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C.P. acknowledges the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S018069/1; BB/W019795/1) for funding. cwEPR data were collected at the Pliotas Laboratory (BB/S018069/1) and PELDOR data were collected at the University of St Andrews, supported by equipment funding from the Wellcome Trust (WT) (099149/Z/12/Z) and BBSRC (BB/R013780/1). CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and the WT (108466/Z/15/Z; 221524/Z/20/Z). J.E.H. and K.F. acknowledge funding from the Medical Research Council (MRC) (MR/P018491/1). S.F.H. is funded by the White Rose BBSRC DTP (BB/M011151/1). J.M. is funded by the WT (222373/Z/21/Z). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). T.F.S and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centrefor Infection Research (DZIF) 09.918) The Β-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. Publisher PDF

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 polyketides, termed veramycins, were isolated from a Streptomyces sp. from the Sanofi microbial strain collection along with their known congeners NFAT-133 and TM-123. Veramycin A, an α-pyrone congener of TM-123 and NFAT-133 showed an increased baseline deoxy-glucose uptake in the absence of insulin in a modified L6 rat skeletal muscle cell line (L6 GLUT4 AS160-like cells). In addition, both compounds slightly increased the sensitivity to insulin in this cell line. Total syntheses of NFAT-133, TM-123 and veramycin A were accomplished starting from a central building block, which bears the three contiguous stereogenic centers of this polyketide family. Our approach enables an efficient, selective and flexible access to all possible isomers of the stereotriad for further exploration of this series as a potential anti-diabetic lead structure as exemplified by the synthesis of an NFAT-133 epimer. Finally, the corresponding biosynthetic gene cluster (BGC) was identified by genome sequencing and gene inactivation. Based on feeding experiments, a biosynthetic pathway was proposed, which enabled access to new veramycin A analogs by precursor-directed biosynthesis.

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.

International audience; 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,β-desaturation. Based on these observations, possible mechanisms of the DarE-catalyzed reactions are discussed.

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.

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.

Investigations on the biochemical relationship between Doriprismatica stellata (Chromodorididae, Doridoidea) nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina (Demospongiae, Porifera) led to the isolation of the structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin, with an unprecedented position of the cyclopropane ring annelated to the ring A. Unlike other scalaranes, which are most often functionalized at C-12 of ring C, it bears two acetoxy groups at C-11 and C-24 instead. The compound was present in all three samples, supporting the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. The results also indicate that D. stellata passes the scalarane metabolite on to its egg ribbons, most likely for protective purposes. The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32).

Indonesia is famous for its underwater biodiversity, which attracts many tourists, especially divers. This is also true for Sangihe Islands Regency, an area composed of several islands in the northern part of North Sulawesi. However, Sangihe Islands Regency is much less known than, e.g., Bunaken National Park (BNP, North Sulawesi). The main island, Sangihe, has recently experienced an increase in tourism and mining activities with potentially high impact on the environment. Recently, monitoring projects began around BNP using marine Heterobranchia as indicators for coral reef health. No information about this taxon exists from the remote islands in North Sulawesi. The present study represents the first monitoring study ever and focuses on marine Heterobranchia around Sangihe. In total, 250 specimens were collected, which could be assigned to Sacoglossa (3), Anthobranchia (19), and Cladobranchia (1). Despite the low number (23 versus 172 in BNP), at least eight species (35%) are not recorded from BNP, probably indicating differences in habitat, but also influence of a strong El Ni&ntilde, o year in 2016. Here we also report for the first time a Chromodoris annae specimen mimicking C. elisabethina, and the discovery of a new Phyllidia species.

We describe a new species, Moridilla jobeli sp. nov., belonging to the marine heterobranch group Aeolidioidea. Up to now, it is only recorded from Bunaken National Park, North Sulawesi, Indonesia. A combination of histological, computer tomographic and scanning electron microscopic methods was applied in order to describe and illustrate the anatomy of Moridilla jobeli sp. nov. in detail. Furthermore, we conducted molecular analyses which include available partial COI and 16S rRNA sequences, as well as the nuclear gene Histone 3 (H3) of Facelinidae and Aeolidiidae. NeighborNet analyses, species delimitation tests and phylogenetic reconstruction methods show the distinctiveness of the new species from the type species Moridilla brockii Bergh, 1888 and the two recently described species Moridilla fifo Carmona & Wilson, 2018 and Moridilla hermanita Carmona & Wilson, 2018, as well as the monophyly of the genus. A phylogenetic analysis of the Facelinidae and Aeolidiidae does not result in a resolved tree, therefore relationship of former assumed closely related genera, Noumeaella Risbec, 1937 and Palisa Edmunds, 1964, cannot be discussed in detail.

Overexpression of a putative type III polyketide synthase (PKSIII) from the marine myxobacterium Enhygromyxa salina SWB007 in Streptoinyces coelicolor MI 146 led to the accumulation of a novel monoketopiperazine consisting of phenylalanine and isoleucine. This compound was named phileucin and shows high structural similarity to phevalin (aureusimine B). The protease inhibiting activity was tested against human cathepsin L, human leukocyte elastase; bovine trypsin and bovine chymotrypsin. In contrast to phevalin, no protease inhibition was observed.

Background Bunaken National Park (BNP) is one of the most famous marine national parks in Indonesia with an extraordinary diversity in marine life forms. However, this diversity is threatened by an increasing population on the islands, ongoing destructive fishing techniques and lately by an increase in tourism. Protecting and managing the future use of BNP resources will require the assessment of both, local marine biodiversity through monitoring efforts and the identification and subsequent reduction of any threats or changes in the park. A high diversity in marine Heterobranchia indicates a high diversity of metazoan life forms and a diverse habitat structure. Surveying the complete biological diversity across taxonomic groups found in BNP would be an extensive undertaking, so focus on heterobranch diversity as an indicator of coral reef health was initiated and a model group on which future monitoring and conservation efforts can be based is provided. This study follows up the first investigation of marine Heterobranchia in BNP, conducted 12 years ago, while assessing molluscan diversity, and intends to present a base line for future monitoring programs. Results The diversity of marine heterobranchs around BNP was surveyed with an emphasis on Bunaken Island by diving and snorkeling at nearly 20 sites. Species are listed with photographic documentation (81 species) and results compared with the former study on molluscan species diversity in BNP. Taking these two studies into account 135 species are now recorded from BNP. The low overlap of described species (21) between the two BNP studies illustrates the gap of knowledge about overall species diversity in this particular area. A comparison with other studies from the region and Indo-Pacific also provides evidence for undersampling, but show similar taxa composition except of a somewhat higher cladobranch number in relation to Anthobranchia. Conclusions BNP is still under-sampled with regard to sea slug diversity. Thus conclusions as to whether or not a shift in species has occurred during the 12 years since the first study cannot be drawn. More and extensive studies are necessary to completely document the species richness in this area.

Xylanases are in the focus of research due to their potential to replace many current polluting chemical technologies by biochemical conversion. The field of application for xylanases is vast; it comprises industrial applications like wood pulp bio-bleaching, papermaking and bioethanol production. In addition, these enzymes can be applied as additives in food and beverage industry, and animal nutrition. However, considering the potential applications for these enzymes, the market share of xylanases is still low. Thus, the search for promising xylanases which tolerate relevant processing conditions and therefore can be used in industrial settings is an ongoing task. This review provides an overview of the enzymes reported from 2012 to mid 2014. Further, legal restrictions for the use of (genetically modified) organisms and enzymes are considered. This review provides an integrated perspective on the potential of specific xylanases for industrial applications.

Overexpression of a putative type III polyketide synthase (PKSIII) from the marine myxobacterium Enhygromyxa salina SWB007 in Streptomyces coelicolor M1146 led to the accumulation of a novel monoketopiperazine consisting of phenylalanine and isoleucine. This compound was named phileucin and shows high structural similarity to phevalin (aureusimine B). The protease inhibiting activity was tested against human cathepsin L, human leukocyte elastase, bovine trypsin and bovine chymotrypsin. In contrast to phevalin, no protease inhibition was observed.

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.