Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues.

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A1 , C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A1 - and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A1 -ring phenol and H-ring C-4' hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.

Bifunctional Nitrone-Conjugated Secondary Metabolite Targeting the Ribosome.

Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer Micromonospora carbonacea var. aurantiaca, we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 Å from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.

Mapping Microbial Response Metabolomes for Induced Natural Product Discovery.

Intergeneric microbial interactions may originate a significant fraction of secondary metabolic gene regulation in nature. Herein, we expose a genomically characterized Nocardiopsis strain, with untapped polyketide biosynthetic potential, to intergeneric interactions via coculture with low inoculum exposure to Escherichia, Bacillus, Tsukamurella, and Rhodococcus. The challenge-induced responses of extracted metabolites were characterized via multivariate statistical and self-organizing map (SOM) analyses, revealing the magnitude and selectivity engendered by the limiting case of low inoculum exposure. The collected inventory of cocultures revealed substantial metabolomic expansion in comparison to monocultures with nearly 14% of metabolomic features in cocultures undetectable in monoculture conditions and many features unique to coculture genera. One set of SOM-identified responding features was isolated, structurally characterized by multidimensional NMR, and revealed to comprise previously unreported polyketides containing an unusual pyrrolidinol substructure and moderate and selective cytotoxicity. Designated ciromicin A and B, they are detected across mixed cultures with intergeneric preferences under coculture conditions. The structural novelty of ciromicin A is highlighted by its ability to undergo a diastereoselective photochemical 12-π electron rearrangement to ciromicin B at visible wavelengths. This study shows how organizing trends in metabolomic responses under coculture conditions can be harnessed to characterize multipartite cultures and identify previously silent secondary metabolism.

Structuring Microbial Metabolic Responses to Multiplexed Stimuli via Self-Organizing Metabolomics Maps.

Secondary metabolite biosynthesis in microorganisms responds to discrete chemical and biological stimuli; however, untargeted identification of these responses presents a significant challenge. Herein we apply multiplexed stimuli to Streptomyces coelicolor and collect the resulting response metabolomes via ion mobility-mass spectrometric analysis. Self-organizing map (SOM) analytics adapted for metabolomic data demonstrate efficient characterization of the subsets of primary and secondary metabolites that respond similarly across stimuli. Over 60% of all metabolic features inventoried from responses are either not observed under control conditions or produced at greater than 2-fold increase in abundance in response to at least one of the multiplexing conditions, reflecting how metabolites encode phenotypic changes in an organism responding to multiplexed challenges. Using abundance as an additional filter, each of 16 known S. coelicolor secondary metabolites is prioritized via SOM and observed at increased levels (1.2- to 22-fold compared with unperturbed) in response to one or more challenge conditions.

Structure and Stereochemical Determination of Hypogeamicins from a Cave-Derived Actinomycete.

Culture extracts from the cave-derived actinomycete Nonomuraea specus were investigated, resulting in the discovery of a new S-bridged pyronaphthoquinone dimer and its monomeric progenitors designated hypogeamicins A-D (1-4). The structures were elucidated using NMR spectroscopy, and the relative stereochemistries of the pyrans were inferred using NOE and comparison to previously reported compounds. Absolute stereochemistry was determined using quantum chemical calculations of specific rotation and vibrational and electronic circular dichroism spectra, after an extensive conformational search and including solute-solvent polarization effects, and comparing with the corresponding experimental data for the monomeric congeners. Interestingly, the dimeric hypogeamicin A (1) was found to be cytotoxic to the colon cancer derived cell line TCT-1 at low micromolar ranges, but not bacteria, whereas the monomeric precursors possessed antibiotic activity but no significant TCT-1 cytotoxicity.

Antimicrobial drug resistance affects broad changes in metabolomic phenotype in addition to secondary metabolism.

Bacteria develop resistance to many classes of antibiotics vertically, by engendering mutations in genes encoding transcriptional and translational apparatus. These severe adaptations affect global transcription, translation, and the correspondingly affected metabolism. Here, we characterize metabolome scale changes in transcriptional and translational mutants in a genomically characterized Nocardiopsis, a soil-derived actinomycete, in stationary phase. Analysis of ultra-performance liquid chromatography-ion mobility-mass spectrometry metabolomic features from a cohort of streptomycin- and rifampicin-resistant mutants grown in the absence of antibiotics exhibits clear metabolomic speciation, and loadings analysis catalogs a marked change in metabolic phenotype. Consistent with derepression, up to 311 features are observed in antibiotic-resistant mutants that are not detected in their progenitors. Mutants demonstrate changes in primary metabolism, such as modulation of fatty acid composition and the increased production of the osmoprotectant ectoine, in addition to the presence of abundant emergent potential secondary metabolites. Isolation of three of these metabolites followed by structure elucidation demonstrates them to be an unusual polyketide family with a previously uncharacterized xanthene framework resulting from sequential oxidative carbon skeletal rearrangements. Designated as "mutaxanthenes," this family can be correlated to a type II polyketide gene cluster in the producing organism. Taken together, these data suggest that biosynthetic pathway derepression is a general consequence of some antibiotic resistance mutations.

Structural mass spectrometry: rapid methods for separation and analysis of peptide natural products.

A significant challenge in natural product discovery is the initial discrimination of discrete secondary metabolites alongside functionally similar primary metabolic cellular components within complex biological samples. A property that has yet to be fully exploited for natural product identification and characterization is the gas-phase collision cross section, or, more generally, the mobility-mass correlation. Peptide natural products possess many of the properties that distinguish natural products, as they are frequently characterized by a high degree of intramolecular bonding and possess extended and compact conformations among other structural modifications. This report describes a rapid structural mass spectrometry technique based on ion mobility-mass spectrometry for the comparison of peptide natural products to their primary metabolic congeners using mobility-mass correlation. This property is empirically determined using ion mobility-mass spectrometry, applied to the analysis of linear versus modified peptides, and used to discriminate peptide natural products in a crude microbial extract. Complementary computational approaches are utilized to understand the structural basis for the separation of primary metabolism derived linear peptides from secondary metabolite cyclic and modified cyclic species. These findings provide a platform for enhancing the identification of secondary metabolic peptides with distinct mobility-mass ratios within complex biological samples.

Biosynthesis of the Apoptolidins in Nocardiopsis sp. FU 40.

The apoptolidins are 20/21-membered macrolides produced by Nocardiopsis sp. FU40. Several members of this family are potent and remarkably selective inducers of apoptosis in cancer cell lines, likely via a distinct mitochondria associated target. To investigate the biosynthesis of this natural product, the complete genome of the apoptolidin producer Nocardiopsis sp. FU40 was sequenced and a 116 Kb region was identified containing a putative apoptolidin biosynthetic gene cluster. The apoptolidin gene cluster comprises a type I polyketide synthase, with 13 homologating modules, apparently initiated in an unprecedented fashion via transfer from a methoxymalonyl-acyl carrier protein loading module. Spanning approximately 39 open reading frames, the gene cluster was cloned into a series of overlapping cosmids and functionally validated by targeted gene disruption experiments in the producing organism. Disruption of putative PKS and P(450) genes delineated the roles of these genes in apoptolidin biosynthesis and chemical complementation studies demonstrated intact biosynthesis peripheral to the disrupted genes. This work provides insight into details of the biosynthesis of this biologically significant natural product and provides a basis for future mutasynthetic methods for the generation of non-natural apopotolidins.