A metabolomics pipeline highlights microbial metabolism in bloodstream infections.

The growth of antimicrobial resistance (AMR) highlights an urgent need to identify bacterial pathogenic functions that may be targets for clinical intervention. Although severe infections profoundly alter host metabolism, prior studies have largely ignored microbial metabolism in this context. Here, we describe an iterative, comparative metabolomics pipeline to uncover microbial metabolic features in the complex setting of a host and apply it to investigate gram-negative bloodstream infection (BSI) in patients.

Accelerating the discovery of alkyl halide-derived natural products using halide depletion.

Even in the genomic era, microbial natural product discovery workflows can be laborious and limited in their ability to target molecules with specific structural features. Here we leverage an understanding of biosynthesis to develop a workflow that targets the discovery of alkyl halide-derived natural products by depleting halide anions, a key biosynthetic substrate for enzymatic halogenation, from microbial growth media. By comparing the metabolomes of bacterial cultures grown in halide-replete and deficient media, we rapidly discovered the nostochlorosides, the products of an orphan halogenase-encoding gene cluster from Nostoc punctiforme ATCC 29133.

Identification and targeting of microbial putrescine acetylation in bloodstream infections.

The growth of antimicrobial resistance (AMR) has highlighted an urgent need to identify bacterial pathogenic functions that may be targets for clinical intervention. Although severe bacterial infections profoundly alter host metabolism, prior studies have largely ignored alterations in microbial metabolism in this context. Performing metabolomics on patient and mouse plasma samples, we identify elevated levels of bacterially-derived -acetylputrescine during gram-negative bloodstream infections (BSI), with higher levels associated with worse clinical outcomes.

Gut microbial metabolism of 5-ASA diminishes its clinical efficacy in inflammatory bowel disease.

For decades, variability in clinical efficacy of the widely used inflammatory bowel disease (IBD) drug 5-aminosalicylic acid (5-ASA) has been attributed, in part, to its acetylation and inactivation by gut microbes. Identification of the responsible microbes and enzyme(s), however, has proved elusive. To uncover the source of this metabolism, we developed a multi-omics workflow combining gut microbiome metagenomics, metatranscriptomics and metabolomics from the longitudinal IBDMDB cohort of 132 controls and patients with IBD.

Biosynthesis of the Unusual Carbon Skeleton of Nocuolin A.

Nocuolin A is a cytotoxic cyanobacterial metabolite that is proposed to be produced by enzymes of the biosynthetic gene cluster. Nocuolin A features a 1,2,3-oxadiazine moiety, a structural feature unique among natural products and, so far, inaccessible through organic synthesis, suggesting that novel enzymatic chemistry might be involved in its biosynthesis. This heterocycle is substituted with two alkyl chains and a 3-hydroxypropanoyl moiety.

Structural basis for an unprecedented enzymatic alkylation in cylindrocyclophane biosynthesis.

The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon-carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology, and the structure and mechanism of CylK are unknown. Here, we report X-ray crystal structures of CylK, revealing a distinctive fusion of a Ca-binding domain and a β-propeller fold.

Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria.

Background: Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC - the first predicted dimetal-carboxylate halogenase to be characterized - was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis.

Refinement of metabolite detection in cystic fibrosis sputum reveals heme correlates with lung function decline.

The bacterial growth environment within cystic fibrosis (CF) sputum is complex, dynamic, and shaped by both host and microbial processes. Characterization of the chemical parameters within sputum that stimulate the in vivo growth of airway pathogens (e.g.

Chemistry, bioactivity and biosynthesis of cyanobacterial alkylresorcinols.

Covering: up to 2019 Alkylresorcinols are amphiphilic metabolites, well-known for their diverse biological activities, produced by both prokaryotes and eukaryotes. A few classes of alkylresorcinol scaffolds have been reported from the photoautotrophic cyanobacteria, ranging from the relatively simple hierridins to the more intricate cylindrocyclophanes. Recently, it has emerged that cyanobacteria employ two different biosynthetic pathways to produce unique alkylresorcinol scaffolds.

Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations.

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.

The Colorful World of Extracellular Electron Shuttles.

Descriptions of the changeable, striking colors associated with secreted natural products date back well over a century. These molecules can serve as extracellular electron shuttles (EESs) that permit microbes to access substrates at a distance. In this review, we argue that the colorful world of EESs has been too long neglected.

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions.

Pyocyanin degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa biofilms.

The opportunistic pathogen Pseudomonas aeruginosa produces colorful redox-active metabolites called phenazines, which underpin biofilm development, virulence, and clinical outcomes. Although phenazines exist in many forms, the best studied is pyocyanin. Here, we describe pyocyanin demethylase (PodA), a hitherto uncharacterized protein that oxidizes the pyocyanin methyl group to formaldehyde and reduces the pyrazine ring via an unusual tautomerizing demethylation reaction.

Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force.

While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown.

Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity.

Eukaryotic yeast-based DNA damage cellular sensors offer many advantages to traditional prokaryotic-based mutagenicity assays. The HUG1P-GFP promoter-reporter construct has proven to be an effective method to selectively screen for multiple types of DNA damage. To enhance the sensitivity and selectivity of the system to different types of DNA damage, two genes involved in distinct DNA damage responses were deleted.

The utilization of a Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct for the selective detection of DNA damage.

In this study, we report the creation and characterization of a yeast-based promoter-reporter construct for the detection of genotoxic compounds within a cell's local environment. We have synthesized a fusion containing the HUG1 promoter and GFP and incorporated this cassette into the yeast genome creating a stable, sensitive genotoxicity indicator. To quantify biosensor performance, HUG1P-GFP cells were exposed to multiple doses of a wide variety of genotoxins, including alkylating agents, an oxidative agent, a ribonucleotide reductase inhibitor, a UV mimetic agent, an agent that causes double strand breaks, a topoisomerase I inhibitor, and ionizing radiation, all of which triggered a detectable and reproducible level of GFP production by the HUG1P-GFP strain.