Targeted Capture and Heterologous Expression of the <i>Pseudoalteromonas</i> Alterochromide Gene Cluster in <i>Escherichia coli</i> Represents a Promising Natural Product Exploratory Platform

Marine pseudoalteromonads represent a very promising source of biologically important natural product molecules. To access and exploit the full chemical capacity of these cosmopolitan Gram-(−) bacteria, we sought to apply universal synthetic biology tools to capture, refactor, and express biosynthetic gene clusters for the production of complex organic compounds in reliable host organisms. Here, we report a platform for the capture of proteobacterial gene clusters using a transformation-associated recombination (TAR) strategy coupled with direct pathway manipulation and expression in <i>Escherichia coli</i>. The ∼34 kb pathway for production of alterochromide lipopeptides by <i>Pseudoalteromonas piscicida</i> JCM 20779 was captured and heterologously expressed in <i>E. coli</i> utilizing native and <i>E. coli</i>-based T7 promoter sequences. Our approach enabled both facile production of the alterochromides and <i>in vivo</i> interrogation of gene function associated with alterochromide’s unusual brominated lipid side chain. This platform represents a simple but effective strategy for the discovery and biosynthetic characterization of natural products from marine proteobacteria

Weighting Low-Intensity MS/MS Ions and <i>m</i>/<i>z</i> Frequency for Spectral Library Annotation

Calculating spectral similarity is a fundamental step in MS/MS data analysis in untargeted metabolomics experiments, as it facilitates the identification of related spectra and the annotation of compounds. To improve matching accuracy when querying an experimental mass spectrum against a spectral library, previous approaches have proposed increasing peak intensities for high m/z ranges. These high m/z values tend to be smaller in magnitude, yet they offer more crucial information for identifying the chemical structure. Here, we evaluate the impact of using these weights for identifying structurally related compounds and mass spectral library searches. Additionally, we propose a weighting approach that (i) takes into account the frequency of the m/z values within a spectral library in order to assign higher importance to the most common peaks and (ii) increases the intensity of lower peaks, similar to previous approaches. To demonstrate our approach, we applied weighting preprocessing to modified cosine, entropy, and fidelity distance metrics and benchmarked it against previously reported weights. Our results demonstrate how weighting-based preprocessing can assist in annotating the structure of unknown spectra as well as identifying structurally similar compounds. Finally, we examined scenarios in which the utilization of weights resulted in diminished performance, pinpointing spectral features where the application of weights might be detrimental

Expanding the Chemical Repertoire of the Endophyte <i>Streptomyces albospinus</i> RLe7 Reveals Amphotericin B as an Inducer of a Fungal Phenotype

During an investigation of the chemistry of the endophytic actinobacterium <i>Streptomyces albospinus</i> RLe7, which was isolated from the roots of the Brazilian medicinal plant <i>Lychnophora ericoides</i>, three new natural products, (2<i>R</i>*,4<i>S</i>*)-2-((1′<i>S</i>*)-hydroxy-4′-methylpentyl)-4-(hydroxymethyl)­butanolide (<b>1</b>), (3<i>R</i>*,4<i>S</i>*,5<i>R</i>*,6<i>S</i>*)-tetrahydro-4-hydroxy-3,5,6-trimethyl-2-pyranone (<b>2</b>), and 1-<i>O</i>-(phenylacetyl)­glycerol (<b>3</b>), together with known secondary metabolites (<i>S</i>)-4-benzyl-3-oxo-3,4-dihydro-1<i>H</i>-pyrrolo­[2,1-<i>c</i>]­[1,4]­oxazine-6-carbaldehyde (<b>4</b>), (<i>S</i>)-4-isobutyl-3-oxo-3,4-dihydro-1<i>H</i>-pyrrolo­[2,1-<i>c</i>]­[1,4]­oxazine-6-carbaldehyde (<b>5</b>), and the diketopiperazines cyclo­(l-Tyr-l-Pro) (<b>6</b>) and cyclo­(l-Val-l-Pro) (<b>7</b>), were isolated. The role of isolated natural products in the interaction between <i>S. albospinus</i> RLe7 and the fungus <i>Coniochaeta</i> sp. FLe4, an endophyte from the same plant, was investigated. None of these isolated actinobacterial compounds were able to inhibit the fungus or induce the fungal red pigmentation observed when both endophytes interact. Further investigation using mass spectrometry approaches enabled identifying the well-known antifungal compound amphotericin B (<b>9</b>) as a microbial metabolite of <i>S. albospinus</i> RLe7. Finally, compound <b>9</b> was demonstrated as at least one of the agents responsible for both the antifungal activity and induction of red-pigmented fungal phenotype

Metabolic Profiling Directly from the Petri Dish Using Nanospray Desorption Electrospray Ionization Imaging Mass Spectrometry

Understanding molecular interaction pathways in complex biological systems constitutes a treasure trove of knowledge that might facilitate the specific, chemical manipulation of the countless microbiological systems that occur throughout our world. However, there is a lack of methodologies that allow the direct investigation of chemical gradients and interactions in living biological systems, in real time. Here, we report the use of nanospray desorption electrospray ionization (nanoDESI) imaging mass spectrometry for in vivo metabolic profiling of living bacterial colonies directly from the Petri dish with absolutely no sample preparation needed. Using this technique, we investigated single colonies of <i>Shewanella oneidensis</i> MR-1, <i>Bacillus subtilis</i> 3610, and <i>Streptomyces coelicolor</i> A3(2) as well as a mixed biofilm of <i>S. oneidensis</i> MR-1 and <i>B. subtilis</i> 3610. Data from <i>B. subtilis</i> 3610 and <i>S. coelicolor</i> A3(2) provided a means of validation for the method while data from <i>S. oneidensis</i> MR-1 and the mixed biofilm showed a wide range of compounds that this bacterium uses for the dissimilatory reduction of extracellular metal oxides, including riboflavin, iron-bound heme and heme biosynthetic intermediates, and the siderophore putrebactin

Homospermidine Lipids: A Compound Class Specifically Formed during Fruiting Body Formation of <i>Myxococcus xanthus</i> DK1622

The fascinating ability of myxobacteria to form multicellular spore filled fruiting bodies under starvation conditions was widely studied as a model for cooperative microbial behavior. The potential of a life cycle induced change of secondary metabolism, as a means to discover novel natural products, remains largely underexplored. We therefore studied the model organism <i>Myxococcus xanthus</i> DK1622 under submersed and solid cultivation conditions to find putatively life-cycle related compounds by applying statistical analysis on analytical data. Utilizing the advantageous characteristics of LC-MS, LC-MS/MS, and MALDI-MSI allowed the identification of compounds unambiguously associated with myxobacterial fruiting bodies. Our screening effort resulted in the purification and structure elucidation of a novel compound, the homospermidine lipid, from cultures that had undergone the fruiting process. A combination of molecular networking and targeted LC-MS/MS in conjunction with our in-house metabolomics database subsequently revealed alternative producers of the respective compound as well as a number of compounds belonging to the same structural class. Three further members of this compound class were isolated from an alternative producer and structurally elucidated by NMR. Insights into the biosynthesis of this novel compound class was gained by feeding of isotopically labeled substrates and <i>in silico</i> analysis

Imaging Mass Spectrometry and Genome Mining via Short Sequence Tagging Identified the Anti-Infective Agent Arylomycin in <i>Streptomyces roseosporus</i>

Here, we described the discovery of anti-infective agent arylomycin and its biosynthetic gene cluster in an industrial daptomycin producing strain <i>Streptomyces roseosporus</i>. This was accomplished via the use of MALDI imaging mass spectrometry (IMS) along with peptidogenomic approach in which we have expanded to short sequence tagging (SST) described herein. Using IMS, we observed that prior to the production of daptomycin, a cluster of ions (<b>1</b>–<b>3</b>) was produced by <i>S. roseosporus</i> and correlated well with the decreased staphylococcal cell growth. With a further adopted SST peptidogenomics approach, which relies on the generation of sequence tags from tandem mass spectrometric data and query against genomes to identify the biosynthetic genes, we were able to identify these three molecules (<b>1</b>–<b>3</b>) to arylomycins, a class of broad-spectrum antibiotics that target type I signal peptidase. The gene cluster was then identified. This highlights the strength of IMS and MS guided genome mining approaches in effectively bridging the gap between phenotypes, chemotypes, and genotypes

Experimental Chagas disease-induced perturbations of the fecal microbiome and metabolome

<div><p><i>Trypanosoma cruzi</i> parasites are the causative agents of Chagas disease. These parasites infect cardiac and gastrointestinal tissues, leading to local inflammation and tissue damage. Digestive Chagas disease is associated with perturbations in food absorption, intestinal traffic and defecation. However, the impact of <i>T</i>. <i>cruzi</i> infection on the gut microbiota and metabolome have yet to be characterized. In this study, we applied mass spectrometry-based metabolomics and 16S rRNA sequencing to profile infection-associated alterations in fecal bacterial composition and fecal metabolome through the acute-stage and into the chronic stage of infection, in a murine model of Chagas disease. We observed joint microbial and chemical perturbations associated with <i>T</i>. <i>cruzi</i> infection. These included alterations in conjugated linoleic acid (CLA) derivatives and in specific members of families <i>Ruminococcaceae</i> and <i>Lachnospiraceae</i>, as well as alterations in secondary bile acids and members of order Clostridiales. These results highlight the importance of multi-‘omics’ and poly-microbial studies in understanding parasitic diseases in general, and Chagas disease in particular.</p></div

Cholic acid/deoxycholic acid-related molecules correlated with parasite burden.

<p>Cholic acid/deoxycholic acid-related molecules correlated with parasite burden.</p

Top-Down Atmospheric Ionization Mass Spectrometry Microscopy Combined With Proteogenomics

Mass spectrometry-based protein analysis has become an important methodology for proteogenomic mapping by providing evidence for the existence of proteins predicted at the genomic level. However, screening and identification of proteins directly on tissue samples, where histological information is preserved, remain challenging. Here we demonstrate that the ambient ionization source, nanospray desorption electrospray ionization (nanoDESI), interfaced with light microscopy allows for protein profiling directly on animal tissues at the microscopic scale. Peptide fragments for mass spectrometry analysis were obtained directly on ganglia of the medicinal leech (Hirudo medicinalis) without in-gel digestion. We found that a hypothetical protein, which is predicted by the leech genome, is highly expressed on the specialized neural cells that are uniquely found in adult sex segmental ganglia. Via this top-down analysis, a post-translational modification (PTM) of tyrosine sulfation to this neuropeptide was resolved. This three-in-one platform, including mass spectrometry, microscopy, and genome mining, provides an effective way for mappings of proteomes under the lens of a light microscope

Conjugated linoleic acid-related molecules correlated to parasite burden.

<p>Conjugated linoleic acid-related molecules correlated to parasite burden.</p