Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Bioinformatics-guided discovery of biaryl-linked lasso peptides.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Using mass spectrometry, stable isotope incorporation, and extensive 2D-NMR spectrometry, we report the structural characterization of two unique examples of (C-N) biaryl-linked lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with a Trp-Tyr crosslink, while longipepetin A, from Longimycelium tulufanense, features a Trp-Trp linkage. Besides the unusual bicyclic frame, a Met of longipepetin A undergoes S-methylation to yield a trivalent sulfonium, a heretofore unprecedented RiPP modification. A bioinformatic survey revealed additional lasso peptide BGCs containing P450 enzymes which await future characterization. Lastly, nocapeptin A bioactivity was assessed against a panel of human and bacterial cell lines with modest growth-suppression activity detected towards Micrococcus luteus.

Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms.

The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum. Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Moreover, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum results in quantitatively lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

Genome Sequences of the Two Telluria Type Strains, Telluria chitinolytica ACM 3522T and Telluria mixta DSM 29330T.

High-quality draft genome sequences were obtained for the two type strains Telluria chitinolytica ACM 3522T and Telluria mixta DSM 29330T. The genomes of both strains show a considerable biosynthetic potential to produce secondary metabolites.

Bioinformatics-Guided Discovery of Biaryl-Tailored Lasso Peptides.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.

Heme b inhibits class III adenylyl cyclases.

Acidic lipid extracts from mouse liver, kidney, heart, brain, and lung inhibited human pseudoheterodimeric adenylyl cyclases (hACs) expressed in HEK293 cells. Using an acidic lipid extract from bovine lung, a combined MS- and bioassay-guided fractionation identified heme b as inhibitor of membrane-bound ACs. IC50 concentrations were 8-12 µM for the hAC isoforms. Hemopexin and bacterial hemophore attenuated heme b inhibition of hAC5. Structurally related compounds, such as hematin, protoporphyrin IX, and biliverdin, were significantly less effective. Monomeric bacterial class III ACs (mycobacterial ACs Rv1625c; Rv3645; Rv1264; cyanobacterial AC CyaG) were inhibited by heme b with similar efficiency. Surprisingly, structurally related chlorophyll a similarly inhibited hAC5. Heme b inhibited isoproterenol-stimulated cAMP accumulation in HEK293 cells. Using cortical membranes from mouse brain hemin efficiently and reversibly inhibited basal and Gsα-stimulated AC activity. The physiological relevance of heme b inhibition of the cAMP generating system in certain pathologies is discussed.

Molecular Network Guided Cataloging of the Secondary Metabolome of Selected Egyptian Red Sea Soft Corals.

Soft corals are recognized as an abundant source of diverse secondary metabolites with unique chemical features and physiologic capabilities. However, the discovery of these metabolites is usually hindered by the traditional protocol which requires a large quantity of living tissue for isolation and spectroscopic investigations. In order to overcome this problem, untargeted metabolomics protocols have been developed. The latter have been applied here to study the chemodiversity of common Egyptian soft coral species, using only minute amounts of coral biomass. Spectral similarity networks, based on high-resolution tandem mass spectrometry data, were employed to explore and highlight the metabolic biodiversity of nine Egyptian soft coral species. Species-specific metabolites were highlighted for future prioritization of soft coral species for MS-guided chemical investigation. Overall, 79 metabolites were tentatively assigned, encompassing diterpenes, sesquiterpenes, and sterols. Simultaneously, the methodology assisted in shedding light on newly-overlooked chemical diversity with potential undescribed scaffolds. For instance, glycosylated fatty acids, nitrogenated aromatic compounds, and polyketides were proposed in Sinularia leptoclados, while alkaloidal terpenes and N-acyl amino acids were proposed in both Sarcophyton roseum and Sarcophyton acutum.

Discovery of Ircinianin Lactones B and C-Two New Cyclic Sesterterpenes from the Marine Sponge Ircinia wistarii.

Two new ircinianin-type sesterterpenoids, ircinianin lactone B and ircinianin lactone C (7 and 8), together with five known entities from the ircinianin compound family (1, 3-6) were isolated from the marine sponge Ircinia wistarii. Ircinianin lactones B and C (7 and 8) represent new ircinianin terpenoids with a modified oxidation pattern. Despite their labile nature, the structures could be established using a combination of spectroscopic data, including HRESIMS and 1D/2D NMR techniques, as well as computational chemistry and quantum-mechanical calculations. In a broad screening approach for biological activity, the class-defining compound ircinianin (1) showed moderate antiprotozoal activity against Plasmodium falciparum (IC50 25.4 µM) and Leishmania donovani (IC50 16.6 µM).

Versatile synthesis of pathogen specific bacterial cell wall building blocks.

Full details on the design, strategies and tactics for development of a novel synthetic sequence to farnesyl lipid I and II analogs is reported. The modular route was based on a three coupling strategy involving an efficient solid phase synthesis of the elaborate peptide fragment, which proceeded with excellent yield and stereoselectivity and was efficiently applied for the convergent synthesis of 3-lipid I and II. Furthermore, the generality of this route was demonstrated by synthesis of 3-lipid I congeners that are characteristic for S. aureus and E. faecalis. All 3-lipid I and II building blocks were obtained in high purity revealing high spectroscopic resolution.

Mycothiol Peroxidase Activity as a Part of the Self-Resistance Mechanisms against the Antitumor Antibiotic Cosmomycin D.

Antibiotic-producing microorganisms usually require one or more self-resistance determinants to survive antibiotic production. The effectors of these mechanisms are proteins that inactivate the antibiotic, facilitate its transport, or modify the target to render it insensitive to the molecule. Streptomyces bacteria biosynthesize various bioactive natural products and possess resistance systems for most metabolites, which are coregulated with antibiotic biosynthesis genes. Streptomyces olindensis strain DAUFPE 5622 produces the antitumor antibiotic cosmomycin D (COSD), a member of the anthracycline family. In this study, we propose three self-resistance mechanisms, anchored or based in the COSD biosynthetic gene cluster. These include cosIJ (an ABC transporter), cosU (a UvrA class IIa protein), and a new self-resistance mechanism encoded by cosP, which shows response against peroxides by the enzyme mycothiol peroxidase (MPx). Activity-based investigations of MPx and its mutant enzyme confirmed peroxidation during the production of COSD. Overexpression of the ABC transporter, the UvrA class IIa protein, and the MPx led to an effective response against toxic anthracyclines, such as cosmomycins. Our findings help to understand how thiol peroxidases play an antioxidant role in the anthracycline producer S. olindensis DAUFPE 5622, a mechanism which has been reported for neoplastic cells that are resistant to doxorubicin (DOX). IMPORTANCE Anthracycline compounds are DNA intercalating agents widely used in cancer chemotherapeutic protocols. This work focused on the self-resistance mechanisms developed by the cosmomycin-producing bacterium Streptomyces olindensis. Our findings showed that cysteine peroxidases, such as mycothiol peroxidase, encoded by the gene cosP, protected S. olindensis against peroxidation during cosmomycin production. This observation can contribute to much better understanding of resistance both in the producers, eventually enhancing production, and in some tumoral cell lines.

High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin.

A chemical reinvestigation of the Indonesian strain Streptomyces sp. SHP 22-7 led to the isolation of three new pyrimidine nucleosides, along with six known analogues and zincphyrin. The structures of the new compounds (6, 7, 10) were elucidated by employing spectroscopic techniques (NMR, MS, CD, and IR) as well as enantioselective analyses of methyl branched side chain configurations. Application of the precursor-directed feeding approach led to the production and partial isolation of nine further pyrimidine analogues. The new compounds 6, 7, and 11 and three of the known compounds (2-4) were found to possess antimycobacterial and cytotoxic properties.

Pseudomonas Lipopeptide-Mediated Biocontrol: Chemotaxonomy and Biological Activity.

Pseudomonas lipopeptides (Ps-LPs) play crucial roles in bacterial physiology, host-microbe interactions and plant disease control. Beneficial LP producers have mainly been isolated from the rhizosphere, phyllosphere and from bulk soils. Despite their wide geographic distribution and host range, emerging evidence suggests that LP-producing pseudomonads and their corresponding molecules display tight specificity and follow a phylogenetic distribution. About a decade ago, biocontrol LPs were mainly reported from the P. fluorescens group, but this has drastically advanced due to increased LP diversity research. On the one hand, the presence of a close-knit relationship between Pseudomonas taxonomy and the molecule produced may provide a startup toolbox for the delineation of unknown LPs into existing (or novel) LP groups. Furthermore, a taxonomy-molecule match may facilitate decisions regarding antimicrobial activity profiling and subsequent agricultural relevance of such LPs. In this review, we highlight and discuss the production of beneficial Ps-LPs by strains situated within unique taxonomic groups and the lineage-specificity and coevolution of this relationship. We also chronicle the antimicrobial activity demonstrated by these biomolecules in limited plant systems compared with multiple in vitro assays. Our review further stresses the need to systematically elucidate the roles of diverse Ps-LP groups in direct plant-pathogen interactions and in the enhancement of plant innate immunity.

Towards enantioselective ultrahigh performance liquid chromatography-mass spectrometry-based metabolomics of branched-chain fatty acids and anteiso-fatty acids under reversed-phase conditions using sub-2-µm amylose- and cellulose-derived chiral stationary phases.

Branched-chain fatty acids (BCFAs) are mostly saturated fatty acids with one or more methyl, seldom ethyl, branches in the alkyl chain. They are derived from branched-chain amino acids, ruminant-derived food, or biosynthetic side products of acetyl-CoA carboxylase. They possess iso- (branching at penultimate carbon) and anteiso-fatty acid structure (branching at antepenultimate carbon) or are branched at any other position of the carbon chain. Except for iso-fatty acids, BCFAs are chiral. They are commonly analyzed by GC-MS, while there is a lack of enantioselective LC-MS methods. In this work, we present a methodology for targeted enantioselective UHPLC-ESI-MS/MS metabolomics of BCFAs. It makes use of precolumn derivatization with 1-naphthylamine and reversed-phase elution conditions. A homologous series of short BCFA analytes with distinct chain lengths (having up to eight carbon atoms), branching type (methyl or ethyl), and position of branching (2, 3, and 4, anteiso and iso) has been systematically studied on six commercially available polysaccharide UHPLC columns. Chiralpak IB-U exhibited the highest and broadest enantioselectivity while IH-U maintained enantioselectivity also for BCFAs with chirality distant from the carboxylic function (i.e., with other branching than in 2-position). The method was used to assign the absolute configuration of a 4-methylhexanoic acid side chain of a natural product from Streptomyces sp. SHP 22-7. The potential of the corresponding UHPLC-ESI-QTOF-MS/MS assay for analyzing stereoselectively BCFAs and other short organic acids by untargeted analysis in human urine was further elucidated in a preliminary proof-of-principle test.

Burkholderia in the genomic era: from taxonomy to the discovery of new antimicrobial secondary metabolites.

Species of Burkholderia are highly versatile being found not only abundantly in soil, but also as plants and animals' commensals or pathogens. Their complex multireplicon genomes harbour an impressive number of polyketide synthase (PKS) and nonribosomal peptide-synthetase (NRPS) genes coding for the production of antimicrobial secondary metabolites (SMs), which have been successfully deciphered by genome-guided tools. Moreover, genome metrics supported the split of this genus into Burkholderia sensu stricto (s.s.) and five new other genera. Here, we show that the successful antimicrobial SMs producers belong to Burkholderia s.s. Additionally, we reviewed the occurrence, bioactivities, modes of action, structural, and biosynthetic information of thirty-eight Burkholderia antimicrobial SMs shedding light on their diversity, complexity, and uniqueness as well as the importance of genome-guided strategies to facilitate their discovery. Several Burkholderia NRPS and PKS display unusual features, which are reflected in their structural diversity, important bioactivities, and varied modes of action. Up to now, it is possible to observe a general tendency of Burkholderia SMs being more active against fungi. Although the modes of action and biosynthetic gene clusters of many SMs remain unknown, we highlight the potential of Burkholderia SMs as alternatives to fight against new diseases and antibiotic resistance.

Genome Sequence of Lysobacter sp. Strain BMK333-48F3, the Producer Strain of Potent Lipopeptide Antibiotics of the Tripropeptin Family.

Lysobacter sp. strain BMK333-48F3 is known primarily for its production of the antibiotically active tripropeptins. Here, we report its draft genome sequence, which will give insight into the biosynthesis of tripropeptins and enable genome mining for further secondary metabolites.

Genome Sequence of Escherichia coli Stbl4, a Versatile Genetic Tool for Heterologous Expression.

Escherichia coli Stbl4 is widely used as a laboratory strain for heterologous expression of large gene clusters. Since no genome sequence has been publicly available, we here report the draft sequence of Stbl4, including its F-plasmid. It should serve as a useful reference for researchers working with Stbl4.

Draft Genome Sequence of the Halophilic Strain Citrobacter braakii AN-PRR1, Isolated from Rhizospheric Soil of Rice (Oryza sativa L.) from Pakistan.

Citrobacter braakii AN-PRR1 is a potential salt-tolerant, plant growth-promoting rice rhizobacterium isolated from Pakistani soil. The 4.9-Mb draft genome sequence contributes to its taxonomic classification and will reveal the genes putatively responsible for its osmoprotectant and plant growth-promoting activity.

Mining Indonesian Microbial Biodiversity for Novel Natural Compounds by a Combined Genome Mining and Molecular Networking Approach.

Indonesia is one of the most biodiverse countries in the world and a promising resource for novel natural compound producers. Actinomycetes produce about two thirds of all clinically used antibiotics. Thus, exploiting Indonesia's microbial diversity for actinomycetes may lead to the discovery of novel antibiotics. A total of 422 actinomycete strains were isolated from three different unique areas in Indonesia and tested for their antimicrobial activity. Nine potent bioactive strains were prioritized for further drug screening approaches. The nine strains were cultivated in different solid and liquid media, and a combination of genome mining analysis and mass spectrometry (MS)-based molecular networking was employed to identify potential novel compounds. By correlating secondary metabolite gene cluster data with MS-based molecular networking results, we identified several gene cluster-encoded biosynthetic products from the nine strains, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Moreover, 16 putative ion clusters and numerous gene clusters were detected that could not be associated with any known compound, indicating that the strains can produce novel secondary metabolites. Our results demonstrate that sampling of actinomycetes from unique and biodiversity-rich habitats, such as Indonesia, along with a combination of gene cluster networking and molecular networking approaches, accelerates natural product identification.