Acetate Uptake Research Articles

Short-term supplementation of celecoxib-shifted butyrate production on a simulated model of the gut microbial ecosystem and ameliorated in vitro inflammation

Celecoxib has been effective in the prevention and treatment of chronic inflammatory disorders through inhibition of altered cyclooxygenase-2 (COX-2) pathways. Despite the benefits, continuous administration may increase risk of cardiovascular events. Understanding microbiome-drug-host interactions is fundamental for improving drug disposition and safety responses of colon-targeted formulations, but little information is available on the bidirectional interaction between individual microbiomes and celecoxib. Here, we conducted in vitro batch incubations of human faecal microbiota to obtain a mechanistic proof-of-concept of the short-term impact of celecoxib on activity and composition of colon bacterial communities. Celecoxib-exposed microbiota shifted metabolic activity and community composition, whereas total transcriptionally active bacterial population was not significantly changed. Butyrate production decreased by 50% in a donor-dependent manner, suggesting that celecoxib impacts in vitro fermentation. Microbiota-derived acetate has been associated with inhibition of cancer markers and our results suggest uptake of acetate for bacterial functions when celecoxib was supplied, which potentially favoured bacterial competition for acetyl-CoA. We further assessed whether colon microbiota modulates anti-inflammatory efficacy of celecoxib using a simplified inflammation model, and a novel in vitro simulation of the enterohepatic metabolism. Celecoxib was responsible for only 5% of the variance in bacterial community composition but celecoxib-exposed microbiota preserved barrier function and decreased concentrations of IL-8 and CXCL16 in a donor-dependent manner in our two models simulating gut inflammatory milieu. Our results suggest that celecoxib-microbiome-host interactions may not only elicit adaptations in community composition but also in microbiota functionality, and these may need to be considered for guaranteeing efficient COX-2 inhibition.

Goethite supplemented natural clay ceramic as an alternative proton exchange membrane and its application in microbial fuel cell

Microbial fuel cell (MFC) is a bioelectrochemical system, which treats wastewater with simultaneous recovery of bioelectricity. Costly proton exchange membrane, like Nafion-117, brings a serious bottleneck towards the sustainable scaled-up application of this technology. For an appropriate field-scale demonstration of MFC, the fabrication cost needs to be reduced drastically. This can be achieved by using low-cost proton exchange membrane (PEM) in MFCs, which would demonstrate equivalent performance as commercially available expensive PEMs. Such a novel PEM constituting 5% goethite and natural clay as a base material was synthesized, named as G-5, and it was used in MFC as a separator. This G-5 membrane was estimated to be five folds cheaper than the commercially available Nafion-117 membrane, which is popularly used in MFC. Membrane properties, like water and acetate uptake, and proton conductivity of the G-5 membrane, were evaluated and compared with Nafion-117. A power density obtained from the MFC using G-5 as PEM (112.81 ± 8.74 mW/m2) was slightly higher than the MFC with Nafion-117 as PEM (106.95 ± 5.52 mW/m2). Chemical oxygen demand removal and Coulombic efficiency for MFC with G-5 membrane were found to be 22% and 8.6% higher, respectively, in comparison with that of the MFC with Nafion-117 as PEM. Thus, low-cost G-5 membrane demonstrated the potential to be used for scaling-up of MFCs to reduce fabrication cost for successful field-scale implementation of MFCs.

Biological phosphorus removal in seawater-adapted aerobic granular sludge

Seawater can be introduced or intrude in sewer systems and can thereby negatively influence biological wastewater treatment processes. Here we studied the impact of artificial seawater on the enhanced biological phosphate removal (EBPR) process performance by aerobic granular sludge (AGS) with synthetic wastewater. Process performance, granule stability and characteristics as well as microbial community of a seawater-adapted AGS system were observed. In seawater conditions strong and stable granules formed with an SVI5 of 20 mL/g and a lower abrasion coefficient than freshwater-adapted granules. Complete anaerobic uptake of acetate, anaerobic phosphate release of 59.5 ± 4.0 mg/L PO43--P (0.35 mg P/mg HAc), and an aerobic P-uptake rate of 3.1 ± 0.2 mg P/g VSS/h were achieved. The dominant phosphate accumulating organisms (PAO) were the same as for freshwater-based aerobic granular sludge systems with a very high enrichment of Ca. Accumulibacter phosphatis clade I, and complete absence of glycogen accumulating organisms. The effect of osmotic downshocks was tested by replacing influent seawater-based medium by demineralized water-based medium. A temporary decrease of the salinity in the reactor led to a decreased phosphate removal activity, while it also induced a rapid release of COD by the sludge, up to 45.5 ± 1.7 mg COD/g VSS. This is most likely attributed to the release of osmolytes by the cells. Recovery of activity was immediately after restoring the seawater feeding. This work shows that functioning of aerobic granular sludge in seawater conditions is as stable as in freshwater conditions, while past research has shown a negative effect on operation of AGS processes with NaCl-based wastewater at the same salinity as seawater.

Metabolic model of nitrite reduction to nitrous oxide coupled to alternating consumption and storage of glycogen and polyhydroxyalkanoate

In the coupled aerobic−anoxic nitrous decomposition operation (CANDO), nitrogen removal occurs in two bioreactors: in the first, ammonia is oxidized to nitrite; in the second, nitrite is reduced to N2O for use as a biogas oxidant. We describe here a N2O-producing Defluviicoccus-dominated microbial community that alternately accumulates glycogen and polyhydroxyalkanoate (PHA). 13C-labelled acetate experiments confirmed the role of glycogen in PHA synthesis, and a metabolic model was developed to simulate this process. Batch experiments enabled estimation of model parameters. Key anaerobic reactions are uptake of acetate and fermentation of glycogen to poly(3-hydroxybutyrate-co-3-hydrovalerate). Key anoxic transformations are growth with PHA oxidation coupled to nitrite reduction to N2O, and conversion of PHA back to glycogen. With this simple model, we predicted and confirmed improved bioreactor performance at higher nitrogen loading rates. The model suggests strategies to either maximize N2O production or limit N2O emissions in systems designed for biological nutrient removal.

The identification and pharmacological evaluation of potent, selective and orally available ACC1 inhibitor.

In our effort to explore the potential of ACC1-selective inhibitor as in vivo probe molecule, a series of 1,3-benzoxazole derivatives was synthesized. Previously, we reported a series of novel bicyclic and monocyclic ACC1-selective inhibitors. Among them, compound 1a exhibited highly potent cellular activity (acetate uptake IC50 = 0.76 nM) as well as promising in vivo PD efficacy. However, compound 1a caused severe body weight reduction in repeated dose administration in the mouse model. Since 1a showed potent inhibitory activity against mouse ACC1 as well as strong inhibition of mouse ACC2, we further examined a series of 1a analogues in order to reduce undesirable body weight change. The replacement of acetamide moiety with ureido moiety dramatically improved selectivity of mouse ACC1 against ACC2. In addition, analogue 1b displayed favorable bioavailability in mouse cassette dosing PK study, hence in vivo PD studies were also carried out. Oral administration of 1b significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of more than 30 mg/kg. Furthermore, compound 1b showed significant antitumor efficacy in 786-O xenograft mice at an oral dose of 30 mg/kg (T/C = 0.5%). Accordingly, our novel potent ACC1-selective inhibitor represents a set of useful orally-available research tools, as well as potential therapeutic agents particularly in terms of new cancer therapies.

Uptake and trophic transfer of nitrogen and carbon in a temperate forested headwater stream

In temperate headwater streams, riparian forests hinder the development of algae by reducing light availability and generate large inputs of detritus. Microbial assemblages associated with this detritus are expected to strongly influence in-stream elemental cycling. However, most research has focused on quantifying nitrogen (N) cycling while we know little about the coupling of N and carbon (C) cycling. We conducted a simultaneous whole-reach tracer addition of 15N-ammonium and 13C-acetate in a forested headwater stream to examine the importance of different primary uptake compartments (e.g. epilithic biofilm, leaves, small wood) on N and C uptake, storage, and transfer to consumers. We predicted high whole-reach uptake of N and C from the water column to satisfy requirements of microbial decomposers. We also predicted a dominant role of the abundant detrital compartments, especially leaf litter, in the uptake, storage and trophic transfer of these labile forms of N and C. Our results show efficient immobilization of both ammonium and acetate along the study reach. Leaf litter showed the highest percentage of contribution among all compartments to whole-reach ammonium and acetate uptake. We also found evidence of rapid transfer of N and C to higher trophic levels, thereby extending the retention time of these elements within the ecosystem. Overall, our study provides relevant insights into the influence of detritus on N and C cycling in headwater streams.

Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica

Acetyl-CoA is the central metabolic node connecting glycolysis, Krebs cycle and fatty acids synthase. Plant-derived polyketides, are assembled from acetyl-CoA and malonyl-CoA, represent a large family of biological compounds with diversified bioactivity. Harnessing microbial bioconversion is considered as a feasible approach to large-scale production of polyketides from renewable feedstocks. Most of the current polyketide production platform relied on the lengthy glycolytic steps to provide acetyl-CoA, which inherently suffers from complex regulation with metabolically-costly cofactor/ATP requirements. Using the simplest polyketide triacetic acid lactone (TAL) as a testbed molecule, we demonstrate that acetate uptake pathway in oleaginous yeast (Yarrowia lipolytica) could function as an acetyl-CoA shortcut to achieve metabolic optimality in producing polyketides. We identified the metabolic bottlenecks to rewire acetate utilization for efficient TAL production in Y. lipolytica, including generation of the driving force for acetyl-CoA, malonyl-CoA and NADPH. The engineered strain, with the overexpression of endogenous acetyl-CoA carboxylase (ACC1), malic enzyme (MAE1) and a bacteria-derived cytosolic pyruvate dehydrogenase (PDH), affords robust TAL production with titer up to 4.76 g/L from industrial glacier acetic acid in shake flasks, representing 8.5-times improvement over the parental strain. The acetate-to-TAL conversion ratio (0.149 g/g) reaches 31.9% of the theoretical maximum yield. The carbon flux through this acetyl-CoA metabolic shortcut exceeds the carbon flux afforded by the native glycolytic pathways. Potentially, acetic acid could be manufactured in large-quantity at low-cost from Syngas fermentation or heterogenous catalysis (methanol carbonylation). This alternative carbon sources present a metabolic advantage over glucose to unleash intrinsic pathway limitations and achieve high carbon conversion efficiency and cost-efficiency. This work also highlights that low-cost acetic acid could be sustainably upgraded to high-value polyketides by oleaginous yeast species in an eco-friendly and cost-efficient manner.

Nutritional requirements for Lactobacillus vini growth in sugarcane derivative substrate of ethanol fermentation.

Lactobacillus vini is a bacterial contaminant found in industrial environments of winemaking and fuel-ethanol fermentation. However, there has been no standard analysis of its physiology that can pinpoint its adaptive traits to these kinds of environments. In view of this lack of information, the aim of this study is to determine the nutritional factors that lead to the growth of L. vini in the industrial plants of fuel-ethanol. First of all, the limited growth of this bacterium was studied in the industrial substrate, which was improved by nutritional supplementation with amino acids, and its homofermentative status was confirmed. Metabolite analysis showed that citrate is a growth factor of paramount importance for this bacterium in industrial processes through pyruvate metabolization, and increases ATP production and biomass formation. Furthermore,e acetate uptake, either from the medium or generated from citrate metabolism, was assimilated for biomass production. Hence, a metabolic model was designed to describe the role of citrate and acetate in the growth of L. vini that could be tested on other lactobacilli.

Contrasting effects of isocitrate dehydrogenase deletion on fluxes through enzymes of central metabolism in Escherichia coli.

Flux analysis is central to understanding cellular metabolism and successful manipulation of metabolic fluxes in microbial cell-factories. Isocitrate dehydrogenase (ICDH) deletion conferred contrasting effects on fluxes through substrate-level phosphorylation (SLP) reactions. While significantly increasing flux through pyruvate kinase, it diminishes flux through succinyl CoA synthetase and upregulates phosphotransacetylase (PTA) and acetate kinase (AK). In addition to acetate, the ICDH-less strain excretes pyruvate, citrate and isocitrate. While efflux to acetate excretion by the Escherichia coli parental strain and its ICDH-less derivative is a reflection of high throughput of glycolytic intermediates, excretion of pyruvate is a reflection of high throughput via pyruvate kinase. On the other hand, citrate and isocitrate excretion is a reflection of truncating the Krebs cycle at the level of ICDH. Furthermore, another striking finding is the inability of the ICDH-less cultures to utilize acetate as a source of carbon despite the availability of an adequate supply of extracellular glutamate (for biosynthesis) and elevated levels of AK and PTA (for acetate uptake). This striking observation is now explicable in the light of the newly proposed hypothesis that the expression of the ace operon enzymes is controlled in response to a minimum threshold signal (ATP), which could not be achieved in the ICDH-less strain.

Plantwide modelling – anaerobic digestion of waste sludge from parent nutrient (N and P) removal systems

Wastewater treatment plant (WWTP) mathematical models are based on the behavioural patterns of microorganisms involved in the treatment process. These microorganisms are assumed incapable of thinking or planning but simply act according to the capabilities afforded to them by their surrounding conditions – hence different microorganisms pre-dominate different WWTP zones according to how well the conditions suit them. When waste activated sludge (WAS) from biological nutrient removal (BNR) activated sludge (AS) systems, containing phosphorus-accumulating organisms (PAOs), is fed to an anaerobic digester, there is a release of high quantities of metals, phosphorus (P) and nitrogen (N). The manner in which we model the release of these metals and nutrients significantly affects the accuracy of predicted anaerobic digestion (AD) outcomes. Previous studies of PAOs show that in the anaerobic zone of the AS system, they can form energy-rich poly3-hydroxybutyrate (PHB) at the expense of their aerobically generated polyphosphate (PP). Thus, it is expected that the PAOs containing PP sent into an anaerobic digester with volatile fatty acids (VFAs) present, would utilize their PP reserves as they would in the anaerobic zone of an AS process ending up with formation and storage of some PHB. Ultimately, all the stored products of the PAO get released, since there is no alternating aerobic environment to cater for their growth. Since it has been established that the PP release in the AD occurs much faster than the PAO biomass hydrolysis rate, it is modelled as a separate process. Steps are presented in the development of this PP release mass-balanced stoichiometries that occur with AD of PAOs. By comparing outcomes from these proposed stoichiometries against measured experimental data, it is noticed that better predictions are obtained with acetate uptake for PHB formation than when modelling the AD PP release to occur with PAO death and hydrolysis.

Ferredoxin5 Deletion Affects Metabolism of Algae during the Different Phases of Sulfur Deprivation.

Ferredoxin5 (FDX5), a minor ferredoxin protein in the alga Chlamydomonas (Chlamydomonas reinhardtii), helps maintain thylakoid membrane integrity in the dark. Sulfur (S) deprivation has been used to achieve prolonged hydrogen production in green algae. Here, we propose that FDX5 is involved in algal responses to S-deprivation as well as to the dark. Specifically, we tested the role of FDX5 in both the initial aerobic and subsequent anaerobic phases of S-deprivation. Under S-deprived conditions, absence of FDX5 causes a distinct delay in achieving anoxia by affecting photosynthetic O2 evolution, accompanied by reduced acetate uptake, lower starch accumulation, and delayed/lower fermentative metabolite production, including photohydrogen. We attribute these differences to transcriptional and/or posttranslational regulation of acetyl-CoA synthetase and ADP-Glc pyrophosphorylase, and increased stability of the PSII D1 protein. Interestingly, increased levels of FDX2 and FDX1 were observed in the mutant under oxic, S-replete conditions, strengthening our previously proposed hypothesis that other ferredoxins compensate in response to a lack of FDX5. Taken together, the results of our omics and pull-down experiments confirmed biochemical and physiological results, suggesting that FDX5 may have other effects on Chlamydomonas metabolism through its interaction with multiple redox partners.

“Candidatus Accumulibacter delftensis”: A clade IC novel polyphosphate-accumulating organism without denitrifying activity on nitrate

Populations of “Candidatus Accumulibacter”, a known polyphosphate-accumulating organism, within clade IC have been proposed to perform anoxic P-uptake activity in enhanced biological phosphorus removal (EBPR) systems using nitrate as electron acceptor. However, no consensus has been reached on the ability of “Ca. Accumulibacter” members of clade IC to reduce nitrate to nitrite. Discrepancies might relate to the diverse operational conditions which could trigger the expression of the Nap and/or Nar enzyme and/or to the accuracy in clade classification. This study aimed to assess whether and how certain operational conditions could lead to the enrichment and enhance the denitrification capacity of “Ca. Accumulibacter” within clade IC. To study the potential induction of the denitrifying enzyme, an EBPR culture was enriched under anaerobic–anoxic–oxic (A2O) conditions that, based on fluorescence in situ hybridization and ppk gene sequencing, was composed of around 97% (on a biovolume basis) of affiliates of “Ca. Accumulibacter” clade IC. The influence of the medium composition, sludge retention time (SRT), polyphosphate content of the biomass (poly-P), nitrate dosing approach, and minimal aerobic SRT on potential nitrate reduction were studied. Despite the different studied conditions applied, only a negligible anoxic P-uptake rate was observed, equivalent to maximum 13% of the aerobic P-uptake rate. An increase in the anoxic SRT at the expenses of the aerobic SRT resulted in deterioration of P-removal with limited aerobic P-uptake and insufficient acetate uptake in the anaerobic phase. A near-complete genome (completeness = 100%, contamination = 0.187%) was extracted from the metagenome of the EBPR biomass for the here-proposed “Ca. Accumulibacter delftensis” clade IC. According to full-genome-based phylogenetic analysis, this lineage was distant from the canonical “Ca. Accumulibacter phosphatis”, with closest neighbor “Ca. Accumulibacter sp. UW-LDO-IC” within clade IC. This was cross-validated with taxonomic classification of the ppk1 gene sequences. The genome-centric metagenomic analysis highlighted the presence of genes for assimilatory nitrate reductase (nas) and periplasmic nitrate reductase (nap) but no gene for respiratory nitrate reductases (nar). This suggests that “Ca. Accumulibacter delftensis” clade IC was not capable to generate the required energy (ATP) from nitrate under strict anaerobic-anoxic conditions to support an anoxic EBPR metabolism. Definitely, this study stresses the incongruence in denitrification abilities of “Ca. Accumulibacter” clades and reflects the true intra-clade diversity, which requires a thorough investigation within this lineage.

NanoSIMS reveals unusual enrichment of acetate and propionate by an anammox consortium dominated by Jettenia asiatica

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into N2 in a chemolithoautotrophic way, meaning that they utilize CO2/HCO3 solely as their carbon sources. Such autotrophic behavior limits their competitiveness with heterotrophic microorganisms in both natural environments and engineered systems. Recently, environmental metagenomic results have indicated the capability of anammox bacteria to metabolize short-chain fatty acids, further confirmed by limited experimental evidence based on highly enriched cultures. However, clear evidence is difficult to get because of the limits of traditional methodologies which rely on the availability of a pure anammox culture. In this study, we identified and quantified the uptake of acetate and propionate, on a single-cell level, by an anammox consortium that was dominated by Candidatus Jettenia asiatica (relative abundance of 96%). The consortium, growing in granular form with an average relative abundance of anammox bacteria of 96.0%, was firstly incubated in a13C-labelled acetate or propionate medium; then microtome sections were scanned by a nanometer-scale secondary ion mass spectrometer (NanoSIMS). The NanoSIMS scannings revealed that the consortium enriched acetate and propionate at a >10 times higher efficiency than bicarbonate incorporation. Our results also suggest that acetate or propionate was likely not assimilated by J. asiatica directly, but firstly oxidized to CO2, which then served as carbon sources for the follow-up autotrophy in J. asiatica cells. Furthermore, more [15N]ammonium was enriched by the propionate-fed consortium than the acetate-fed consortium despite that exactly the same amount of 13C atoms were supplied. Our study strongly indicates an alternative lifestyle, namely organotrophy, in addition to chemolithoautotrophy of anammox bacteria, making it more versatile than often expected. It suggests that the niche of anammox bacteria in both natural and engineered ecosystems can be much broader than usual assumed. Recognising this is important for their role in wastewater treatment and the global nitrogen turn-over rates.

Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate

BackgroundAcetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production.ResultsHere, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain.ConclusionsTyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5′ untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.

Significance of Acetate as a Microbial Carbon and Energy Source in the Water Column of Gulf of Mexico: Implications for Marine Carbon Cycling

AbstractAcetate 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.

Uncultured Gammaproteobacteria and Desulfobacteraceae Account for Major Acetate Assimilation in a Coastal Marine Sediment.

Acetate is a key intermediate in anaerobic mineralization of organic matter in marine sediments. Its turnover is central to carbon cycling, however, the relative contribution of different microbial populations to acetate assimilation in marine sediments is unknown. To quantify acetate assimilation by in situ abundant bacterial populations, we incubated coastal marine sediments with 14C-labeled acetate and flow-sorted cells that had been labeled and identified by fluorescence in situ hybridization. Subsequently, scintillography determined the amount of 14C-acetate assimilated by distinct populations. This approach fostered a high-throughput quantification of acetate assimilation by phylogenetically identified populations. Acetate uptake was highest in the oxic-suboxic surface layer for all sorted bacterial populations, including deltaproteobacterial sulfate-reducing bacteria (SRB), which accounted for up to 32% of total bacterial acetate assimilation. We show that the family Desulfobulbaceae also assimilates acetate in marine sediments, while the more abundant Desulfobacteraceae dominated acetate assimilation despite lower uptake rates. Unexpectedly, members of Gammaproteobacteria accounted for the highest relative acetate assimilation in all sediment layers with up to 31–62% of total bacterial acetate uptake. We also show that acetate is used to build up storage compounds such as polyalkanoates. Together, our findings demonstrate that not only the usual suspects SRB but a diverse bacterial community may substantially contribute to acetate assimilation in marine sediments. This study highlights the importance of quantitative approaches to reveal the roles of distinct microbial populations in acetate turnover.

Crystal structure of the bacterial acetate transporter SatP reveals that it forms a hexameric channel

Acetate is found ubiquitously in the natural environment and can be used as an exogenous carbon source by bacteria, fungi, and mammalian cells. A representative member of the acetate uptake transporter (AceTr) family named SatP (also yaaH) has been preliminarily identified as a succinate-acetate/proton symporter in Escherichia coli However, the molecular mechanism of acetate uptake by SatP still remains elusive. Here, we report the crystal structure of SatP from E. coli at 2.8 Å resolution, determined with a molecular replacement approach using a previously developed predicted model algorithm, which revealed a hexameric UreI-like channel structure. Structural analysis identified six transmembrane (TM) helices surrounding the central channel pore in each protomer and three conserved hydrophobic residues, FLY, located in the middle of the TM region for pore constriction. According to single-channel conductance recordings, performed with purified SatP reconstituted into lipid bilayer, three conserved polar residues in the TM1 facing to the periplasmic side are closely associated with acetate translocation activity. These analyses provide critical insights into the mechanism of acetate translocation in bacteria and a first glimpse of a structure of an AceTr family transporter.

The physiological effect of heavy metals and volatile fatty acids on Methanococcus maripaludis S2

BackgroundMethanogenic archaea are of importance to the global C-cycle and to biological methane (CH4) production through anaerobic digestion and pure culture. Here, the individual and combined effects of copper (Cu), zinc (Zn), acetate, and propionate on the metabolism of the autotrophic, hydrogenotrophic methanogen Methanococcus maripaludis S2 were investigated. Cu, Zn, acetate, and propionate may interfere directly and indirectly with the acetyl-CoA synthesis and biological CH4 production. Thus, these compounds can compromise or improve the performance of M. maripaludis, an organism which can be applied as biocatalyst in the carbon dioxide (CO2)-based biological CH4 production (CO2-BMP) process or of methanogenic organisms applied in anaerobic digestion.ResultsHere, we show that Cu concentration of 1.9 µmol L−1 reduced growth of M. maripaludis, whereas 4.4 and 6.3 µmol L−1 of Cu even further retarded biomass production. However, 1.0 mmol L−1 of Zn enhanced growth, but at Zn concentrations > 2.4 mmol L−1 no growth could be observed. When both, Cu and Zn, were supplemented to the medium, growth and CH4 production could even be observed at the highest tested concentration of Cu (6.3 µmol L−1). Hence, it seems that the addition of 1 mmol L−1 of Zn enhanced the ability of M. maripaludis to counteract the toxic effect of Cu. The physiological effect to rising concentrations of acetate (12.2, 60.9, 121.9 mmol L−1) and/or propionate (10.3, 52.0, 104.1 mmol L−1) was also investigated. When instead of acetate 10.3 mmol L−1 propionate was provided in the growth medium, M. maripaludis could grow without reduction of the specific growth rate (µ) or the specific CH4 productivity (qCH4). A combination of inorganic and/or organic compounds resulted in an increase of µ and qCH4 for Zn/Cu and Zn/acetate beyond the values that were observed if only the individual concentrations of Zn, Cu, acetate were used.ConclusionsOur study sheds light on the physiological effect of VFAs and heavy metals on M. maripaludis. Differently from µ and qCH4, MER was not influenced by the presence of these compounds. This indicated that each of these compounds directly interacted with the C-fixation machinery of M. maripaludis. Until now, the uptake of VFAs other than acetate was not considered to enhance growth and CH4 production of methanogens. The finding of propionate uptake by M. maripaludis is important for the interpretation of VFA cycling in anaerobic microenvironments. Due to the importance of methanogens in natural and artificial anaerobic environments, our results help to enhance the understanding the physiological and biotechnological importance with respect to anaerobic digestion, anaerobic wastewater treatment, and CO2-BMP. Finally, we propose a possible mechanism for acetate uptake into M. maripaludis supported by in silico analyses.

Microbial activity response to hydrogen injection in thermophilic anaerobic digesters revealed by genome-centric metatranscriptomics

BackgroundThe expansion of renewable energy produced by windmills and photovoltaic panels has generated a considerable electricity surplus, which can be utilized in water electrolysis systems for hydrogen production. The resulting hydrogen can then be funneled to anaerobic digesters for biogas upgrading (biomethanation) purposes (power-to-methane) or to produce high value-added compounds such as short-chain fatty acids (power-to-chemicals).Genome-centric metagenomics and metatranscriptomic analyses were performed to better understand the metabolic dynamics associated with H2 injection in two different configurations of anaerobic digesters treating acidic wastes, specifically cheese manufacturing byproducts. These approaches revealed the key-genes involved in methanation and carbon fixation pathways at species level.ResultsThe biogas upgrading process in the single-stage configuration increased the CH4 content by 7%. The dominant methanogenic species responsible for the upregulation of the hydrogenotrophic pathway in this reactor was Methanothermobacter wolfeii UC0008. In the two-stage configuration, H2 injection induced an upregulation of CO2 fixation pathways producing short-chain fatty acids, mainly acetate and butyrate. In this configuration, the abundant species Anaerobaculum hydrogeniformans UC0046 and Defluviitoga tunisiensis UC0050 primarily upregulated genes related to electron transport chains, suggesting putative syntrophisms with hydrogen scavenger microbes. Interestingly, Tepidanaerobacter acetatoxydans UC0018 did not act as an acetate-oxidizer in either reactor configurations, and instead regulated pathways involved in acetate production and uptake. A putative syntrophic association between Coprothermobacter proteolyticus UC0011 and M. wolfeii UC0008 was proposed in the two-stage reactor. In order to support the transcriptomic findings regarding the hydrogen utilization routes, an advanced bioconversion model was adapted for the simulation of the single- and two-stage reactor setups.ConclusionsThis is the first study investigating biogas reactor metatranscriptome dynamics following hydrogen injection for biomethanation and carbon fixation to short-chain fatty acids purposes. The same microbes showed different patterns of metabolic regulation in the two reactor configurations. It was observed an effect of the specialized acidogenic reactor on the overall microbial consortium composition and activity in the two-stage digester. There were also suggested the main species responsible for methanation, short-chain fatty acids production, and electron transport chain mechanisms, in both reactor configurations.

The acetate uptake transporter family motif “NPAPLGL(M/S)” is essential for substrate uptake

Organic acids are recognized as one of the most prevalent compounds in ecosystems, thus the transport and assimilation of these molecules represent an adaptive advantage for organisms. The AceTr family members are associated with the active transport of organic acids, namely acetate and succinate. The phylogenetic analysis shows this family is dispersed in the tree of life. However, in eukaryotes, it is almost limited to microbes, though reaching a prevalence close to 100% in fungi, with an essential role in spore development. Aiming at deepening the knowledge in this family, we studied the acetate permease AceP from Methanosarcina acetivorans, as the first functionally characterized archaeal member of this family. Furthermore, we demonstrate that the yeast Gpr1 from Yarrowia lipolytica is an acetate permease, whereas the Ady2 closest homologue in Saccharomyces cerevisiae, Fun34, has no role in acetate uptake. In this work, we describe the functional role of the AceTr conserved motif NPAPLGL(M/S). We further unveiled the role of the amino acid residues R122 and Q125 of SatP as essential for protein activity.