Author Correction: A new antibiotic selectively kills Gram-negative pathogens.

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Cofunctioning of bacterial exometabolites drives root microbiota establishment.

Soil-dwelling microbes are the principal inoculum for the root microbiota, but our understanding of microbe-microbe interactions in microbiota establishment remains fragmentary. We tested 39,204 binary interbacterial interactions for inhibitory activities in vitro, allowing us to identify taxonomic signatures in bacterial inhibition profiles. Using genetic and metabolomic approaches, we identified the antimicrobial 2,4-diacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites whose combined functions explain most of the inhibitory activity of the strongly antagonistic Pseudomonas brassicacearum R401. Microbiota reconstitution with a core of Arabidopsis thaliana root commensals in the presence of wild-type or mutant strains revealed a root niche-specific cofunction of these exometabolites as root competence determinants and drivers of predictable changes in the root-associated community. In natural environments, both the corresponding biosynthetic operons are enriched in roots, a pattern likely linked to their role as iron sinks, indicating that these cofunctioning exometabolites are adaptive traits contributing to pseudomonad pervasiveness throughout the root microbiota.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells.

The ß-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

Characterization of a Radical SAM Oxygenase for the Ether Crosslinking in Darobactin Biosynthesis.

Darobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C-C crosslinking. However, the specific mechanism of the crosslink formation, especially the ether crosslink, remains elusive. Here, using in vitro enzyme assays, we demonstrate that both crosslinks are formed by the DarE radical S-adenosylmethionine (SAM) enzyme in an O2-dependent manner. The relevance of the observed activity to darobactin A biosynthesis was demonstrated by proteolytic transformation of the DarE product into darobactin A. Furthermore, DarE assays in the presence of 18O2 or [18O]water demonstrated that the oxygen of the ether crosslink originates from O2 and not from water. These results demonstrate that DarE is a radical SAM enzyme that uses oxygen as a co-substrate in its physiologically relevant function. Since radical SAM enzymes are generally considered to function under anaerobic environments, the discovery of a radical SAM oxygenase represents a significant change in the paradigm and suggests that these radical SAM enzymes function in aerobic cells. Also, the study revealed that DarE catalyzes the formation of three distinct modifications on DarA; ether and C-C crosslinks and α,ß-desaturation. Based on these observations, possible mechanisms of the DarE-catalyzed reactions are discussed.

Bioactive natural products from Bacteroidetes.

Covering: up to end of January 2022Bacteria representing the phylum Bacteroidetes produce a diverse range of natural products, including polyketides, peptides and lactams. Here, we discuss unique aspects of the bioactive compounds discovered thus far, and the corresponding biosynthetic pathways if known, providing a comprehensive overview of the Bacteroidetes as a natural product reservoir.

Trichoderma-Derived Pentapeptides from the Infected Nest Mycobiome of the Subterranean Termite Coptotermes testaceus.

Termites live in a dynamic environment where colony health is strongly influenced by surrounding microbes. However, little is known about the mycobiomes of lower termites and their nests, and how these change in response to disease. Here we compared the individual and nest mycobiomes of a healthy subterranean termite colony (Coptotermes testaceus) to one infected and ultimately eradicated by a fungal pathogen. We identified Trichoderma species in the materials of both nests, but they were also abundant in the infected termites. Methanolic extracts of Trichoderma sp. FHG000531, isolated from the infected nest, were screened for secondary metabolites by UHPLC-HR MS/MS-guided molecular networking. We identified many bioactive compounds with potential roles in the eradication of the infected colony, as well as a cluster of six unknown peptides. The novel peptide FE011 was isolated and characterized by NMR spectroscopy. The function of this novel peptide family as well as the role of Trichoderma species in dying termite colonies therefore requires further investigation.

Discovery and Biosynthesis of Antimicrobial Phenethylamine Alkaloids from the Marine Flavobacterium Tenacibaculum discolor sv11.

The bacterial genus Tenacibaculum has been associated with various ecological roles in marine environments. Members of this genus can act, for example, as pathogens, predators, or episymbionts. However, natural products produced by these bacteria are still unknown. In the present work, we investigated a Tenacibaculum strain for the production of antimicrobial metabolites. Six new phenethylamine (PEA)-containing alkaloids, discolins A and B (1 and 2), dispyridine (3), dispyrrolopyridine A and B (4 and 5), and dispyrrole (6), were isolated from media produced by the predatory bacterium Tenacibaculum discolor sv11. Chemical structures were elucidated by analysis of spectroscopic data. Alkaloids 4 and 5 exhibited strong activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600, and Staphylococcus aureus ATCC25923, with minimum inhibitory concentration (MIC) values ranging from 0.5 to 4 µg/mL, and moderate activity against Candida albicans FH2173 and Aspergillus flavus ATCC9170. Compound 6 displayed moderate antibacterial activities against Gram-positive bacteria. Dispyrrolopyridine A (4) was active against efflux pump deficient Escherichia coli ATCC25922 ΔtolC, with an MIC value of 8 µg/mL, as well as against Caenorhabditis elegans N2 with an MIC value of 32 µg/mL. Other compounds were inactive against these microorganisms. The biosynthetic route toward discolins A and B (1 and 2) was investigated using in vivo and in vitro experiments. It comprises an enzymatic decarboxylation of phenylalanine to PEA catalyzed by DisA, followed by a nonenzymatic condensation to form the central imidazolium ring. This spontaneous formation of the imidazolium core was verified by means of a synthetic one-pot reaction using the respective building blocks. Six additional strains belonging to three Tenacibaculum species were able to produce discolins, and several DisA analogues were identified in various marine flavobacterial genera, suggesting the widespread presence of PEA-derived compounds in marine ecosystems.

Genome-Mining-Guided Discovery and Characterization of the PKS-NRPS-Hybrid Polyoxyperuin Produced by a Marine-Derived Streptomycete.

The azinothricin family comprises several cyclic hexadepsipeptides with diverse pharmacological bioactivities, including antimicrobial, antitumoral, and apoptosis induction. In this work, using a genome mining approach, a biosynthetic gene cluster encoding an azinothricin-like compound was identified from the Streptomyces sp. s120 genome sequence (pop BGC). Comparative MS analysis of extracts from the native producer and a knockout mutant led to the identification of metabolites corresponding to the pop BGC. Furthermore, regulatory elements of the BGC were identified. By overexpression of an LmbU-like transcriptional activator, the production yield of 1 and 2 was increased, enabling isolation and structure elucidation of polyoxyperuin A seco acid (1) and polyoxyperuin A (2) using high-resolution mass spectrometry and NMR spectroscopy. Compound 1 exhibited a low antibiotic effect against Micrococcus luteus, while 2 showed a strong Gram-positive antibiotic effect in a micro-broth-dilution assay.

Seven New Alkaloids Isolated from Marine Flavobacterium Tenacibaculum discolor sv11.

Marine flavobacterium Tenacibaculum discolor sv11 has been proven to be a promising producer of bioactive nitrogen-containing heterocycles. A chemical investigation of T. discolor sv11 revealed seven new heterocycles, including the six new imidazolium-containing alkaloids discolins C-H (1−6) and one pyridinium-containing alkaloid dispyridine A (7). The molecular structure of each compound was elucidated by analysis of NMR and HR-ESI-MS data. Furthermore, enzymatic decarboxylation of tryptophan and tyrosine to tryptamine and tyramine catalyzed by the decarboxylase DisA was investigated using in vivo and in vitro experiments. The antimicrobial activity of the isolated compounds (1−7) was evaluated. Discolin C and E (1 and 3) exhibited moderate activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600 and Staphylococcus aureus ATCC25923, with MIC values ranging from 4 µg/mL to 32 µg/mL.

Discovery of Marine Natural Products as Promising Antibiotics against Pseudomonas aeruginosa.

Pseudomonas aeruginosa, one of the most intractable Gram-negative bacteria, has become a public health threat due to its outer polysaccharide layer, efflux transporter system, and high level of biofilm formation, all of which contribute to multi-drug resistance. Even though it is a pathogen of the highest concern, the status of the antibiotic development pipeline is unsatisfactory. In this review, we summarize marine natural products (MNPs) isolated from marine plants, animals, and microorganisms which possess unique structures and promising antibiotic activities against P. aeruginosa. In the last decade, nearly 80 such MNPs, ranging from polyketides to alkaloids, peptides, and terpenoids, have been discovered. Representative compounds exhibited impressive in vitro anti-P. aeruginosa activities with MIC values in the single-digit nanomolar range and in vivo efficacy in infectious mouse models. For some of the compounds, the preliminary structure-activity-relationship (SAR) and anti-bacterial mechanisms of selected compounds were introduced. Compounds that can disrupt biofilm formation or membrane integrity displayed potent inhibition of multi-resistant clinical P. aeruginosa isolates and could be considered as lead compounds for future development. Challenges on how to translate hits into useful candidates for clinical development are also proposed and discussed.

Optimization of heterologous Darobactin A expression and identification of the minimal biosynthetic gene cluster.

Darobactin A (DAR) is a ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotic, which was initially identified from bacteria belonging to the genus Photorhabdus. In addition, the corresponding biosynthetic gene cluster (BGC) was identified and subsequently detected in several bacteria genera. DAR represents a highly promising lead structure for the development of novel antibacterial therapeutic agents. It targets the outer membrane protein BamA and is therefore specific for Gram-negative bacteria. This, together with the convincing in vivo activities in mouse infection models, makes it a particular promising candidate for further research. To improve compound supply for further investigation of DAR and to enable production of novel derivatives, establishment of an efficient and versatile microbial production platform for these class of RiPP antibiotics is highly desirable. Here we describe design and construction of a heterologous production and engineering platform for DAR, which will ensure production yield and facilitates structure modification approaches. The known Gram-negative workhorses Escherichia coli and Vibrio natriegens were tested as heterologous hosts. In addition to that, DAR producer strains were generated and optimization of the expression constructs yielded production titers of DAR showing around 10-fold increase and 5-fold decrease in fermentation time compared to the original product description. We also report the identification of the minimal DAR BGC, since only two genes were necessary for heterologous production of the RiPP.

In vitro characterization of 3-chloro-4-hydroxybenzoic acid building block formation in ambigol biosynthesis.

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.

A new antibiotic kills pathogens without detectable resistance.

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster.

Pseudouridimycin (PUM) was recently discovered from Streptomyces sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of Streptococcus pyogenes peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host Streptomyces coelicolor M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC.

Novel Glycerophospholipid, Lipo- and N-acyl Amino Acids from Bacteroidetes: Isolation, Structure Elucidation and Bioactivity.

The 'core' metabolome of the Bacteroidetes genus Chitinophaga was recently discovered to consist of only seven metabolites. A structural relationship in terms of shared lipid moieties among four of them was postulated. Here, structure elucidation and characterization via ultra-high resolution mass spectrometry (UHR-MS) and nuclear magnetic resonance (NMR) spectroscopy of those four lipids (two lipoamino acids (LAAs), two lysophosphatidylethanolamines (LPEs)), as well as several other undescribed LAAs and N-acyl amino acids (NAAAs), identified during isolation were carried out. The LAAs represent closely related analogs of the literature-known LAAs, such as the glycine-serine dipeptide lipids 430 (2) and 654. Most of the here characterized LAAs (1, 5-11) are members of a so far undescribed glycine-serine-ornithine tripeptide lipid family. Moreover, this study reports three novel NAAAs (N-(5-methyl)hexanoyl tyrosine (14) and N-(7-methyl)octanoyl tyrosine (15) or phenylalanine (16)) from Olivibacter sp. FHG000416, another Bacteroidetes strain initially selected as best in-house producer for isolation of lipid 430. Antimicrobial profiling revealed most isolated LAAs (1-3) and the two LPE 'core' metabolites (12, 13) active against the Gram-negative pathogen M. catarrhalis ATCC 25238 and the Gram-positive bacterium M. luteus DSM 20030. For LAA 1, additional growth inhibition activity against B. subtilis DSM 10 was observed.

Halogenation-Guided Chemical Screening Provides Insight into Tjipanazole Biosynthesis by the Cyanobacterium Fischerella ambigua.

Halogenated natural products (HNPs) show a wide range of interesting biological activities. Chemistry-guided screening with a software tool dedicated to identifying halogenated compounds in HPLC-MS data indicated the presence of several uncharacterised HNPs in an extract of the cyanobacterium Fischerella ambigua (Näg.) Gomont 108b. Three new natural products, tjipanazoles K, L, and M, were isolated from this strain together with the known tjipanazoles D and I. Taking into account the structures of all tjipanazole derivatives detected in this strain, reanalysis of the tjipanazole biosynthetic gene cluster allowed us to propose a biosynthetic pathway for the tjipanazoles. As the isolated tjipanazoles show structural similarity to arcyriaflavin A, an inhibitor of the clinically relevant multidrug-transporter ABCG2 overexpressed by different cancer cell lines, the isolated compounds were tested for ABCG2 inhibitory activity. Only tjipanazole K showed appreciable transporter inhibition, whereas the compounds lacking the pyrrolo[3,4-c] ring or featuring additional chloro substituents were found to be much less active.

Quintaquinone, a Merosesquiterpene from the Yellow Sponge Verongula cf. rigida Esper.

A chemical investigation of the sponge Verongula cf. rigida led to the isolation of 13 merosesquiterpenes, among which quintaquinone (2), 5-epi-nakijiquinone L (3), and 3-farnesyl-2-hydroxy-5-methoxyquinone (4) were isolated and reported here for the first time. Particularly, compound 2 is the first member of merosesquiterpenes with a polyketide side chain substituted on C-19. All of the isolated compounds were examined for steroid 5α-reductase inhibitory activity. Cyclospongiaquinone 1 (5) showed a strong activity in the same range as that of standard finasteride.

Molecular Networking-Guided Discovery and Characterization of Stechlisins, a Group of Cyclic Lipopeptides from a Pseudomonas sp.

Increasingly sensitive analytical instruments and robust downstream data processing tools have revolutionized natural product research over the past decade. A molecular networking-guided survey led to the identification of 33 new cyclic lipopeptides (CLPs) from the culture broth of the proteobacterium Pseudomonas sp. FhG100052. The compound family resembles members of the amphisin group of CLPs that possess a 3-hydroxy fatty acid linked to the N-terminus of an undecapeptide core. Culture optimization led to the isolation and subsequent structure elucidation of one known and five new derivatives by extensive MS/MS and NMR experiments in combination with Marfey's analysis. The data were in agreement with in silico analysis of the corresponding biosynthetic gene cluster. Most strikingly, the length of the incorporated fatty acid defined the growth inhibitory effects against Moraxella catarrhalis FH6810, as observed by MIC values ranging from no inhibition (>128 µg/mL) to 4 µg/mL.

Sustainable Low-Volume Analysis of Environmental Samples by Semi-Automated Prioritization of Extracts for Natural Product Research (SeaPEPR).

The discovery of novel natural products (NPs) that will serve as lead structures has to be an ongoing effort to fill the respective development pipelines. However, identification of NPs, which possess a potential for application in e.g., the pharma or agro sector, must be as cost effective and fast as possible. Furthermore, the amount of sample available for initial testing is usually very limited, not least because of the fact that the impact on the environment, i.e., the sampled biosystem, should be kept minimal. Here, our pipeline SeaPEPR is described, in which a primary bioactivity screening of crude extracts is combined with the analysis of their metabolic fingerprint. This enabled prioritization of samples for subsequent microfractionation and dereplication of the active compounds early in the workflow. As a case study, 76 marine sponge-derived extracts were screened against a microbial screening panel. Thereunder, human pathogenic bacteria (Escherichia coli ATCC35218 and Staphylococcus aureus ATCC33592) and yeast (Candida albicans FH2173), as well as the phytopathogenic fungus Septoria tritici MUCL45407. Overall, nine extracts revealed activity against at least one test organism. Metabolic fingerprinting enabled assigning four active extracts into one metabolic group; therefore, one representative was selected for subsequent microfractionation. Dereplication of the active fractions showed a new dibrominated aplysinopsin and a hypothetical chromazonarol stereoisomer derivative. Furthermore, inhibitory activity against the common plant pest Septoria tritici was discovered for NPs of marine origin.