Genomic Analysis of Novel Sulfitobacter Bacterial Strains Isolated from Marine Biofilms.

Bacteria from the genus Sulfitobacter are distributed across various marine habitats and play a significant role in sulfur cycling. However, the metabolic features of Sulfitobacter inhabiting marine biofilms are still not well understood. Here, complete genomes and paired metatranscriptomes of eight Sulfitobacter strains, isolated from biofilms on subtidal stones, have been analyzed to explore their central energy metabolism and potential of secondary metabolite biosynthesis. Based on average nucleotide identity and phylogenetic analysis, the eight strains were classified into six novel species and two novel strains. The reconstruction of the metabolic pathways indicated that all strains had a complete Entner-Doudoroff pathway, pentose phosphate pathway, and diverse pathways for amino acid metabolism, suggesting the presence of an optimized central carbon metabolism. Pangenome analysis further revealed the differences between the gene cluster distribution patterns among the eight strains, suggesting significant functional variation. Moreover, a total of 47 biosynthetic gene clusters were discovered, which were further classified into 37 gene cluster families that showed low similarity with previously documented clusters. Furthermore, metatranscriptomic analysis revealed the expressions of key functional genes involved in the biosynthesis of ribosomal peptides in in situ marine biofilms. Overall, this study sheds new light on the metabolic features, adaptive strategies, and value of genome mining in this group of biofilm-associated Sulfitobacter bacteria.

Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses.

Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana, Nicotiana tabacum, and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

Modelling metabolic fluxes of tomato stems reveals that nitrogen shapes central metabolism for defence against Botrytis cinerea.

Among plant pathogens, the necrotrophic fungus Botrytis cinerea is one of the most prevalent, leading to severe crop damage. Studies related to its colonization of different plant species have reported variable host metabolic responses to infection. In tomato, high N availability leads to decreased susceptibility. Metabolic flux analysis can be used as an integrated method to better understand which metabolic adaptations lead to effective host defence and resistance. Here, we investigated the metabolic response of tomato infected by B. cinerea in symptomless stem tissues proximal to the lesions for 7 d post-inoculation, using a reconstructed metabolic model constrained by a large and consistent metabolic dataset acquired under four different N supplies. An overall comparison of 48 flux solution vectors of Botrytis- and mock-inoculated plants showed that fluxes were higher in Botrytis-inoculated plants, and the difference increased with a reduction in available N, accompanying an unexpected increase in radial growth. Despite higher fluxes, such as those involved in cell wall synthesis and other pathways, fluxes related to glycolysis, the tricarboxylic acid cycle, and amino acid and protein synthesis were limited under very low N, which might explain the enhanced susceptibility. Limiting starch synthesis and enhancing fluxes towards redox and specialized metabolism also contributed to defence independent of N supply.

Primary Human M2 Macrophage Subtypes Are Distinguishable by Aqueous Metabolite Profiles.

The complexity of macrophage (MΦ) plasticity and polarization states, which include classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ phenotypes, is becoming increasingly appreciated. Within the M2 MΦ polarization state, M2a, M2b, M2c, and M2d MΦ subcategories have been defined based on their expression of specific cell surface receptors, secreted cytokines, and specialized immune effector functions. The importance of immunometabolic networks in mediating the function and regulation of MΦ immune responses is also being increasingly recognized, although the exact mechanisms and extent of metabolic modulation of MΦ subtype phenotypes and functions remain incompletely understood. In this study, proton (1H) nuclear magnetic resonance (NMR) metabolomics was employed to determine the polar metabolomes of M2 MΦ subtypes and to investigate the relationship between aqueous metabolite profiles and M2 MΦ functional phenotypes. Results from this study demonstrate that M2a MΦs are most distinct from M2b, M2c, and M2d MΦ subtypes, and that M2b MΦs display several metabolic traits associated with an M1-like MΦ phenotype. The significance of metabolome differences for metabolites implicated in glycolysis, the tricarboxylic acid (TCA) cycle, phospholipid metabolism, and creatine-phosphocreatine cycling is discussed. Altogether, this study provides biochemical insights into the role of metabolism in mediating the specialized effector functions of distinct M2 MΦ subtypes and supports the concept of a continuum of macrophage activation states rather than two well-separated and functionally distinct M1/M2 MΦ classes, as originally proposed within a classical M1/M2 MΦ framework.

mSphere of Influence: Targeting bacterial signaling and metabolism to overcome antimicrobial resistance.

Dr Merve Suzan Zeden works in the field of molecular bacteriology and antibiotic resistance. In this mSphere of Influence article, she reflects on how three papers, entitled "c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation," "Amino acid catabolism in Staphylococcus aureus and the function of carbon catabolite repression," and "Evolving MRSA: high-level ß-lactam resistance in Staphylococcus aureus is associated with RNA polymerase alterations and fine tuning of gene expression," made an impact on her work on bacterial metabolism and antimicrobial resistance and how it shaped her research in understanding the link in between.

Investigating inhibitors of 1-deoxy-d-xylulose 5-phosphate synthase in a mouse model of UTI.

The rising rate of antimicrobial resistance continues to threaten global public health. Further hastening antimicrobial resistance is the lack of new antibiotics against new targets. The bacterial enzyme, 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), is thought to play important roles in central metabolism, including processes required for pathogen adaptation to fluctuating host environments. Thus, impairing DXPS function represents a possible new antibacterial strategy. We previously investigated a DXPS-dependent metabolic adaptation as a potential target in uropathogenic Escherichia coli (UPEC) associated with urinary tract infection (UTI), using the DXPS-selective inhibitor butyl acetylphosphonate (BAP). However, investigations of DXPS inhibitors in vivo have not been conducted. The goal of the present study is to advance DXPS inhibitors as in vivo probes and assess the potential of inhibiting DXPS as a strategy to prevent UTI in vivo. We show that BAP was well-tolerated at high doses in mice and displayed a favorable pharmacokinetic profile for studies in a mouse model of UTI. Further, an alkyl acetylphosphonate prodrug (homopropargyl acetylphosphonate, pro-hpAP) was significantly more potent against UPEC in urine culture and exhibited good exposure in the urinary tract after systemic dosing. Prophylactic treatment with either BAP or pro-hpAP led to a partial protective effect against UTI, with the prodrug displaying improved efficacy compared to BAP. Overall, our results highlight the potential for DXPS inhibitors as in vivo probes and establish preliminary evidence that inhibiting DXPS impairs UPEC colonization in a mouse model of UTI.IMPORTANCENew antibiotics against new targets are needed to prevent an antimicrobial resistance crisis. Unfortunately, antibiotic discovery has slowed, and many newly FDA-approved antibiotics do not inhibit new targets. Alkyl acetylphosphonates (alkyl APs), which inhibit the enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), represent a new possible class of compounds as there are no FDA-approved DXPS inhibitors. To our knowledge, this is the first study demonstrating the in vivo safety, pharmacokinetics, and efficacy of alkyl APs in a urinary tract infection mouse model.

Plant Heterotrophic Cultures: No Food, No Growth.

Plant cells are capable of uptaking exogenous organic substances. This inherited trait allows the development of heterotrophic cell cultures in various plants. The most common of them are Nicotiana tabacum and Arabidopsis thaliana. Plant cells are widely used in academic studies and as factories for valuable substance production. The repertoire of compounds supporting the heterotrophic growth of plant cells is limited. The best growth of cultures is ensured by oligosaccharides and their cleavage products. Primarily, these are sucrose, raffinose, glucose and fructose. Other molecules such as glycerol, carbonic acids, starch, and mannitol have the ability to support growth occasionally, or in combination with another substrate. Culture growth is accompanied by processes of specialization, such as elongation growth. This determines the pattern of the carbon budget. Culture ageing is closely linked to substrate depletion, changes in medium composition, and cell physiological rearrangements. A lack of substrate leads to starvation, which results in a decrease in physiological activity and the mobilization of resources, and finally in the loss of viability. The cause of the instability of cultivated cells may be the non-optimal metabolism under cultural conditions or the insufficiency of internal regulation.

Lighting up Pyruvate Metabolism in Saccharomyces cerevisiae by a Genetically Encoded Fluorescent Biosensor.

Monitoring intracellular pyruvate is useful for the exploration of fundamental metabolism and for guiding the construction of yeast cell factories for chemical production. Here, we employed a genetically encoded fluorescent Pyronic biosensor to light up the pyruvate metabolic state in the cytoplasm, nucleus, and mitochondria of Saccharomyces cerevisiae BY4741. A strong correlation was observed between the pyruvate fluctuation in mitochondria and cytoplasm when exposed to different metabolites. Further metabolic analysis of pyruvate uptake and glycolytic dynamics showed that glucose and fructose dose-dependently activated cytoplasmic pyruvate levels more effectively than direct exposure to pyruvate. Meanwhile, the Pyronic biosensor could visually distinguish phenotypes of the wild-type S. cerevisiae BY4741 and the pyruvate-hyperproducing S. cerevisiae TAM at a single-cell resolution, having the potential for high-throughput screening. Overall, Pyronic biosensors targeting different suborganelles contribute to mapping and studying the central carbon metabolism in-depth and guide the design and construction of yeast cell factories.

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

A Desmethylphosphinothricin Dipeptide Derivative Effectively Inhibits Escherichia coli and Bacillus subtilis Growth.

New antibiotics are unquestionably needed to fight the emergence and spread of multidrug-resistant bacteria. To date, antibiotics targeting bacterial central metabolism have been poorly investigated. By determining the minimal inhibitory concentration (MIC) of desmethylphosphinothricin (Glu-γ-PH), an analogue of glutamate with a phosphinic moiety replacing the γ-carboxyl group, we previously showed its promising antibacterial activity on Escherichia coli. Herein, we synthetized and determined the growth inhibition exerted on E. coli by an L-Leu dipeptide derivative of Glu-γ-PH (L-Leu-D,L-Glu-γ-PH). Furthermore, we compared the growth inhibition obtained with this dipeptide with that exerted by the free amino acid, i.e., Glu-γ-PH, and by their phosphonic and non-desmethylated analogues. All the tested compounds were more effective when assayed in a chemically-defined minimal medium. The dipeptide L-Leu-D,L-Glu-γ-PH had a significantly improved antibacterial activity (2 µg/mL), at a concentration between the non-desmethytaled (0.1 µg/mL) and the phosphonic (80 µg/mL) analogues. Also, in Bacillus subtilis, the dipeptide L-Leu-D,L-Glu-γ-PH displayed an activity comparable to that of the antibiotic amoxicillin. This work highlights the antibacterial relevance of the phosphinic pharmacophore and proposes new avenues for the development of novel antimicrobial drugs containing the phosphinic moiety.

Acinetobacter baumannii coordinates central metabolism, plasmid dissemination, and virulence by sensing nutrient availability.

Plasmid conjugation plays an important role in the dissemination of antibiotic-resistance genes. The emergence of multidrug-resistant isolates of Acinetobacter baumannii poses grave challenges in treating infections caused by this notorious nosocomial pathogen. Yet, the composition, functionality, and regulation of conjugative machinery utilized by A. baumannii remain poorly understood. Here, we found that conjugation of the major plasmid pAB3 of A. baumannii is mediated by a type IVB secretion system similar to the Dot/Icm transporter of Legionella pneumophila. Furthermore, the expression of the structural genes of the Dot/Icm-like system is co-regulated with genes involved in central metabolism by the GacS/GacA two-component system in response to various metabolites, including intermediates of the tricarboxylic acid cycle. Loss of GacS/A also severely impaired bacterial virulence. These results establish that A. baumannii coordinates metabolism with plasmid conjugation and virulence by sensing nutrient availability, which may be exploited to develop inhibitory agents for controlling the spread of drug-resistance genes and virulence factors. IMPORTANCE Plasmid conjugation is known to be an energy-expensive process, but our understanding of the molecular linkage between conjugation and metabolism is limited. Our finding reveals that Acinetobacter baumannii utilizes a two-component system to co-regulate metabolism, plasmid transfer, and virulence by sensing reaction intermediates of key metabolic pathways, which suggests that nutrient availability dictates not only bacterial proliferation but also horizontal gene transfer. The identification of Dot/Icm-like proteins as components of a conjugation system involved in the dissemination of antibiotic-resistance genes by A. baumannii has provided important targets for the development of agents capable of inhibiting virulence and the spread of anti-microbial-resistance genes in bacterial communities.

Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering.

The capability to manipulate and analyze hard-wired metabolic pathways sets the pace at which we can engineer cellular metabolism. Here, we present a framework to extensively rewrite the central metabolic pathway for malonyl-CoA biosynthesis in yeast and readily assess malonyl-CoA output based on pathway-scale DNA reconstruction in combination with colorimetric screening (Pracs). We applied Pracs to generate and test millions of enzyme variants by introducing genetic mutations into the whole set of genes encoding the malonyl-CoA biosynthetic pathway and identified hundreds of beneficial enzyme mutants with increased malonyl-CoA output. Furthermore, the synthetic pathways reconstructed by randomly integrating these beneficial enzyme variants generated vast phenotypic diversity, with some displaying higher production of malonyl-CoA as well as other metabolites, such as carotenoids and betaxanthin, thus demonstrating the generic utility of Pracs to efficiently orchestrate central metabolism to optimize the production of different chemicals in various metabolic pathways. Pracs will be broadly useful to advance our ability to understand and engineer cellular metabolism.

Transcriptome Metabolic Characterization of Tuber borchii SP1-A New Spanish Strain for In Vitro Studies of the Bianchetto Truffle.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

How Bacterial Pathogens Coordinate Appetite with Virulence.

Cells adjust growth and metabolism to nutrient availability. Having access to a variety of carbon sources during infection of their animal hosts, facultative intracellular pathogens must efficiently prioritize carbon utilization. Here, we discuss how carbon source controls bacterial virulence, with an emphasis on Salmonella enterica serovar Typhimurium, which causes gastroenteritis in immunocompetent humans and a typhoid-like disease in mice, and propose that virulence factors can regulate carbon source prioritization by modifying cellular physiology. On the one hand, bacterial regulators of carbon metabolism control virulence programs, indicating that pathogenic traits appear in response to carbon source availability. On the other hand, signals controlling virulence regulators may impact carbon source utilization, suggesting that stimuli that bacterial pathogens experience within the host can directly impinge on carbon source prioritization. In addition, pathogen-triggered intestinal inflammation can disrupt the gut microbiota and thus the availability of carbon sources. By coordinating virulence factors with carbon utilization determinants, pathogens adopt metabolic pathways that may not be the most energy efficient because such pathways promote resistance to antimicrobial agents and also because host-imposed deprivation of specific nutrients may hinder the operation of certain pathways. We propose that metabolic prioritization by bacteria underlies the pathogenic outcome of an infection.

Knocking out central metabolism genes to identify new targets and alternating substrates to improve lipid synthesis in Y. lipolytica.

Introduction: Systematic gene knockout studies may offer us novel insights on cell metabolism and physiology. Specifically, the lipid accumulation mechanism at the molecular or cellular level is yet to be determined in the oleaginous yeast Y. lipolytica. Methods: Herein, we established ten engineered strains with the knockout of important genes involving in central carbon metabolism, NADPH generation, and fatty acid biosynthetic pathways. Results: Our result showed that NADPH sources for lipogenesis include the OxPP pathway, POM cycle, and a trans-mitochondrial isocitrate-α-oxoglutarate NADPH shuttle in Y. lipolytica. Moreover, we found that knockout of mitochondrial NAD+ isocitrate dehydrogenase IDH2 and overexpression of cytosolic NADP+ isocitrate dehydrogenase IDP2 could facilitate lipid synthesis. Besides, we also demonstrated that acetate is a more favorable carbon source for lipid synthesis when glycolysis step is impaired, indicating the evolutionary robustness of Y. lipolytica. Discussion: This systematic investigation of gene deletions and overexpression across various lipogenic pathways would help us better understand lipogenesis and engineer yeast factories to upgrade the lipid biomanufacturing platform.

Control of central metabolism's architecture in Escherichia coli: An overview.

Understanding metabolic networks' architecture is central to successfully manipulating metabolic fluxes in microbial cell factories. The transition of central metabolism's architecture from acetogenic to gluconeogenic and from the canonical monocyclic architecture of the Krebs tricarboxylic acids (TCA) cycle to a bicyclic architecture in which the TCA and the dicarboxylic acids (DCA) cycles work in unison, with the glyoxylate bypass fulfilling the anaplerotic function, has been the subject of much debate and remains elusive. In this article, the author sheds light on the intricacies surrounding the transition of central metabolism from one architecture to another and shows that the transition from the monocyclic architecture to the bicyclic architecture is triggered in response to a minimum threshold signal of growth rate (≲0.40h-1) and is a consequence of competitions, on the one hand. between phosphotransacetylase (PTA) and α-ketoglutarate dehydrogenase (α-KGDH) for their common co-factor, free HS-CoA, and, on the other hand, between catabolic and anaplerotic routs for acetyl phosphate. Further restriction of carbon supply in the bioreactor to the point of starvation forces E. coli to further modify its central metabolism to the PEP-glyoxylate architecture to maintain the redox balance. Interestingly the sudden change from hunger ('famine') to carbon excess ('feast') leads to yet another architecture in which the methylglyoxal pathway figure prominently to maintain the adenylate energy charge. Moreover, the author sheds light on the biochemical implications of each architecture.

Systems-Wide Dissection of Organic Acid Assimilation in Pseudomonas aeruginosa Reveals a Novel Path To Underground Metabolism.

The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of 'omics approaches (transcriptomics, proteomics, metabolomics, and 13C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how "underground metabolism," facilitated by enzyme substrate promiscuity, "rewires" Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that, due to its unrivalled resistance to antibiotics, ubiquity in the built environment, and aggressiveness in infection scenarios, has acquired the somewhat dubious accolade of being designated a "critical priority pathogen" by the WHO. In this work, we uncover the pathways and mechanisms used by P. aeruginosa to grow on a substrate that is abundant at many infection sites: propionate. We found that if the organism is prevented from metabolizing propionate, the substrate turns from being a convenient nutrient source into a potent poison, preventing bacterial growth. We further show that one of the enzymes involved in these reactions, 2-methylcitrate synthase (PrpC), is promiscuous and can moonlight for another essential enzyme in the cell (citrate synthase). Indeed, mutations that abolish citrate synthase activity (which would normally prevent the cell from growing) can be readily overcome if the cell acquires additional mutations that increase the expression of PrpC. This is a nice example of the evolutionary utility of so-called "underground metabolism."

Geobacter sulfurreducens metabolism at different donor/acceptor ratios.

Geobacter species have great application potential in remediation processes and electrobiotechnology. In all applications, understanding the metabolism will enable target-oriented optimization of the processes. The typical electron donor and carbon source of the Geobacter species is acetate, while fumarate is the usual electron acceptor. Here, we could show that depending on the donor/acceptor ratio in batch cultivation of Geobacter sulfurreducens different product patterns occur. With a donor/acceptor ratio of 1:2.5 malate accumulated as an intermediate product but was metabolized to succinate subsequently. At the end of the cultivation, the ratio of fumarate consumed and succinate produced was approximately 1:1. When fumarate was added in excess, malate accumulated in the fermentation broth without further metabolization. After the addition of acetate to stationary cells, malate concentration decreased immediately and additional succinate was synthesized. Finally, it was shown that also resting cells of G. sulfurreducens could efficiently convert fumarate to malate without an additional electron donor. Overall, it was demonstrated that by altering the donor/acceptor ratio, targeted optimization of the metabolite conversion by G. sulfurreducens can be realized.

An expanded role for the transcription factor WRINKLED1 in the biosynthesis of triacylglycerols during seed development.

The transcription factor WRINKLED1 (WRI1) is known as a master regulator of fatty acid synthesis in developing oilseeds of Arabidopsis thaliana and other species. WRI1 is known to directly stimulate the expression of many fatty acid biosynthetic enzymes and a few targets in the lower part of the glycolytic pathway. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors is controlled by WRI1. To shortlist possible gene targets for future in-planta experimental validation, here we present a strategy that combines phylogenetic foot printing of cis-regulatory elements with additional layers of evidence. Upstream regions of protein-encoding genes in A. thaliana were searched for the previously described DNA-binding consensus for WRI1, the ASML1/WRI1 (AW)-box. For about 900 genes, AW-box sites were found to be conserved across orthologous upstream regions in 11 related species of the crucifer family. For 145 select potential target genes identified this way, affinity of upstream AW-box sequences to WRI1 was assayed by Microscale Thermophoresis. This allowed definition of a refined WRI1 DNA-binding consensus. We find that known WRI1 gene targets are predictable with good confidence when upstream AW-sites are phylogenetically conserved, specifically binding WRI1 in the in vitro assay, positioned in proximity to the transcriptional start site, and if the gene is co-expressed with WRI1 during seed development. When targets predicted in this way are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in glycolysis and fatty acid biosynthesis by WRI1. Several of the newly predicted targets are in the upper glycolysis pathway and the pentose phosphate pathway. Of these, plastidic isoforms of fructokinase (FRK3) and of phosphoglucose isomerase (PGI1) are particularly corroborated by previously reported seed phenotypes of respective null mutations.

Manganese Utilization in Salmonella Pathogenesis: Beyond the Canonical Antioxidant Response.

The metal ion manganese (Mn2+) is equally coveted by hosts and bacterial pathogens. The host restricts Mn2+ in the gastrointestinal tract and Salmonella-containing vacuoles, as part of a process generally known as nutritional immunity. Salmonella enterica serovar Typhimurium counteract Mn2+ limitation using a plethora of metal importers, whose expression is under elaborate transcriptional and posttranscriptional control. Mn2+ serves as cofactor for a variety of enzymes involved in antioxidant defense or central metabolism. Because of its thermodynamic stability and low reactivity, bacterial pathogens may favor Mn2+-cofactored metalloenzymes during periods of oxidative stress. This divalent metal catalyzes metabolic flow through lower glycolysis, reductive tricarboxylic acid and the pentose phosphate pathway, thereby providing energetic, redox and biosynthetic outputs associated with the resistance of Salmonella to reactive oxygen species generated in the respiratory burst of professional phagocytic cells. Combined, the oxyradical-detoxifying properties of Mn2+ together with the ability of this divalent metal cation to support central metabolism help Salmonella colonize the mammalian gut and establish systemic infections.