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[Image: see text] Toxoplasma gondii causes a prevalent human infection for which only the acute stage has an FDA-approved therapy. To find inhibitors of both the acute stage parasites and the persistent cyst stage that causes a chronic infection, we repurposed a compound library containing known inhibitors of parasitic hexokinase, the first step in the glycolysis pathway, along with a larger collection of new structural derivatives. The focused screen of 22 compounds showed a 77% hit rate (>50% multistage inhibition) and revealed a series of aminobenzamide-linked picolinic acids with submicromolar potency against both T. gondii parasite forms. Picolinic acid 23, designed from an antiparasitic benzamidobenzoic acid class with challenging ADME properties, showed 60-fold-enhanced solubility, a moderate LogD(7.4), and a 30% improvement in microsomal stability. Furthermore, isotopically labeled glucose tracing revealed that picolinic acid 23 does not function by hexokinase inhibition. Thus, we report a new probe scaffold to interrogate dual-stage inhibition of T. gondii.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K1/2 value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Gut bacteria influence human physiology by chemically modifying host-synthesized primary bile acids. These modified bile acids, known as secondary bile acids, can act as signaling molecules that modulate host lipid, glucose, and energy metabolism and affect gut microbiota composition via selective antimicrobial properties. However, knowledge regarding the bile acid-transforming capabilities of individual gut microbes remains limited. To help address this knowledge gap, we screened 72 bacterial isolates, spanning seven major phyla commonly found in the human gut, for their ability to chemically modify unconjugated bile acids. We found that 43 isolates, representing 41 species, were capable of in vitro modification of one or more of the three most abundant unconjugated bile acids in humans: cholic acid, chenodeoxycholic acid, and deoxycholic acid. Of these, 32 species have not been previously described as bile acid transformers. The most prevalent bile acid transformations detected were oxidation of 3α-, 7α-, or 12α-hydroxyl groups on the steroid core, a reaction catalyzed by hydroxysteroid dehydrogenases. In addition, we found 7α-dehydroxylation activity to be distributed across various bacterial genera, and we observed several other complex bile acid transformations. Finally, our screen revealed widespread bacterial conjugation of primary and secondary bile acids to glycine, a process that was thought to only occur in the liver, and to 15 other amino acids, resulting in the discovery of 44 novel microbially conjugated bile acids. IMPORTANCE Our current knowledge regarding microbial bile acid transformations comes primarily from biochemical studies on a relatively small number of species or from bioinformatic predictions that rely on homology to known bile acid-transforming enzyme sequences. Therefore, much remains to be learned regarding the variety of bile acid transformations and their representation across gut microbial species. By carrying out a systematic investigation of bacterial species commonly found in the human intestinal tract, this study helps better define the gut bacteria that impact composition of the bile acid pool, which has implications in the context of metabolic disorders and cancers of the digestive tract. Our results greatly expand upon the list of bacterial species known to perform different types of bile acid transformations. This knowledge will be vital for assessing the causal connections between the microbiome, bile acid pool composition, and human health.

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA., eLife digest Living organisms need energy to stay alive; in cells, this energy is supplied in the form of a small molecule called adenosine triphosphate, or ATP, a nucleotide that stores energy in the bonds between its three phosphate groups. ATP is present in all living cells and is often referred to as the energy currency of the cell, because it can be easily stored and transported to where it is needed. However, it is unknown why cells rely so heavily on ATP when a highly similar nucleotide called guanosine triphosphate, or GTP, could also act as an energy currency. There are several examples of proteins that originally used GTP and have since evolved to use ATP, but it is not clear why this switch occurred. One suggestion is that ATP is the more readily available nucleotide in the cell. To test this hypothesis, Updegrove, Harke et al. studied a protein that helps bacteria transition into spores, which are hardier and can survive in extreme environments until conditions become favorable for bacteria to grow again. In modern bacteria, this protein uses ATP to provide energy, but it evolved from an ancestral protein that used GTP instead. First, Updegrove, Harke et al. engineered the protein so that it became more similar to the ancestral protein and used GTP instead of ATP. When this was done, the protein gained the ability to break down GTP and release energy from it, but it no longer performed its enzymatic function. This suggests that both the energy released and the source of that energy are important for a protein’s activity. Further analysis showed that the modern version of the protein has evolved to briefly hold on to ATP after releasing its energy, which did not happen with GTP in the modified protein. Updegrove, Harke et al. also discovered that the levels of GTP in a bacterial cell fall as it transforms into a spore, while ATP levels remain relatively high. This suggests that ATP may indeed have become the source of energy of choice because it was more available. These findings provide insights into how ATP became the energy currency in cells, and suggest that how ATP is bound by proteins can impact a protein’s activity. Additionally, these experiments could help inform the development of drugs targeting proteins that bind nucleotides: it may be essential to consider the entirety of the binding event, and not just the release of energy.

The assembly of microbial communities and functions emerge from a complex and dynamic web of interactions. A major challenge in microbiome engineering is identifying organism configurations with community-level behaviors that achieve a desired function. The number of possible subcommunities scales exponentially with the number of species in a system, creating a vast experimental design space that is challenging to even sparsely traverse. We develop a model-guided experimental design framework for microbial communities and apply this method to explore the functional landscape of the health-relevant metabolite butyrate using a 25-member synthetic human gut microbiome community. Based on limited experimental measurements, our model accurately forecasts community assembly and butyrate production at every possible level of complexity. Our results elucidate key ecological and molecular mechanisms driving butyrate production including inter-species interactions, pH and hydrogen sulfide. Our model-guided iterative approach provides a flexible framework for understanding and predicting community functions for a broad range of applications.

This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi., Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum. In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi → 2 pyruvate + 5 NTP + Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi → ADP + PPi. Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids. IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.

(p)ppGpp is a highly conserved bacterial alarmone which regulates many aspects of cellular physiology and metabolism. In Gram-positive bacteria such as B. subtilis, cellular (p)ppGpp level is determined by the bifunctional (p)ppGpp synthetase/hydrolase RelA and two small alarmone synthetases (SASs) YjbM (SasB) and YwaC (SasA). However, it is less clear whether these enzymes are also involved in regulation of alarmones outside of (p)ppGpp. Here we developed an improved LC-MS-based method to detect a broad spectrum of metabolites and alarmones from bacterial cultures with high efficiency. By characterizing the metabolomic signatures of SasA expressing B. subtilis, we identified strong accumulation of the (p)ppGpp analog pGpp, as well as accumulation of ppApp and AppppA. The induced accumulation of these alarmones is abolished in the catalytically dead sasA mutant, suggesting that it is a consequence of SasA synthetase activity. In addition, we also identified depletion of specific purine nucleotides and their precursors including IMP precursors FGAR, SAICAR and AICAR (ZMP), as well as GTP and GDP. Furthermore, we also revealed depletion of multiple pyrimidine precursors such as orotate and orotidine 5′-phosphate. Taken together, our work shows that induction of a single (p)ppGpp synthetase can cause concomitant accumulation and potential regulatory interplay of multiple alarmones.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+., Author summary The obligate intracellular parasite T. gondii is commonly found among human populations worldwide and poses severe health risks to fetuses and individuals with AIDS. While some treatments are available they are limited in scope. A possible target for new therapies is T. gondii’s incomplete metabolism, which makes it heavily reliant on its host. In this study, we generated a joint host/parasite metabolome to better understand host manipulation by the parasite and to discover unique aspects of T. gondii metabolism that could serve as the next generation of drug targets. Metabolomic analysis of T. gondii infection over time found broad alterations to host metabolism by the parasite in both energetic and biosynthetic pathways. We discovered a new T. gondii enzyme, sedoheptulose bisphosphatase, which redirects carbon from glycolysis into the pentose phosphate pathway. The wholesale remodeling of host metabolism for optimal parasite growth is also of interest, although the mechanisms behind this host manipulation must be further studied before therapeutic targets can be identified.

Background Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. Results Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. Conclusions Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism., Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology.

Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C(5) and C(6) sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H(2) concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how Toxoplasma manipulates host metabolism for its overall growth rate and non-essential metabolites. To address this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in metabolic enzyme levels. Toxoplasma infection increased activity in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, derive from the parasite, while changes in others, like the pentose phosphate pathway, were host and parasite driven. Further experiments led to the discovery of a Toxoplasma enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into ribose synthesis through a energetically driven dephosphorylation reaction. This second route for ribose synthesis resolves a conflict between the Toxoplasma tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. During periods of high energetic and ribose need, the competition for NADP+ could result in lethal redox imbalances. Sedoheptulose bisphosphatase represents a novel step in Toxoplasma central carbon metabolism that allows Toxoplasma to satisfy its ribose demand without using NADP+. Sedoheptulose bisphosphatase is not present in humans, highlighting its potential as a drug target.Author SummaryThe obligate intracellular parasite Toxoplasma is commonly found among human populations worldwide and poses severe health risks to fetuses and individuals with AIDS. While some treatments are available they are limited in scope. A possible target for new therapies is Toxoplasma’s limited metabolism, which makes it heavily reliant in its host. In this study, we generated a joint host/parasite metabolome to better understand host manipulation by the parasite and to discover unique aspects of Toxoplasma metabolism that could serve as the next generation of drug targets. Metabolomic analysis of Toxoplasma during an infection time course found broad activation of host metabolism by the parasite in both energetic and biosynthetic pathways. We discovered a new Toxoplasma enzyme, sedoheptulose bisphosphatase, which redirects carbon from glycolysis into ribose synthesis. Humans lack sedoheptulose bisphosphatase, making it a potential drug target. The wholesale remodeling of host metabolism for optimal parasite growth is also of interest, although the mechanisms behind this host manipulation must be further studied before therapeutic targets can be identified.

Glycolysis plays a central role in producing ATP and biomass. Its control principles, however, remain incompletely understood. Here, we develop a method that combines (2)H and (13)C tracers to determine glycolytic thermodynamics. Using this method, we show that, in conditions and organisms with relatively slow fluxes, multiple steps in glycolysis are near to equilibrium, reflecting spare enzyme capacity. In Escherichia coli, nitrogen or phosphorus upshift rapidly increases the thermodynamic driving force, deploying the spare enzyme capacity to increase flux. Similarly, respiration inhibition in mammalian cells rapidly increases both glycolytic flux and the thermodynamic driving force. The thermodynamic shift allows flux to increase with only small metabolite concentration changes. Finally, we find that the cellulose-degrading anaerobe Clostridium cellulolyticum exhibits slow, near-equilibrium glycolysis due to the use of pyrophosphate rather than ATP for fructose-bisphosphate production, resulting in enhanced per-glucose ATP yield. Thus, near-equilibrium steps of glycolysis promote both rapid flux adaptation and energy efficiency.

Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI Actinobacteria from three clades (acI-A, acI-B, acI-C). Members of this cosmopolitan lineage are proposed to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins (actR). This model has been difficult to experimentally validate because acI are not consistently culturable. In this study, using genomes from single cells and metagenomes, we provide a detailed biosynthetic route for many acI-A and -B members to synthesize retinal and its carotenoid precursors. Accordingly, these acI should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, in contrast to acI-C members and other bacteria that encode opsins but lack retinal-production machinery. Moreover, we show that all acI clades contain genes for a complex carotenoid pathway that starts with retinal precursors. Transcription analysis of acI in a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and that actR is among the most highly-transcribed of all acI genes. Furthermore, heterologous expression of retinal pathway genes shows that lycopene, retinal, and ActR can be made. Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed acI-holo-ActR and acidified solution during illumination. Our results prove that acI containing both ActR and retinal-production enzymes have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin.IMPORTANCEMicrobes play critical roles in determining the quality of freshwater ecosystems that are vital to human civilization. Because acI Actinobacteria are ubiquitous and abundant in freshwater lakes, clarifying their ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production, and energy cycling in freshwater habitats.

Background Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application. Results Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum. Conclusions We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work. Electronic supplementary material The online version of this article (10.1186/s13068-017-0961-3) contains supplementary material, which is available to authorized users.

The effect of dietary nitrate supplementation on rumen bacterial community composition was examined in beef steers fed either a nitrate-N diet or urea-N diet. An automated method of ribosomal intergenic spacer analysis was applied to solid and liquid fractions of ruminal contents to allow comparison of bacterial communities. Supplemental N source affected relative population size of four amplicon lengths (ALs) in the liquid fraction and three ALs in the solid fraction. Five ALs were more prevalent after adaptation to nitrate. Correspondence analysis indicated that feeding the steers the nitrate-N diet versus urea-N diet changed the bacterial community composition in the liquid but not in the solid fraction. This led to an investigation of the relative sizes of potential nitrate-reducing populations. Mannheimia succiniciproducens, Veillonella parvula, and Campylobacter fetus were obtained from nitrate enrichment culture and quantified by real-time PCR based on 16S rRNA sequence. Nitrate supplementation increased the percentage of C. fetus in the liquid and solid phases, and in solid phase, the percentage of M. succiniciproducens increased. No change in species prevalence was observed for V. parvula. However, even after adaptation to dietary nitrate, the relative population sizes for all three putative nitrate-reducing species were very low (

a b s t r a c t Mixed ruminal microbes were incubated for 24 h in vitro with mixed forage and concentrate rations under conditions that differed in starch level (200 or 300 g/kg dry matter (DM)), initial pH (6.3 or 6.7) and corn oil (0 or 10 g/kg DM), in order to determine effects on fermentation (fiber digestibility, volatile fatty acid (VFA) production) and relative population sizes (RPS) of various bacterial taxa (expressed as a percentage of 16S ribosomal RNA gene copies determined by quantitative polymerase chain reaction (qPCR)). Within the range of in vitro conditions tested, monensin-adapted inocula incubated in the presence of monensin did not differ in in vitro fiber digestibility relative to inocula from the same cows prior to adaptation and incubated in the absence of monensin. Although total VFA production did not differ among in vitro treatments, a shift from acetate toward propionate production was generally observed at the higher starch level, lower pH, and presence of corn oil. Surprisingly, monensin cultures displayed slightly decreased proportions of propionate and increased proportions of butyrate and valerate. After 24 h in vitro incubations containing 300 g starch/kg DM and in the absence of monensin, the RPS of 13 of the 16 taxa examined differed (P

The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1). This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol,5.0 g l(-1), were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.

Background Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. Here, we used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. Results A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. Conclusions Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1066) contains supplementary material, which is available to authorized users.

A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium containing ethanol as an electron donor and acetate and succinate (common products of rumen fermentation) as electron acceptors. The isolate displayed a narrow substrate range but wide temperature and pH ranges atypical of ruminal bacteria and a maximum specific growth rate near the typical liquid dilution rate of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread among bovine ruminal samples but was present at only very low levels (0.00002% to 0.0002% of bacterial 16S rRNA gene copy number). However, the species was present in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne silage (but not maize silage) that comprised much of the cows' diet. While C. kluyveri may account for several observations regarding ethanol utilization and volatile fatty acid production in the rumen, its population size and growth characteristics suggest that it is not a significant contributor to ruminal metabolism in typical dairy cattle, although it may be a significant contributor to silage fermentation. The ability of unadapted cultures to produce substantial levels (12.8 g L(-1)) of caproic (hexanoic) acid in vitro suggests that this strain may have potential for industrial production of caproic acid.

A microarray study of chemostat growth on insoluble cellulose or soluble cellobiose has provided substantial new information on Clostridium thermocellum gene expression. This is the first comprehensive examination of gene expression in C. thermocellum under defined growth conditions. Expression was detected from 2,846 of 3,189 genes, and regression analysis revealed 348 genes whose changes in expression patterns were growth rate and/or substrate dependent. Successfully modeled genes included those for scaffoldin and cellulosomal enzymes, intracellular metabolic enzymes, transcriptional regulators, sigma factors, signal transducers, transporters, and hypothetical proteins. Unique genes encoding glycolytic pathway and ethanol fermentation enzymes expressed at high levels simultaneously with previously established maximal ethanol production were also identified. Ranking of normalized expression intensities revealed significant changes in transcriptional levels of these genes. The pattern of expression of transcriptional regulators, sigma factors, and signal transducers indicates that response to growth rate is the dominant global mechanism used for control of gene expression in C. thermocellum.

Real-time polymerase chain reaction (PCR) was used to quantify 16 procaryotic taxa in the rumina of two lactating dairy cows following supply and subsequent withdrawal of the feed additive monensin (13.9 mg/kg of diet dry matter) in a high-starch, silage-based ration. PCR was conducted on DNA from rumen samples collected 6 h post feeding on two successive days before monensin supplementation, after 30 days of monensin supplementation, and at six weekly intervals after monensin withdrawal. Mean values of relative population size (RPS, the percent of bacterial 16S rRNA copy number) for genus Prevotella increased (P0.05) from 41.8% without monensin to 49.2% with monensin and declined to 42.5% after monensin withdrawal. Mean RPS values for two biohydrogenating species (Megasphaera elsdenii and Butyrivibrio fibrisolvens) were low (0.4%) and declined several-fold in response to monensin. Mean RPS values for the biohydrogenating species Eubacterium ruminantium, four cellulolytic species, four starch- or dextrin-fermenting species, and Domain Archaea were not altered (P0.10) upon monensin feeding or withdrawal. The data suggest that monensin in high-starch diets does not suppress populations of classical ruminal Gram-positive bacteria or the availability of H2, though it may affect bacteria involved in biohydrogenation of lipids that regulate bovine mammary lipogenesis.

Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in C. thermocellum ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a ∼10-fold range of dilution rates (0.013 to 0.16 h −1 ). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes ( recA and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h −1 : cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.

In this NASA-funded study, the oxidation kinetics of methanol and ammonia in supercritical water have been experimentally determined in an isothermal plug flow reactor. Theoretical studies have also been carried out to characterize key reaction pathways. Methanol oxidation rates were found to be proportional to the first power of methanol concentration and independent of oxygen concentration and were highly activated with an activation energy of approximately 98 kcal/mole over the temperature range 480 to 540 C at 246 bar. The oxidation of ammonia was found to be catalytic with an activation energy of 38 kcal/mole over temperatures ranging from 640 to 700 C. An elementary reaction model for methanol oxidation was applied after correction for the effect of high pressure on the rate constants. The conversion of methanol predicted by the model was in good agreement with experimental data.