate and GABA but did not have a similar effect in the cytosolic compartment where labelling of glutamine was reduced at 1.25 mmol/L [U-C] D-βOHB. At higher concentrations (2.5 mmol/L) [U-C] D-βOHB inhibited metabolism of [1-C] d-glucose, and reduced total label incorporation and total metabolite pools. When glucose levels were reduced, βOHB was able to partially restore the loss of glutamate and GABA caused by hypoglycaemia, but was not able to supplement levels of lactate, glutamine or alanine or to prevent the increase in aspartate. Under depolarizing conditions glucose was the preferred substrate over βOHB, even in hypoglycaemic conditions where comparatively less βOHB was incorporated except into aspartate isotopomers. Inhibition of glutamine synthesis with MSO had no significant effect on incorporation of label from [U-C] D-βOHB into GABA C2,1 indicating that the majority of this GABA was synthesized in GABAergic neurons from [U-C] D-βOHB rather than from Gln C4,5 imported from astrocytes. [ABSTRACT FROM AUTHOR]

[13C]Acetate is known to label metabolites preferentially in astrocytes rather than neurons and it has consequently been used as a marker for astrocytic activity. Recent discoveries suggest that control of acetate metabolism and its contributions to the synthesis of metabolites in brain is not as simple as first thought. Here, using a Guinea pig brain cortical tissue slice model metabolizing [1-13C]D-glucose and [1,2-13C]acetate, we investigated control of acetate metabolism and the degree to which it reflects astrocytic activity. Using a range of [1,2-13C]acetate concentrations, we found that acetate is a poor substrate for metabolism and will inhibit metabolism of itself and of glucose at concentrations in excess of 2 mmol/L. By activating astrocytes using potassium depolarization, we found that use of [1,2-13C]acetate to synthesize glutamine decreases significantly under these conditions showing that acetate metabolism does not necessarily reflect astrocytic activity. By blocking synthesis of glutamine using methionine sulfoximine, we found that significant amount of [1,2-13C]acetate are still incorporated into GABA and its metabolic precursors in neurons, with around 30% of the GABA synthesized from [1,2-13C]acetate likely to be made directly in neurons rather than from glutamine supplied by astrocytes. Finally, to test whether activity of the acetate metabolizing enzyme acetyl-CoA synthetase is under acetylation control in the brain, we incubated slices with the Ace CS1 deacetylase silent information regulator 1 ( SIRT1) activator SRT 1720 and showed consequential increased incorporation of [1,2-13C]acetate into metabolites. Taken together, these data show that acetate metabolism is not directly nor exclusively related to astrocytic metabolic activity, that use of acetate is related to enzyme acetylation and that acetate is directly metabolized to a significant degree in GABAergic neurons. Changes in acetate metabolism should be interpreted as modulation of metabolism through changes in cellular energetic status via altered enzyme acetylation levels rather than simply as an adjustment of glial-neuronal metabolic activity. [ABSTRACT FROM AUTHOR]

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d -lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35–40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d -lactate plus [14C]- l -lactate, the radioactive label was found in methane, CO 2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d -lactate oxidation was coupled to O 2 -consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d -lactate as carbon source and showed membrane-bound d -lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d -lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd : methanophenazine (quinol) oxidoreductase. Biomass generation and O 2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen. [Display omitted] • Air-adapted Methanosarcina acetivorans can grow in presence of acetate + lactate. • Lactate was oxidized in the presence of O 2 , producing methane, glycogen and biomass. • D-iLDH and cyt bd oxidase transcripts increased in cells showing high O 2 -consumption. • Respiration linked to lactate oxidation was sensitive to HQNO and partially to azide. • Lactate oxidation potentially through cytochromes, consumed the otherwise toxic O 2. [ABSTRACT FROM AUTHOR]

Volatile fatty acids (VFAs) are an inexpensive and renewable carbon source that can be generated from gas fermentation and anaerobic digestion of fermentable wastes. The oleaginous yeast Yarrowia lipolytica is a promising biocatalyst that can utilize VFAs and convert them into triacylglycerides (TAGs). However, currently there is limited knowledge on the metabolism of Y. lipolytica when cultured on VFAs. To develop a better understanding, we used acetate as the sole carbon source to culture two strains, a control strain and a previously engineered strain for lipid overaccumulation. For both strains, metabolism during the growth phase and lipid production phase were investigated by metabolic flux analysis using two parallel sodium acetate tracers. The resolved flux distributions demonstrate that the glyoxylate shunt pathway is constantly active and the flux through gluconeogenesis varies depending on strain and phase. In particular, by regulating the activities of malate transport and pyruvate kinase, the cells divert only a portion of the glyoxylate shunt flux required to satisfy the needs for anaplerotic reactions and NADPH production through gluconeogenesis and the oxidative pentose phosphate pathway (PPP). Excess flux flows back to the tricarboxylic acid (TCA) cycle for energy production. As with the case of glucose as the substrate, the primary source for lipogenic NADPH is derived from the oxidative PPP. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii has recently emerged as a viable alternative source of fossil fuel. However, the metabolic flow of carbon in C. reinhardtii towards making the carbon reserve is not yet understood. In addressing this issue, we have monitored the assimilation of 13 C-labeled acetate by the wild-type ( cw15 ) and the starch deficient ( sta6 ) strains of C. reinhardtii using 13 C NMR. The dynamics of starch and lipid reserves was studied under nitrogen starvation and supplement conditions. The starch was found to accumulate and mobilize faster than triacylglycerol (TAG) through the formation of CO 2 aq and bicarbonate. Addition of exogenous acetate significantly slowed down mobilization of both labeled starch and TAG reserves, suggesting that cells preferred incoming acetate to internal reserve as a source of carbon. In parallel, we undertook the analyses of cellular free amino acid pool changes using LC/MS to investigate how specific amino acid changes contribute towards “nitrogen sparing effect” during nitrogen starvation. Bulk of remainder amino acids do not exhibit any discernible changes in their steady state pool sizes, suggesting the possibility that carbon flow leading to starch, followed by lipid is homeostatically balanced during nitrogen starvation such that the amino acid carbon levels in the cells stay largely regulated. Taken all together, this study thus describes an excellent cellular system for probing the nitrogen starvation, nitrogen uptake and the consequent changes in carbon flow in relation to both starch/lipid accumulation and their re-mobilization and the changes in free amino acid pools in C. reinhardtii cells. [ABSTRACT FROM AUTHOR]

Background Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica ; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here. Methods The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae. Results Recombinant ACS showed higher catalytic efficiencies ( Vmax / Km ) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49–93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10–20%) in the hexose phosphates and PPi levels. Conclusions These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis. Significance The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out. [ABSTRACT FROM AUTHOR]

Background: The pstSCAB operon of Corynebacterium glutamicum, which encodes an ABC transport system for uptake of phosphate (Pi), is induced during the Pi starvation response. The two-component regulatory system PhoRS is involved in this response, but partial Pi starvation induction of pstSCAB in a ΔphoRS mutant indicated the involvement of additional regulator(s). Regulation of pstSCAB also involves the global transcriptional regulator GlxR. Results: DNA affinity chromatography identified the regulator of acetate metabolism RamB as a protein binding to pstS promoter DNA in vitro. Gel mobility shift assays and mutational analysis of the pstS promoter region revealed that RamB binds to two sites localized at positions -74 to -88 and -9 to +2 with respect to the transcriptional start site of pstSCAB. Reporter gene studies supported the in vivo relevance of both binding sites for activation of pstSCAB by RamB. DNA microarray analysis revealed that expression of many Pi starvation genes reached higher levels during the Pi starvation response on minimal medium with glucose as sole carbon source than in Pi starved acetate-grown C. glutamicum cells. Conclusions: In C. glutamicum, RamB is involved in expression control of pstSCAB operon. Thus, transcriptional regulation of pstSCAB is complex involving activation by the phosphate-responsive two-component regulatory system PhoSR and the regulators of carbon metabolism GlxR and RamB. [ABSTRACT FROM AUTHOR]

Cells of the green alga Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential—impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii is a unicellular, soil-dwelling (and aquatic) green alga that has significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences hypoxic/anoxic conditions. The diversity of pathways available to ferment sugars is often revealed in mutants in which the activities of specific branches of fermentative metabolism have been eliminated; compensatory pathways that have little activity in parental strains under standard laboratory fermentative conditions are often activated. The ways in which these pathways are regulated and integrated have not been extensively explored. In this review, we primarily discuss the intricacies of dark anoxic metabolism in Chlamydomonas, but also discuss aspects of dark oxic metabolism, the utilization of acetate, and the relatively uncharacterized but critical interactions that link chloroplastic and mitochondrial metabolic networks. [ABSTRACT FROM AUTHOR]

Background: VarS/VarA is one of the global factors regulating diverse aspects of the metabolism and virulence of bacteria including pathogenic Vibrio spp. An experiment to identify the VarS/VarA-regulon in V. vulnificus revealed that a putative LuxR-type transcriptional regulator was down-regulated in ΔvarA mutant. To investigate the roles of this regulatory cascade, the target gene regulated by a LuxR-regulator was identified and its expression was characterized. Results: Transcriptomic analysis of the mutant deficient in this LuxR-type regulator showed that the acsA gene encoding acetyl-CoA synthetase was down-regulated. Thus, this regulator was named AcsR for "regulator of acetyl-CoA synthetase". A putative histidine kinase gene, acsS, was located five ORFs downstream of the acsR gene. Expression of an acsA::luxAB transcriptional fusion was decreased in both ΔacsR and AacsS mutants. Similar to a AacsA mutant, strains carrying deletions either in acsR or acsS grew slowly than wild type in a minimal medium with acetate as a sole carbon source. Growth defect of the ΔacsR strain in acetate-minimal medium was restored by complementation. To investigate if AcsR directly regulates acsA expression, in vitro-gel shift assays were performed using the recombinant AcsR and the regulatory region of the acsA gene, showing that AcsR specifically bound the upstream region of the acsA ORF. Conclusion: This study indicates that the VarS/VarA system plays a role in V. vulnificus metabolism via regulating AcsR, which in turn controls acetate metabolism by activating the transcription of the acetyl-CoA synthetase gene. [ABSTRACT FROM AUTHOR]