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It has recently been shown that in anaerobic microorganisms the tricarboxylic acid (TCA) cycle, including the seemingly irreversible citrate synthase reaction, can be reversed and used for autotrophic fixation of carbon1,2. This reversed oxidative TCA cycle requires ferredoxin-dependent 2-oxoglutarate synthase instead of the NAD-dependent dehydrogenase as well as extremely high levels of citrate synthase (more than 7% of the proteins in the cell). In this pathway, citrate synthase replaces ATP-citrate lyase of the reductive TCA cycle, which leads to the spending of one ATP-equivalent less per one turn of the cycle. Here we show, using the thermophilic sulfur-reducing deltaproteobacterium Hippea maritima, that this route is driven by high partial pressures of CO2. These high partial pressures are especially important for the removal of the product acetyl coenzyme A (acetyl-CoA) through reductive carboxylation to pyruvate, which is catalysed by pyruvate synthase. The reversed oxidative TCA cycle may have been functioning in autotrophic CO2 fixation in a primordial atmosphere that is assumed to have been rich in CO2. In the deltaproteobacterium Hippea maritima, the tricarboxylic acid (TCA) cycle can be reversed by high partial pressures of CO2 for the autotrophic fixation of carbon.

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. Both the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex catalyze this reaction. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygenin vitroand is required for optimal photomixotrophic growth under aerobic conditions while the PDH complex is inactivated under the same conditions. We found that cells rely on a general shift from utilizing NAD(H)-dependent to ferredoxin-dependent enzymes under these conditions.The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

Endozoicomonas, a core bacterial group in corals, may also be a coral symbiont. Endozoicomonas communities often decrease rapidly in corals under heat stress. However, how the bacteria respond to changes in temperature and coral host during heat stress is unknown. Here, we employed the cultivable, dominant species E. montiporae as a working organism to explore how Endozoicomonas responds to heat stress. We designed two experiments to clarify the extent to which E. montiporae is influenced by temperature and coral host. We detected differentially expressed protein (DEP) profiles in this bacterium at 31°C and 33°C compared to 25°C by tandem mass tags-based quantitative proteome analysis. Fifty DEPs, including many heat shock proteins, were detected when the temperature changed. The expression of antioxidant defense proteins and key pyruvate synthase proteins decreased, suggesting that E. montiporae were in a physiology of stress at 33°C. Furthermore, some proteins were differentially expressed because of the heat-stress-treated coral lysate specifically, suggesting that not only heat but also heat-induced host factors can affect the protein expression of the bacterium. This study provides an in-depth analysis of how the molecular mechanisms of Endozoicomonas are affected by heat stress and coral host.

Pyruvate synthase (pyruvate:ferredoxin oxidoreductase, PFOR) catalyzes the interconversion of acetyl-CoA and pyruvate, but the reductive carboxylation activities of heterotetrameric PFORs remain largely unknown. In this study, we cloned, expressed, and purified selected six heterotetrameric PFORs from hyperthermophilic archaea. The reductive carboxylation activities of these heterotetrameric PFORs were measured at 70 °C and the ratio of reductive carboxylation activity to oxidative decarboxylation activity (red/ox ratio) were calculated. Four out of six showed reductive decarboxylation activities. Among them, the PFORpfm from Pyrolobus fumarii showed the highest reductive carboxylation activities and the highest red/ox ratio, which were 54.32 mU/mg and 0.51, respectively. The divergence of the reductive carboxylation activities and the red/ox ratios of heterotetrameric PFORs in hyperthermophilic archaea indicate the diversity of the functions of PFOR over long-term evolution. This can help us better understand the autotrophic CO2 fixation process in thermal vent, or in other CO2-rich high temperature habitat.

11 páginas, 7 figuras, The biorecovery of europium (Eu) from primary (mineral deposits) and secondary (mining wastes) resources is of interest due to its remarkable luminescence properties, important for modern technological applications. In this study, we explored the tolerance levels, reduction and intracellular bioaccumulation of Eu by a site-specific bacterium, Clostridium sp. 2611 isolated from Phalaborwa carbonatite complex. Clostridium sp. 2611 was able to grow in minimal medium containing 0.5 mM Eu3+. SEM-EDX analysis confirmed an association between Eu precipitates and the bacterium, while TEM-EDX analysis indicated intracellular accumulation of Eu. According to the HR-XPS analysis, the bacterium was able to reduce Eu3+ to Eu2+ under growth and non-growth conditions. Preliminary protein characterization seems to indicate that a cytoplasmic pyruvate oxidoreductase is responsible for Eu bioreduction. These findings suggest the bioreduction of Eu3+ by Clostridium sp. as a resistance mechanism, can be exploited for the biorecovery of this metal., This research was conducted with financial support from the Technology and Innovation Agency. The financial assistance of the National Research Foundation of South Africa (88833 and 106460) towards this research is hereby acknowledged.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in marine and terrestrial habitats, playing a major role in the global nitrogen cycle. However, their evolutionary history remains unexplored, which limits our understanding of their adaptation mechanisms. Here, our comprehensive phylogenomic tree of Thaumarchaeota supports three sequential events: origin of AOA from terrestrial non-AOA ancestors, colonization of the shallow ocean, and expansion to the deep ocean. Careful molecular dating suggests that these events coincided with the Great Oxygenation Event around 2300 million years ago (Mya), and oxygenation of the shallow and deep ocean around 800 and 635–560 Mya, respectively. The first transition was likely enabled by the gain of an aerobic pathway for energy production by ammonia oxidation and biosynthetic pathways for cobalamin and biotin that act as cofactors in aerobic metabolism. The first transition was also accompanied by the loss of dissimilatory nitrate and sulfate reduction, loss of oxygen-sensitive pyruvate oxidoreductase, which reduces pyruvate to acetyl-CoA, and loss of the Wood–Ljungdahl pathway for anaerobic carbon fixation. The second transition involved gain of a K(+) transporter and of the biosynthetic pathway for ectoine, which may function as an osmoprotectant. The third transition was accompanied by the loss of the uvr system for repairing ultraviolet light-induced DNA lesions. We conclude that oxygen availability drove the terrestrial origin of AOA and their expansion to the photic and dark oceans, and that the stressors encountered during these events were partially overcome by gene acquisitions from Euryarchaeota and Bacteria, among other sources.

The thermophilic anaerobes Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production. T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70 g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25 g/L. We hypothesize that one or more of the enzymes in the ethanol production pathway in C. thermocellum is not adequate for ethanol production at high titer. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol. In T. saccharolyticum, we replaced all of the genes encoding proteins in this pathway with their homologs from C. thermocellum and examined what combination of gene replacements restricted ethanol titer. We found that a pathway consisting of Ct_nfnAB, Ct_fd, Ct_adhE and Ts_pforA was sufficient to support ethanol titer greater than 50 g/L, however replacement of Ts_pforA by Ct_pfor1 dramatically decreased the maximum ethanol titer to 14 g/L. We then demonstrated that the reason for reduced ethanol production is that the Ct_pfor1 is inhibited by accumulation of ethanol and NADH, while Ts_pforA is not.

Campylobacter jejuni is a microaerophilic zoonotic pathogen with an atypical respiratory Complex I that oxidizes a flavodoxin (FldA) instead of NADH. FldA is essential for viability and is reduced via pyruvate and 2-oxoglutarate oxidoreductases (POR/OOR). Here, we show that FldA can also be reduced by FqrB (Cj0559), an NADPH:FldA reductase. An fqrB deletion mutant was viable but displayed a significant growth defect. FqrB is related to flavoprotein reductases from Gram-positive bacteria that can reduce NrdI, a specialized flavodoxin that is needed for tyrosyl radical formation in NrdF, the beta subunit of class 1b-type (Mn) ribonucleotide reductase (RNR). However, C. jejuni possesses a single class Ia-type (Fe) RNR (NrdAB) that would be expected to be ferredoxin dependent. We show that CjFldA is an unusually high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild-type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in an NADPH, FqrB, and FldA dependent manner, as evidenced by both optical and electron paramagnetic resonance (EPR) spectroscopy. Thus, FldA activates RNR in C. jejuni, partly explaining its essentiality.

Trichomoniasis, is the most prevalent non-viral sexually transmitted disease worldwide. Although metronidazole (MDZ) is the recommended treatment, several strains of the parasite are resistant to MDZ, and new treatments are required. Curcumin (CUR) is a polyphenol with anti-inflammatory, antioxidant and antiparasitic properties. In this study, we evaluated the effects of CUR on two biochemical targets: on proteolytic activity and hydrogenosomal metabolism in Trichomonas vaginalis. We also investigated the role of CUR on pro-inflammatory responses induced in RAW 264.7 phagocytic cells by parasite proteinases on pro-inflammatory mediators such as the nitric oxide (NO), tumor necrosis factor &alpha, (TNF&alpha, ), interleukin-1beta (IL-1&beta, ), chaperone heat shock protein 70 (Hsp70) and glucocorticoid receptor (mGR). CUR inhibited the growth of T. vaginalis trophozoites, with an IC50 value between 117 &plusmn, 7 &mu, M and 173 &plusmn, 15 &mu, M, depending on the culture phase. CUR increased pyruvate:ferredoxin oxidoreductase (PfoD), hydrogenosomal enzyme expression and inhibited the proteolytic activity of parasite proteinases. CUR also inhibited NO production and decreased the expression of pro-inflammatory mediators in macrophages. The findings demonstrate the potential usefulness of CUR as an antiparasitic and anti-inflammatory treatment for trichomoniasis. It could be used to control the disease and mitigate the associated immunopathogenic effects.

Bacteroides thetaiotaomicron was examined to determine whether its obligate anaerobiosis is imposed by endogenous reactive oxygen species or by molecular oxygen itself. Previous analyses established that aerated B. thetaiotaomicron loses some enzyme activities due to a high rate of endogenous superoxide formation. However, the present study establishes that another key step in central metabolism is poisoned by molecular oxygen itself. Pyruvate dissimilation was shown to depend upon two enzymes, pyruvate:formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), that lose activity upon aeration. PFL is a glycyl-radical enzyme whose vulnerability to oxygen is already understood. The rate of PFOR damage was unaffected by the level of superoxide or peroxide, showing that molecular oxygen itself is the culprit. The cell cannot repair PFOR, which amplifies the impact of damage. The rates of PFOR and fumarase inactivation are similar, suggesting that superoxide dismutase is calibrated so the oxygen- and superoxide-sensitive enzymes are equally sensitive to aeration. The physiological purpose of PFL and PFOR is to degrade pyruvate without disrupting the redox balance, and they do so using catalytic mechanisms that are intrinsically vulnerable to oxygen. In this way, the anaerobic excellence and oxygen sensitivity of B. thetaiotaomicron are two sides of the same coin.

Background Bio-based succinic acid holds promise as a sustainable platform chemical. Its production through microbial fermentation concurs with the fixation of CO2, through the carboxylation of phosphoenolpyruvate. Here, we studied the effect of the available CO2 on the metabolism of Pseudoclostridium thermosuccinogenes, the only known succinate producing thermophile. Batch cultivations in bioreactors sparged with 1 and 20% CO2 were conducted that allowed us to carefully study the effect of CO2 limitation. Results Formate yield was greatly reduced at low CO2 concentrations, signifying a switch from pyruvate formate lyase (PFL) to pyruvate:ferredoxin oxidoreductase (PFOR) for acetyl-CoA formation. The corresponding increase in endogenous CO2 production (by PFOR) enabled succinic acid production to be largely maintained as its yield was reduced by only 26%, thus also maintaining the concomitant NADH re-oxidation, essential for regenerating NAD+ for glycolysis. Acetate yield was slightly reduced as well, while that of lactate was slightly increased. CO2 limitation also prompted the formation of significant amounts of ethanol, which is only marginally produced during CO2 excess. Altogether, the changes in fermentation product yields result in increased ferredoxin and NAD+ reduction, and increased NADPH oxidation during CO2 limitation, which must be linked to reshuffled (trans) hydrogenation mechanisms of those cofactors, in order to keep them balanced. RNA sequencing, to investigate transcriptional effects of CO2 limitation, yielded only ambiguous results regarding the known (trans) hydrogenation mechanisms. Conclusions The results hinted at a decreased NAD+/NADH ratio, which could ultimately be responsible for the stress observed during CO2 limitation. Clear overexpression of an alcohol dehydrogenase (adhE) was observed, which may explain the increased ethanol production, while no changes were seen for PFL and PFOR expression that could explain the anticipated switch based on the fermentation results.

The online version of this article (https://doi.org/10.1038/s41396-019-0573-y) contains supplementary material, which is available to authorized users, The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20?mM sulfate or 20?mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2\% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains., We thank Johanna Gutleben and Maryam Chaib de Mares for sediment sampling, W. Irene C. Rijpstra for fatty acid analysis, and Andreas Marquardt (Proteomics Centre of the University of Konstanz) for proteomic analyses. We acknowledge the China Scholarship Council (CSC) for the support to PP and YL. The authors thank BE-BASIC funds (grants F07.001.05 and F08.004.01) from the Dutch Ministry of Economic Affairs, ERC grant (project 323009), the Gravitation grant (project 024.002.002) and the UNLOCK project (NRGWI.obrug.2018.005) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO), and National Natural Science Foundation of China (project No.51709100) for funding., info:eu-repo/semantics/publishedVersion

Background Blastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children. Methods Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity. Results The 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower. Conclusions Although the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs.

Mesaconate, a branched unsaturated dicarboxylic acid, has drawn great interest because of its versatile applications. In this work, we optimized the fermentation efficiency of Escherichia coli to produce mesaconate from glucose. We first drove the carbon flux to 2-ketoglutarate by overexpressing genes involved in TCA precursor pathway and anaplerotic pathways. Then, to increase the pool of phosphoenolpyruvate (PEP), an upstream precursor for 2-ketoglutarate, the phosphotransferase system (PTS) of E. coli was inactivated by deleting glucose PTS permease and the import of glucose was altered by overexpressing galactose/H+ symporter GalP. Further, production optimization was achieved by deleting a class I fumarase (FumA) to block the hydration of mesaconate. Finally, we overexpressed PEP synthase (PpsA) to increase the availability of phosphoenolpyruvate and accelerate the production of mesaconate. These genetic modifications led to mesaconate production with a titer of 23.1 g L−1 and a yield of 0.46 g g−1 glucose (64% of the theoretical maximum). This work demonstrates the possibility of engineering a highly efficient bacteria strain that converts glucose into mesaconate with promising titer, rate, and yield.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a microbial enzyme that uses thiamine pyrophosphate (TPP), three [4Fe-4S] clusters, and coenzyme A (CoA) in the reversible oxidation of pyruvate to generate acetyl-CoA and carbon dioxide. The two electrons that are generated as a result of pyruvate decarboxylation are used in the reduction of low potential ferredoxins, which provide reducing equivalents for central metabolism, including the Wood–Ljungdahl pathway. PFOR is a member of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily, which plays major roles in both microbial redox reactions and carbon dioxide fixation. Here, we present a set of crystallographic snapshots of the best-studied member of this superfamily, the PFOR from Moorella thermoacetica ( Mt PFOR). These snapshots include the native structure, those of lactyl-TPP and acetyl-TPP reaction intermediates, and the first of an OFOR with CoA bound. These structural data reveal the binding site of CoA as domain III, the function of which in OFORs was previously unknown, and establish sequence motifs for CoA binding in the OFOR superfamily. Mt PFOR structures further show that domain III undergoes a conformational change upon CoA binding that seals off the active site and positions the thiolate of CoA directly adjacent to the TPP cofactor. These structural findings provide a molecular basis for the experimental observation that CoA binding accelerates catalysis by 10 5 -fold.

In Europe, up to 90% of isolated Trichomonas vaginalis strains are naturally infected with Mycoplasma hominis, a facultative pathogen of the human genital tract. The consequences of this endosymbiosis are not yet well understood. The aim of the current study was to evaluate the impact of natural and artificial infections with M. hominis on the RNA expression levels of metronidazole susceptibility-associated genes of T. vaginalis. Three T. vaginalis strains (TVSS10-, TVSS25-, G3) without M. hominis, as well as the same strains naturally (TVSS10+, TVSS25+) and artificially (G3-MhSS25, TVSS25-MhSS25) infected with M. hominis, were investigated for their expression profiles of three genes associated with metronidazole resistance (ferredoxin, flavin reductase 1 and pyruvate:ferredoxin oxidoreductase). The minimal inhibitory concentrations (MICs) of metronidazole were evaluated for all combinations and the respective M. hominis-free T. vaginalis strains were used as controls. The sole presence of M. hominis led to a down-regulation of metronidazole susceptibility-associated genes in all T. vaginalis strains tested. Interestingly, the effect was more prominent in the artificial symbioses. Moreover, a twofold enhancement of metronidazole tolerability was observed in three infected T. vaginalis strains, compared to the respective strains without M. hominis. In conclusion, M. hominis had an impact on gene expression in all T. vaginalis strains and on metronidazole MIC in all but one strain tested.

Background: Clostridium pasteurianum as an emerging new microbial cell factory can produce both n-butanol (BuOH) and 1,3-propanediol (1,3-PDO), and the pattern of product formation changes significantly with the composition of the culture medium. Among others iron content in the medium was shown to strongly affect the products selectivity. However, the mechanism behind this metabolic regulation is still unclear. For a better understanding of such metabolic regulation and for process optimization, we carried out fermentation experiments under either iron excess or iron limitation conditions, and performed metabolic, stoichiometric and proteomic analyses.Results: 1,3-PDO is most effectively produced under iron limited condition (Fe−), whereas 1,3-PDO and BuOH were both produced under iron rich condition (Fe+). With increased iron availability the BuOH/1,3-PDO ratio increased significantly from 0.27 mol/mol (at Fe−) to 1.4 mol/mol (at Fe+). Additionally, hydrogen production was enhanced significantly under Fe+ condition. Proteomic analysis revealed differentiated expression of many proteins including several ones of the central carbon metabolic pathway. Among others, pyruvate: ferredoxin oxidoreductase, hydrogenases, and several electron transfer flavoproteins was found to be strongly up-regulated under Fe+ condition, pointing to their strong involvement in the regeneration of the oxidized form of ferredoxin, and consequently their influences on the product selectivity in C. pasteurianum. Of particular significance is the finding that H2 formation in C. pasteurianum is coupled to the ferredoxin-dependent butyryl-CoA dehydrogenase catalyzed reaction, which significantly affects the redox balance and thus the product selectivity.Conclusions: The metabolic, stoichiometric and proteomic results clearly show the key roles of hydrogenases and ferredoxins dependent reactions in determining the internal redox balance and hence product selectivity. Not only the NADH pool but also the regulation of the ferredoxin pool could explain such product variation under different iron conditions.

Pyruvate ferredoxin oxidoreductase from Citrobacter sp. S-77 (PFORS77) was purified in order to develop a method for acetyl-CoA production. Although the purified PFORS77 showed high O2-sensitivity, the activity could be remarkably stabilized in anaerobic conditions. PFORS77 was effectively immobilized on ceramic hydroxyapatite (PFORS77-HA) with an efficiency of more than 96%, however, after encapsulation of PFORS77-HA in alginate, the rate of catalytic acetyl-CoA production was highly reduced to 36% when compared to that of the free enzyme. However, the operational stability of the PFORS77-HA in alginate hydrogels was remarkable, retaining over 68% initial activity even after ten repeated cycles. The results suggested that the PFORS77-HA hydrogels have a high potential for biotechnological application.

Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei. The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYK(Hm) [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPS(Hm) [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pyk(Hm) gene and pps(Hm) was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYK(Hm) catalyzed the conversion from PEP to pyruvate and that PPS(Hm) catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials. IMPORTANCE Archaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea. However, this process is still uncharacterized in the haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.

Two variants of the enzyme family pyruvate:ferredoxin oxidoreductase (PFOR), derived from the anaerobic sulfate-reducing bacterium Desulfovibrio africanus and the extremophilic crenarchaeon Sulfolobus acidocaldarius, respectively, were evaluated for their capacity to fixate CO

Two variants of the enzyme family pyruvate:ferredoxin oxidoreductase (PFOR), derived from the anaerobic sulfate-reducing bacterium Desulfovibrio africanus and the extremophilic crenarchaeon Sulfolobus acidocaldarius, respectively, were evaluated for their capacity to fixate CO2 in vitro. PFOR reversibly catalyzes the conversion of acetyl-CoA and CO2 to pyruvate using ferredoxin as redox partner. The oxidative decarboxylation of pyruvate is thermodynamically strongly favored, and most previous studies only considered the oxidative direction of the enzyme. To assay the pyruvate synthase function of PFOR during reductive carboxylation of acetyl-CoA is more challenging and requires to maintain the reaction far from equilibrium. For this purpose, a biochemical assay was established where low-potential electrons were introduced by photochemical reduction of EDTA/deazaflavin and the generated pyruvate was trapped by chemical derivatization with semicarbazide. The product of CO2 fixation could be detected as pyruvate semicarbazone by HPLC-MS. In a combinatorial approach, both PFORs were tested with ferredoxins from different sources. The pyruvate semicarbazone product could be detected with low-potential ferredoxins of the green sulfur bacterium Chlorobium tepidum and of S. acidocaldarius whereas CO2 fixation was not supported by the native ferredoxin of D. africanus. Methylviologen as an artificial electron carrier also allowed CO2 fixation. For both enzymes, the results are the first demonstration of CO2 fixation in vitro. Both enzymes exhibited high stability in the presence of oxygen during purification and storage. In conclusion, the employed PFOR enzymes in combination with non-native ferredoxin cofactors might be promising candidates for further incorporation in biocatalytic CO2 conversion. ENZYMES: EC1.2.7.1. Pyruvate:Ferredoxin Oxidoreductase.

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Metronidazole is administered in an inactive form then activated to its cytotoxic form within the hydrogenosome of trichomonads. Two hydrogenosomal proteins, pyruvate ferredoxin oxidoreductase (PFOR) and ferredoxin, play a critical role in the reductive activation of metronidazole. The expression of these proteins and other hydrogenosomal proteins are likewise positively regulated by iron. In the present study, the effect of iron on minimal lethal concentration (MLC) of metronidazole on in vitro cultured Trichomonas vaginalis(T. vaginalis) isolates was investigated. Interestingly, Addition of Ferrous ammonium sulphate (FAS) to T. vaginalis culture led to decrease in the MLC of metronidazole. On using aerobic assay, MLC of metronidazole on untreated T. vaginalis of both isolates was 12.5 μg/ml that decreased to 0.38 μg/ml on FAS treated trichomonads. Also anaerobic assay revealed that MLC on untreated parasites was 3.12 μg/ml that decreased to 0.097 μg/ml and 0.19 μg/ml for isolate 1 and isolate 2 respectively after iron addition. It was concluded that, addition of iron to in vitro cultured T. vaginalis decreases metronidazole MLC that was detected by both aerobic and anaerobic assays.

Purpose. The gastrointestinal tract is home to thousands of commensal bacterial species. Therefore, competition for nutrients is paramount for successful bacterial pathogen invasion of intestinal ecosystems. The human pathogen Vibrio cholerae, the causative agent of the severe diarrhoeal disease, cholera, is able to colonize the small intestine, which is protected by mucus. However, it is unclear which metabolic pathways or nutrients V. cholerae utilizes during intestinal colonization and growth. Methodology. In this study, we investigated the effect of various metabolic key genes, including those involved in the gluconeogenesis pathway, on V. cholerae physiology and in vivo colonization. Results. We found that gluconeogenesis is important for infant mouse colonization. Growth assays showed that mutations in the key components of gluconeogenesis pathway, PpsA and PckA, lead to a growth defect in a minimal medium supplemented with mucin as a carbon source. Furthermore, the ppsA/pckA mutants colonized poorly in the adult mouse intestine, particularly when more gut commensal flora are present. Conclusion. Gluconeogenesis biosynthesis is important for the successful colonization of V. cholerae in a niche that is full of competing microbiota.

Mitochondria are not just the powerhouses of the cell; these 'end of function' organelles are crucial components of cellular physiology and influence many central metabolic and signaling pathways that support complex multicellular life. Not surprisingly, these organelles play vital roles in adaptations for extreme survival strategies including hibernation and freeze tolerance, both of which are united by requirements for a strong reduction and reprioritization of metabolic processes. To facilitate metabolic rate depression, adaptations of all aspects of mitochondrial function are required, including; energetics, physiology, abundance, gene regulation, and enzymatic controls. This review discusses these factors with a focus on the stress-specific nature of mitochondrial genes and transcriptional regulators, and processes including apoptosis and chaperone protein responses. We also analyze the regulation of glutamate dehydrogenase and pyruvate dehydrogenase, central mitochondrial enzymes involved in coordinating the shifts in metabolic fuel use associated with extreme survival strategies. Finally, an emphasis is given to the novel mitochondrial research areas of microRNAs, peptides, epigenetics, and gaseous mediators and their potential roles in facilitating hypometabolism. © 2018 IUBMB Life, 70(12):1260-1266, 2018.

The genus Mesotoga, the only described mesophilic Thermotogae lineage, is common in mesothermic anaerobic hydrocarbon-rich environments. Besides mesophily, Mesotoga displays lineage-specific phenotypes, such as no or little H

Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition., Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition. Here, we show that amixicile, a highly selective water-soluble derivative of NTZ diminishes Cryptosporidium infection severity in a malnourished mouse model despite a lack of direct anticryptosporidial activity. We suggest that amixicile, by tamping down anaerobes associated with intestinal inflammation, reverses weight loss and indirectly mitigates infection-associated pathology.

Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr + phenotype). Here, we provide evidence that suggests that the Pyr + mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase ( por ) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr + strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase ( pyc ) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr + strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri . IMPORTANCE The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens.

There is mounting evidence that metabolic reprogramming is critical for the survival of organisms exposed to changing and stressed environments. Using the soil microbe Pseudomonas fluorescens as a model system, we demonstrate that the metabolic networks aimed at the conversion of aspartate into pyruvate are enhanced in the presence of hydrogen peroxide (H2O2). The metabolites pyruvate, oxaloacetate and acetate were increased in the treated cultures as measured by HPLC. Enzymes such as aspartate transaminase and phosphoenolpyruvate carboxylase (PEPC) that mediate the conversion of aspartate to phosphoenolpyruvate (PEP) were up-regulated. This high-energy phosphate was readily converted into ATP, a process facilitated by the increased activity of pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthase (PEPS) as oxidative phosphorylation was severely compromised. The ensuing formation of pyruvate readily detoxified reactive oxygen species with the concomitant formation of acetate. This H2O2-induced metabolic reconfiguration not only helps generate the antioxidants necessary to thwart oxidative stress but also powers the formation of energy.

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3−complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.