Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence.

Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from ß-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.

Ferrous-Iron-Activated Transcriptional Factor AdhR Regulates Redox Homeostasis in Clostridium beijerinckii.

The AdhR regulatory protein is an activator of σ54-dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA+ domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ54 showed large shifts in intracellular redox state indicated by the NADH/NAD+ ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe2+-activated transcriptional regulator AdhR and σ54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckiiIMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a σ54-dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of σ54 in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia.

The cyanobacterial ornithine-ammonia cycle involves an arginine dihydrolase.

Living organisms have evolved mechanisms for adjusting their metabolism to adapt to environmental nutrient availability. Terrestrial animals utilize the ornithine-urea cycle to dispose of excess nitrogen derived from dietary protein. Here, we identified an active ornithine-ammonia cycle (OAC) in cyanobacteria through an approach combining dynamic 15N and 13C tracers, metabolomics, and mathematical modeling. The pathway starts with carbamoyl phosphate synthesis by the bacterial- and plant-type glutamine-dependent enzyme and ends with conversion of arginine to ornithine and ammonia by a novel arginine dihydrolase. An arginine dihydrolase-deficient mutant showed disruption of OAC and severely impaired cell growth when nitrogen availability oscillated. We demonstrated that the OAC allows for rapid remobilization of nitrogen reserves under starvation and a high rate of nitrogen assimilation and storage after the nutrient becomes available. Thus, the OAC serves as a conduit in the nitrogen storage-and-remobilization machinery in cyanobacteria and enables cellular adaptation to nitrogen fluctuations.

Genomic reconstruction of σ54 regulons in Clostridiales.

BACKGROUND: The σ54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ54 remain unknown. RESULTS: For systematic analysis of the regulatory functions of σ54, we performed comparative genomic reconstruction of transcriptional regulons of σ54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ54 regulons between different Clostridiales species. It is proposed that σ54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species. CONCLUSIONS: This study reveals previously unrecognized functions of σ54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species.

PTS regulation domain-containing transcriptional activator CelR and sigma factor σ(54) control cellobiose utilization in Clostridium acetobutylicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of ß-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity, which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity.

Redox-responsive repressor Rex modulates alcohol production and oxidative stress tolerance in Clostridium acetobutylicum.

Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD(+) ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO2/CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2 on intracellular NADH and NAD(+) concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD(+) ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD(+) homeostasis in clostridia.

Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting Clostridium ljungdahlii.

Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO2/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, we identified a RpiR family transcription factor (TF) that can regulate the autotrophic growth and carbon fixation of C. ljungdahlii. After deletion of the corresponding gene, the resulting mutant strain exhibited significantly impaired growth in gas fermentation, thus reducing the production of acetic acid and ethanol. In contrast, the overexpression of this TF gene could promote cell growth, indicating a positive regulatory effect of this TF in C. ljungdahlii. Thus, we named the TF as GssR (growth and solvent synthesis regulator). Through the following comparative transcriptomic analysis and biochemical verification, we discovered three important genes (encoding pyruvate carboxylase, carbon hunger protein CstA, and a BlaI family transcription factor) that were directly regulated by GssR. Furthermore, an upstream regulator, BirA, that could directly bind to gssR was found; thus, these two regulators may form a cascade regulation and jointly affect the physiology and metabolism of C. ljungdahlii. These findings substantively expand our understanding on the metabolic regulation of carbon fixation in gas-fermenting Clostridium species.

A bacterial spermidine biosynthetic pathway via carboxyaminopropylagmatine.

Spermidine, a ubiquitous polyamine, is known to be required for critical physiological functions in bacteria. Two principal pathways are known for spermidine biosynthesis, both of which involve aminopropylation of putrescine. Here, we identified a spermidine biosynthetic pathway via a previously unknown metabolite, carboxyaminopropylagmatine (CAPA), in a model cyanobacterium Synechocystis sp. PCC 6803 through an approach combining 13C and 15N tracers, metabolomics, and genetic and biochemical characterization. The CAPA pathway starts with reductive condensation of agmatine and l-aspartate-ß-semialdehyde into CAPA by a previously unknown CAPA dehydrogenase, followed by decarboxylation of CAPA to form aminopropylagmatine, and ends with conversion of aminopropylagmatine to spermidine by an aminopropylagmatine ureohydrolase. Thus, the pathway does not involve putrescine and depends on l-aspartate-ß-semialdehyde as the aminopropyl group donor. Genomic, biochemical, and metagenomic analyses showed that the CAPA-pathway genes are widespread in 15 different phyla of bacteria distributed in marine, freshwater, and other ecosystems.

Increasing brain N-acetylneuraminic acid alleviates hydrocephalus-induced neurological deficits.

AIMS: This metabolomic study aimed to evaluate the role of N-acetylneuraminic acid (Neu5Ac) in the neurological deficits of normal pressure hydrocephalus (NPH) and its potential therapeutic effect. METHODS: We analyzed the metabolic profiles of NPH using cerebrospinal fluid with multivariate and univariate statistical analyses in a set of 42 NPH patients and 38 controls. We further correlated the levels of differential metabolites with severity-related clinical parameters, including the normal pressure hydrocephalus grading scale (NPHGS). We then established kaolin-induced hydrocephalus in mice and treated them using N-acetylmannosamine (ManNAc), a precursor of Neu5Ac. We examined brain Neu5Ac, astrocyte polarization, demyelination, and neurobehavioral outcomes to explore its therapeutic effect. RESULTS: Three metabolites were significantly altered in NPH patients. Only decreased Neu5Ac levels were correlated with NPHGS scores. Decreased brain Neu5Ac levels have been observed in hydrocephalic mice. Increasing brain Neu5Ac by ManNAc suppressed the activation of astrocytes and promoted their transition from A1 to A2 polarization. ManNAc also attenuated the periventricular white matter demyelination and improved neurobehavioral outcomes in hydrocephalic mice. CONCLUSION: Increasing brain Neu5Ac improved the neurological outcomes associated with the regulation of astrocyte polarization and the suppression of demyelination in hydrocephalic mice, which may be a potential therapeutic strategy for NPH.

Functional dissection and modulation of the BirA protein for improved autotrophic growth of gas-fermenting Clostridium ljungdahlii.

Gas-fermenting Clostridium species can convert one-carbon gases (CO2 /CO) into a variety of chemicals and fuels, showing excellent application prospects in green biological manufacturing. The discovery of crucial genes and proteins with novel functions is important for understanding and further optimization of these autotrophic bacteria. Here, we report that the Clostridium ljungdahlii BirA protein (ClBirA) plays a pleiotropic regulator role, which, together with its biotin protein ligase (BPL) activity, enables an effective control of autotrophic growth of C. ljungdahlii. The structural modulation of ClBirA, combined with the in vivo and in vitro analyses, further reveals the action mechanism of ClBirA's dual roles as well as their interaction in C. ljungdahlii. Importantly, an atypical, flexible architecture of the binding site was found to be employed by ClBirA in the regulation of a lot of essential pathway genes, thereby expanding BirA's target genes to a broader range in clostridia. Based on these findings, molecular modification of ClBirA was performed, and an improved cellular performance of C. ljungdahlii was achieved in gas fermentation. This work reveals a previously unknown potent role of BirA in gas-fermenting clostridia, providing new perspective for understanding and engineering these autotrophic bacteria.

Serum Metabolite Biomarkers Predictive of Response to PD-1 Blockade Therapy in Non-Small Cell Lung Cancer.

Background: Despite remarkable success of immunotherapies with checkpoint blockade antibodies targeting programmed cell death protein 1 (PD-1), the majority of patients with non-small-cell lung cancer (NSCLC) have yet to receive durable benefits. We used the metabolomic profiling of early on-treatment serum to explore predictors of clinical outcomes of anti-PD-1 treatment in patients with advanced NSCLC. Methods: We recruited 74 Chinese patients who had stage IIIB/IV NSCLC-proven tumor progression and were treated with PD-1 inhibitor. The study was comprised of a discovery cohort of patients treated with nivolumab and two validation cohorts of patients receiving tislelizumab or nivolumab. Serum samples were collected 2-3 weeks after the first infusion of PD-1 inhibitor. Metabolomic profiling of serum was performed using ultrahigh performance lipid chromatograph-mass spectrometry. The serum metabolite biomarkers were identified using an integral workflow of nontargeted metabolomic data analysis. Results: A serum metabolite panel consisting of hypoxanthine and histidine was identified and validated as a predictor of response to PD-1 blockade treatment in patients with advanced NSCLC. High levels of both hypoxanthine and histidine in early on-treatment serum were associated with improved progression-free survival [hazard ratio (HR) = 0.078, 95% confidence interval (CI), 0.027-0.221, p < 0.001] and overall survival (HR = 0.124, 95% CI, 0.039-0.397, p < 0.001) in the discovery cohort. The serum metabolite panel showed a high sensitivity and specificity in distinguishing responders and non-responders in the validation cohorts 1 and 2, with an area under the receiver-operating characteristic curve of 0.933 and 1.000, respectively. High levels of serum hypoxanthine and histidine were correlated with improved progression-free survival in the validation cohort 1 (HR = 0.137, 95% CI, 0.040-0.467, p = 0.001) and in the validation cohort 2 (HR = 0.084, 95% CI, 0.009-0.762, p = 0.028). Conclusion: Our results revealed that hypoxanthine and histidine in early on-treatment serum are predictive biomarkers of response to PD-1 blockade therapy in patients with advanced NSCLC. The serum biomarker panel would enable early identification of NSCLC patients who may benefit from PD-1 blockade therapy.

Biological insights into non-model microbial hosts through stable-isotope metabolic flux analysis.

Stable-isotope metabolic flux analysis is an important approach to unravel the metabolic network and its regulation in organisms. It has become a key analytical technology for biotechnological applications. During recent years non-model microorganisms have received increasing attention because they possess unique metabolic capabilities and can serve as a host for production of biofuels and biochemicals. Stable-isotope metabolic flux analysis has been widely used in these microorganisms for exploring novel pathways, elucidating the operation of central metabolic networks, and revealing the metabolic changes that result from genetic manipulations. Here, we review recent applications of stable-isotope metabolic flux analysis in characterizing non-model microbial hosts, guiding the development of rational engineering strategies for enhancement of biochemical production and extension of substrate range, and understanding of industrial production processes.

Control of solvent production by sigma-54 factor and the transcriptional activator AdhR in Clostridium beijerinckii.

Clostridia are obligate anaerobic bacteria that can produce solvents such as acetone, butanol and ethanol. Alcohol dehydrogenases (ADHs) play a key role in solvent production; however, their regulatory mechanisms remain largely unknown. In this study, we characterized the regulatory mechanisms of two ADH-encoding genes in C. beijerinckii. SigL (sigma factor σ54 ) was found to be required for transcription of adhA1 and adhA2 genes. Moreover, a novel transcriptional activator AdhR was identified, which binds to the σ54 promoter and activates σ54 -dependent transcription of adhA1 and adhA2. Clostridia beijerinckii mutants deficient in SigL or AdhR showed severely impaired butanol and ethanol production as well as altered acetone and butyrate synthesis. Overexpression of SigL resulted in significantly improved solvent production by C. beijerinckii when butyrate was added to cultures. Our results reveal SigL as a novel engineering target for improving solvent production by C. beijerinckii and other solvent-producing clostridia. Moreover, this study gains an insight into regulation of alcohol metabolism in diverse clostridia.

Aldolase B suppresses hepatocellular carcinogenesis by inhibiting G6PD and pentose phosphate pathways.

Metabolic reprogramming is a core hallmark of cancer but it remains poorly defined in hepatocellular carcinogenesis (HCC). Here we show that hepatic aldolase B (Aldob) suppresses HCC by directly binding and inhibiting the rate-limiting enzyme in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD). A stage-dependent decrease of Aldob and increase of G6PD in human tumors are correlated with poor prognosis for patients with HCC. Global or liver-specific Aldob knockout promotes tumorigenesis in mice through enhancing G6PD activity and pentose phosphate pathway metabolism, whereas pharmacological inhibition or genetic knockdown of G6PD suppresses HCC. Consistently, restoration of Aldob in Aldob knockout mice attenuates tumorigenesis. We further demonstrate that Aldob potentiates p53-mediated inhibition of G6PD in an Aldob-G6PD-p53 complex. This scaffolding effect is independent of Aldob enzymatic activity. Together, our study reveals a new mode of metabolic reprogramming in HCC due to the loss of Aldob, suggesting a potential therapeutic strategy for HCC treatment.

Metabolic regulation in solventogenic clostridia: regulators, mechanisms and engineering.

Solventogenic clostridia, a group of important industrial microorganisms, have exceptional substrate and product diversity, capable of producing a series of two-carbon and even long-chain chemicals and fuels by using various substrates, including sugars, cellulose and hemicellulose, and C1 gases. For the sake of in-depth understanding and engineering these anaerobic microorganisms for broader applications, studies on metabolic regulation of solventogenic clostridia had been extensively carried out during the past ten years, based on the rapid development of various genetic tools. To date, a number of regulators that are essential for cell physiological and metabolic processes have been identified in clostridia, and the relevant mechanisms have also been dissected, providing a wealth of valuable information for metabolic engineering. Here, we reviewed the latest research progresses on the metabolic regulation for chemical production and substrate utilization in solventogenic clostridia, by focusing on three typical Clostridium species, the saccharolytic C. acetobutylicum and C. beijerinckii, as well as the gas-fermenting C. ljungdahlii. On this basis, future directions in the study and remodeling of clostridial regulation systems, were proposed for effective modification of these industrially important anaerobes.