Draft genome sequence of Galbibacter sp. PAP.153, isolated from a marine sponge in Papua, Indonesia.

Galbibacter sp. PAP.153 was isolated from a marine sponge. Here, we report its 4.12 Mbp draft genome sequence and rate its specialized metabolite production capacity with specific focus on the chemotaxonomic marker flexirubin.

Design, synthesis and antimycobacterial activity of imidazo[1,5-a]quinolines and their zinc-complexes.

Tuberculosis has remained one of the world's deadliest infectious diseases. The complexity and numerous adverse effects of current treatment options as well as the emergence of multi-drug resistant M. tuberculosis (Mtb) demand research and innovation efforts to yield new anti-mycobacterial agents. In this study, we synthesized a series of imidazo[1,5-a]quinolines, including 4 new analogs, and evaluated their activity against Mtb. Inspired by previous studies, we also designed 8 compounds featuring a coordinated metal ion, determined their absolute configuration by single-crystal X-ray diffraction and included them in the bioactivity study. Remarkably, the metal complexation of 5c with either Zn2+ or Fe2+ increased the Mtb inhibitory activity of the compound 12.5-fold and reduced its cytotoxicity. Ultimately, out of the 21 analyzed imidazo[1,5-a]quinoline analogs, two zinc complexes (C1 and C7) showed the strongest, specific activity against Mtb H37Rv in vitro (IC90 = 7.7 and 17.7 µM).

Physiochemical interaction between osmotic stress and a bacterial exometabolite promotes plant disease.

Various microbes isolated from healthy plants are detrimental under laboratory conditions, indicating the existence of molecular mechanisms preventing disease in nature. Here, we demonstrated that application of sodium chloride (NaCl) in natural and gnotobiotic soil systems is sufficient to induce plant disease caused by an otherwise non-pathogenic root-derived Pseudomonas brassicacearum isolate (R401). Disease caused by combinatorial treatment of NaCl and R401 triggered extensive, root-specific transcriptional reprogramming that did not involve down-regulation of host innate immune genes, nor dampening of ROS-mediated immunity. Instead, we identified and structurally characterized the R401 lipopeptide brassicapeptin A as necessary and sufficient to promote disease on salt-treated plants. Brassicapeptin A production is salt-inducible, promotes root colonization and transitions R401 from being beneficial to being detrimental on salt-treated plants by disturbing host ion homeostasis, thereby bolstering susceptibility to osmolytes. We conclude that the interaction between a global change stressor and a single exometabolite from a member of the root microbiome promotes plant disease in complex soil systems.

Draft genome sequences of 21 Pedobacter strains isolated from amphibian specimens.

The genomes of 21 Pedobacter strains isolated from the European salamander Salamandra salamandra and different Madagascan frog species were sequenced using Illumina sequencing. Here, we report their draft genome sequences (~4.7-7.2 Mbp in size) to allow comparative genomics and taxonomic assignment of these strains.

Functional in vitro and in vivo analysis of biosynthetic genes by heterologous expression in E. coli.

Biosynthetic gene clusters of natural products often harbor genes of unknown function, which are difficult to characterize. Here, we present a protocol for the functional analysis in vitro and in vivo of these biosynthetic genes by heterologous expression in E. coli. We describe steps for the expression of genes of interest in an established E. coli strain optimized to heterologously express natural products. We then detail the expression of a His-tagged gene to deduce the specific function of the protein. For complete details on the use and execution of this protocol, please refer to Böhringer et al.1.

Extracts of Talaromyces purpureogenus Strains from Apis mellifera Bee Bread Inhibit the Growth of Paenibacillus spp. In Vitro.

Honey bees coexist with fungi that colonize hive surfaces and pollen. Some of these fungi are opportunistic pathogens, but many are beneficial species that produce antimicrobial compounds for pollen conservation and the regulation of pathogen populations. In this study, we tested the in vitro antimicrobial activity of Talaromyces purpureogenus strains isolated from bee bread against Paenibacillus alvei (associated with European foulbrood disease) and three Aspergillus species that cause stonebrood disease. We found that methanol extracts of T. purpureogenus strains B18 and B195 inhibited the growth of P. alvei at a concentration of 0.39 mg/mL. Bioactivity-guided dereplication revealed that the activity of the crude extracts correlated with the presence of diketopiperazines, a siderophore, and three unknown compounds. We propose that non-pathogenic fungi such as Talaromyces spp. and their metabolites in bee bread could be an important requirement to prevent disease. Agricultural practices involving the use of fungicides can disrupt the fungal community and thus negatively affect the health of bee colonies.

Genome- and metabolome-guided discovery of marine BamA inhibitors revealed a dedicated darobactin halogenase.

Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.). While the organization of the BGC core is highly conserved, a small subset of Pseudoalteromonas carries an extended BGC with additional genes. Here, we report the identification of brominated and dehydrated darobactin derivatives from P. luteoviolacea strains. The marine derivatives are active against multidrug-resistant (MDR) Gram-negative bacteria and showed solubility and plasma protein binding ability different from darobactin A, rendering it more active than darobactin A. The halogenation reaction is catalyzed by DarH, a new class of flavin-dependent halogenases with a novel fold.

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.

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.

Draft Genome Sequence of Sinomicrobium sp. Strain PAP.21, Isolated from a Coast Sample of Papua, Indonesia.

Sinomicrobium sp. strain PAP.21 (EXT111902) was isolated from the coast of Cenderawasih Bay National Park in West Papua, Indonesia. Its genome was assembled into 151 contigs with a total size of 5.439 Mbp, enabling the prediction of its specialized metabolite production capacity.

Draft Genome Sequences of Algoriphagus sp. Strain PAP.12 and Roseivirga sp. Strain PAP.19, Isolated from Marine Samples from Papua, Indonesia.

Algoriphagus sp. strain PAP.12 (EXT111900) and Roseivirga sp. strain PAP.19 (EXT111901) were isolated from marine samples. Here, we report their draft genome sequences, 5.032 Mbp and 4.583 Mbp in size, respectively, and rate their specialized metabolite production capacity. Taxonomic ranks established by genome-based analysis indicate that Algoriphagus sp. strain PAP.12 represents a candidate new species.

Antimicrobial Activity of Ceftazidime-Avibactam, Ceftolozane-Tazobactam, Cefiderocol, and Novel Darobactin Analogs against Multidrug-Resistant Pseudomonas aeruginosa Isolates from Pediatric and Adolescent Cystic Fibrosis Patients.

The emergence and spread of antimicrobial resistance (AMR) in Gram-negative pathogens, such as carbapenem-resistant Pseudomonas aeruginosa, pose an increasing threat to health care. Patients with immunodeficiencies or chronic pulmonary disease, like cystic fibrosis (CF), are particularly vulnerable to Pseudomonas infections and depend heavily on antibiotic therapy. To broaden limited treatment options, this study evaluated the potency of the recently licensed drugs ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), and cefiderocol (FDC) as well as two novel preclinical antibiotics, darobactins B (DAR B) and B9 (DAR B9), against clinical P. aeruginosa isolates derived from respiratory samples of CF patients. We observed high levels of resistance to all three newly licensed drugs, with cefiderocol exhibiting the best activity. From the 66 investigated P. aeruginosa isolates, a total of 53% were resistant to CZA, 49% to C/T, and 30% to FDC. Strikingly, 52 of the evaluated isolates were obtained from CF patients prior to market introduction of the drugs. Thus, our results suggest that resistance to CZA, C/T, and FDC may be due to preexisting resistance mechanisms. On the other hand, our two novel preclinical compounds performed better than (CZA and C/T) or close to (FDC) the licensed drugs-most likely due to the novel mode of action. Thus, our results highlight the necessity of global consistency in the area of antibiotic stewardship to prevent AMR from further impairing the potency of antibiotics in clinical practice. Ultimately, this study demonstrates the urgency to support the development of novel antimicrobials, preferably with a new mode of action such as darobactins B and B9, two very promising antimicrobial compounds for the treatment of critically ill patients suffering from multidrug-resistant Gram-negative (MRGN) infections. IMPORTANCE Antimicrobial resistance (AMR) represents an ever increasing threat to the health care system. Even recently licensed drugs are often not efficient for the treatment of infections caused by Gram-negative bacteria, like Pseudomonas aeruginosa, a causative agent of lung infections. To address this unmet medical need, innovative antibiotics, which possess a new mode of action, need to be developed. Here, the antibiogram of clinical isolates derived from cystic fibrosis patients was generated and new bicyclic heptapeptides, which inhibit the outer membrane protein BamA, exhibited strong activity, also against multidrug-resistant isolates.

Production of Antimicrobial Compounds by Homologous and Heterologous Expression.

Natural product discovery campaigns aim to identify compounds with the desired bioactivity, for example, metabolites with antibiotic activity. The major driver of many projects is still the finding of bioactive extracts, which will be followed up to isolate the activity-causing agent as pure compound. However, nowadays also additional strategies can be used to increase the probability of success. Metabolomic approaches indicate chemical novelty, and genomics allow identification of putative biosynthetic gene clusters (BGCs) of interest, even though the corresponding metabolite is unknown. Whatever the entry to the campaign is, at one point the scientists need to have the desired compound in hand to analyze it in detail. Hence, expression must be achieved to yield the compound of interest, either to link it to the corresponding putative BGC or to overcome the bottleneck of sparse compound supply. Therefore, homologous and heterologous expression approaches are feasible ways forward to increase production yield, shorten fermentation time, or to get BGCs expressed at all for which no suitable fermentation condition was identified.In this chapter, expression approaches in bacteria are described to biosynthesize compounds of interest. Homologous expression, by genetic manipulation of the original Streptomyces producer strain, and heterologous expression in the microbial workhorse Escherichia coli are exemplified.

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.

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.

Isolation, characterization, and bioactivity profiling of oxazoline containing madurastatin siderophores from Actinomadura sp.

Nine new hydroxyphenyloxazolines, madurastatin B4, C2, D3 and D4, E1 and E2, F1 as well as G1 and G2 (8-16), along with two new enantiomers of madurastatin D1 (ent-6) and D2 (ent-7) and two known congeners, madurastatin B1 (2) and C1 (5), were isolated from the liquid culture of Actinomadura sp. ST100801 based on the initial activity against Escherichia coli screened in bicarbonate-supplemented Mueller Hinton II medium and identification via molecular networking. Structure elucidation was achieved by comprehensive 1D and 2D NMR as well as MS/MS fragmentation analyses. Their absolute configuration was determined by Marfey's analysis. Complemented with functionalized hydroxyphenyloxazolines (2, 4, 17-18) obtained by total synthesis, the isolated compounds were evaluated for antibacterial activities revealing MICs down to 4 µg ml-1 against Moraxella catarrhalis. Therefore, this study enlarges the family of madurastatin siderophores.

Genomic and Chemical Decryption of the Bacteroidetes Phylum for Its Potential to Biosynthesize Natural Products.

With progress in genome sequencing and data sharing, 1,000s of bacterial genomes are publicly available. Genome mining-using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis, and rating-has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Mining this data set, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively. IMPORTANCE The development of pipelines for anti-infectives to be applied in plant, animal, and human health management are dried up. However, the resistance development against compounds in use calls for new lead structures. To fill this gap and to enhance the probability of success for the discovery of new bioactive natural products, microbial taxa currently underinvestigated must be mined. This study investigates the potential within the bacterial phylum Bacteroidetes. A combination of omics-technologies revealed taxonomical hot spots for specialized metabolites. Genome- and metabolome-based analyses showed that the phylum covers a new chemical space compared with classic natural product producers. Members of the Bacteroidetes may thus present a promising bioresource for future screening and isolation campaigns.

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.