A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling.

Inspired by natural sideromycins, the conjugation of antibiotics to siderophores is an attractive strategy to facilitate "Trojan horse" delivery of antibiotics into bacteria. Genome analysis of a soil bacterium, Dactylosporangium fulvum, found a "hybrid" biosynthetic gene cluster responsible for the production of both an antibiotic, pyridomycin, and a novel chlorocatechol-containing siderophore named chlorodactyloferrin. While both of these natural products were synthesized independently, analysis of the culture supernatant also identified a conjugate of both molecules. We then found that the addition of ferric iron to purified chlorodactyloferrin and pyridomycin instigated their conjugation, leading to the formation of a covalent bond between the siderophore-catechol and the pyridomycin-pyridine groups. Using model reactants, this iron-based reaction was found to proceed through a Michael-type addition reaction, where ferric iron oxidizes the siderophore-catechol group into its quinone form, which is then attacked by the antibiotic pyridyl-nitrogen to form the catechol-pyridinium linkage. These findings prompted us to explore if other "cargo" molecules could be attached to chlorodactyloferrin in a similar manner, and this was indeed confirmed with a pyridine-substituted TAMRA fluorophore as well as with pyridine-substituted penicillin, rifampicin, and norfloxacin antibiotic analogues. The resultant biomimetic conjugates were demonstrated to effectively enter a number of bacteria, with TAMRA-chlorodactyloferrin conjugates causing fluorescent labeling of the bacteria, and with penicillin and rifampicin conjugates eliciting antibiotic activity. These findings open up new opportunities for the design and facile synthesis of a novel class of biomimetic siderophore conjugates with antibiotic activity.

Dactylosporolides: Glycosylated Macrolides from Dactylosporangium fulvum.

A series of novel macrolides were discovered from the culture supernatant of the rare soil actinobacteria Dactylosporangium fulvum and named dactylosporolides A-C. The structure and absolute configuration of these dactylosporolides were defined using a combination of NMR structural elucidation and analysis of the dactylosporolide biosynthetic gene cluster. Together these data revealed dactylosporolides to be composed of a central 22-membered macrolactone with an internal hemiketal ring and a protruding ketide tail that were (poly)glycosylated at two distal parts. While bearing no antibiotic activity, these dactylosporolides displayed activity against Plasmodium falciparum 3D7.

Rubrolone production by Dactylosporangium vinaceum: biosynthesis, modulation and possible biological function.

Rare actinomycetes are likely treasure troves for bioactive natural products, and it is therefore important that we enrich our understanding of biosynthetic potential of these relatively understudied bacteria. Dactylosporangium are a genus of such rare Actinobacteria that are known to produce a number of important antibacterial compounds, but for which there are still no fully assembled reference genomes, and where the extent of encoded biosynthetic capacity is not defined. Dactylosporangium vinaceum (NRRL B-16297) is known to readily produce a deep wine red-coloured diffusible pigment of unknown origin, and it was decided to define the chemical identity of this natural product pigment, and in parallel use whole genome sequencing and transcriptional analysis to lay a foundation for understanding the biosynthetic capacity of these bacteria. Results show that the produced pigment is made of various rubrolone conjugates, the spontaneous product of the reactive pre-rubrolone, produced by the bacterium. Genome and transcriptome analysis identified the highly expressed biosynthetic gene cluster (BGC) for pre-rubrolone. Further analysis of the fully assembled genome found it to carry 24 additional BGCs, of which the majority were poorly transcribed, confirming the encoded capacity of this bacterium to produce natural products but also illustrating the main bottleneck to exploiting this capacity. Finally, analysis of the potential environmental role of pre-rubrolone found it to react with a number of amine containing antibiotics, antimicrobial peptides and siderophores pointing to its potential role as a "minesweeper" of xenobiotic molecules in the bacterial environment. KEY POINTS: • D. vinaceum encodes many BGC, but the majority are transcriptionally silent. • Chemical screening identifies molecules that modulate rubrolone production. • Pre-rubrolone is efficient at binding and inactivating many natural antibiotics.

Antitumor astins originate from the fungal endophyte Cyanodermella asteris living within the medicinal plant Aster tataricus.

Medicinal plants are a prolific source of natural products with remarkable chemical and biological properties, many of which have considerable remedial benefits. Numerous medicinal plants are suffering from wildcrafting, and thus biotechnological production processes of their natural products are urgently needed. The plant Aster tataricus is widely used in traditional Chinese medicine and contains unique active ingredients named astins. These are macrocyclic peptides showing promising antitumor activities and usually containing the highly unusual moiety 3,4-dichloroproline. The biosynthetic origins of astins are unknown despite being studied for decades. Here we show that astins are produced by the recently discovered fungal endophyte Cyanodermella asteris. We were able to produce astins in reasonable and reproducible amounts using axenic cultures of the endophyte. We identified the biosynthetic gene cluster responsible for astin biosynthesis in the genome of C. asteris and propose a production pathway that is based on a nonribosomal peptide synthetase. Striking differences in the production profiles of endophyte and host plant imply a symbiotic cross-species biosynthesis pathway for astin C derivatives, in which plant enzymes or plant signals are required to trigger the synthesis of plant-exclusive variants such as astin A. Our findings lay the foundation for the sustainable biotechnological production of astins independent from aster plants.

Complete Genome Sequence of the Polymyxin E (Colistin)-Producing Paenibacillus sp. Strain B-LR.

Paenibacillus bacteria are recovered from varied niches, including human lung, rhizosphere, marine sediments, and hemolymph. Paenibacilli can have plant growth-promoting activities and be antibiotic producers. They can produce exopolysaccharides and enzymes of industrial interest. Illumina and PacBio reads were used to produce a complete genome sequence of the colistin producer Paenibacillus sp. strain B-LR.

Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis.

Bacteria of the Bacillus genus are able to synthesize several families of lipopeptides. These small molecules are the product of non-ribosomal peptide synthetases. In 2000, it was found that Bacillus thuringiensis, an entomopathogenic bacterium of the Bacillus cereus group, produced a previously unknown lipopeptide: kurstakin. Genomic analyses reveal that the krs locus, encoding the kurstakin synthetases, is specific to the B. cereus group, but is unevenly distributed within this group. Previous work showed that krs transcription requires the necrotrophism quorum-sensor NprR. Here, we demonstrated that the genes of the krs locus form an operon and we defined its transcription start site. Following krs transcription at the population and single-cell levels in multiple culture conditions, we depicted a condition-dependent transcription pattern, indicating that production of kurstakin is subject to environmental regulation. Consistent with this idea, we found krs transcription to be regulated by another master regulator, Spo0A, suggesting that krs expression is fine-tuned by integrating multiple signals. We also reported an unknown DNA palindrome in the krs promoter region that modulates krs expression. Due to their surfactant properties, lipopeptides could play several physiological roles. We showed that the krs locus was required for proper biofilm structuration.

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus ('Penicillium islandicum').

Talaromyces islandicus ('Penicillium islandicum') is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains the unusual amino acids ß-phenylalanine, 2-aminobutyrate and 3,4-dichloroproline. Although the chemical structure has been known for over five decades, nothing is known about the biosynthetic pathway of cyclochlorotine. Bioinformatic analysis of the recently sequenced genome of T. islandicus identified a wealth of gene clusters potentially coding for the synthesis of secondary metabolites. Here, we show by RNA interference-mediated gene silencing that a nonribosomal peptide synthetase, CctN, is responsible for the synthesis of cyclochlorotine. Moreover, we identified novel cyclochlorotine chemical variants, whose production also depended on cctN expression. Surprisingly, the halogenase required for cyclochlorotine biosynthesis is not encoded in the cct cluster. Nonetheless, our findings enabled us to propose a detailed model for cyclochlorotine biosynthesis. In addition, comparative genomics revealed that cct-like clusters are present in all of the sequenced Talaromyces strains indicating a high prevalence of cyclochlorotine production ability.

Draft genome sequence of Talaromyces islandicus ("Penicillium islandicum") WF-38-12, a neglected mold with significant biotechnological potential.

Talaromyces (Penicillium) islandicus is a common mold found in stored rice or cereals. It has a highly versatile metabolism characterized by the secretion of numerous biopolymer degrading enzymes, mycotoxins, and anthraquinones that altogether offer a broad range of potential industrial applications. Here, we report the draft genome sequence of Talaromyces islandicus, which provides the basis of a biotechnological usage of this species.

Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

Prediction of monomer isomery in Florine: a workflow dedicated to nonribosomal peptide discovery.

Nonribosomal peptides represent a large variety of natural active compounds produced by microorganisms. Due to their specific biosynthesis pathway through large assembly lines called NonRibosomal Peptide Synthetases (NRPSs), they often display complex structures with cycles and branches. Moreover they often contain non proteogenic or modified monomers, such as the D-monomers produced by epimerization. We investigate here some sequence specificities of the condensation (C) and epimerization (E) domains of NRPS that can be used to predict the possible isomeric state (D or L) of each monomer in a putative peptide. We show that C- and E- domains can be divided into 2 sub-regions called Up-Seq and Down-Seq. The Up-Seq region corresponds to an InterPro domain (IPR001242) and is shared by C- and E-domains. The Down-Seq region is specific to the enzymatic activity of the domain. Amino-acid signatures (represented as sequence logos) previously described for complete C-and E-domains have been restricted to the Down-Seq region and amplified thanks to additional sequences. Moreover a new Down-Seq signature has been found for Ct-domains found in fungi and responsible for terminal cyclization of the peptides. The identification of these signatures has been included in a workflow named Florine, aimed to predict nonribosomal peptides from NRPS sequence analyses. In some cases, the prediction of isomery is guided by genus-specific rules. Florine was used on a Pseudomonas genome to allow the determination of the type of pyoverdin produced, the update of syringafactin structure and the identification of novel putative products.

A Novel Depsipeptide Produced by Paenibacillus alvei 32 Isolated from a Cystic fibrosis Patient.

The important viscosity of the respiratory tract mucus of Cystic fibrosis (CF) patients impairs the mucociliary transport system and allows the growth of numerous micro-organisms. Among them, Pseudomonas aeruginosa and Staphylococcus aureus are known to be responsible for pulmonary infections. We imagined that CF microflora could also harbour micro-organisms naturally equipped to compete with these pathogens. A method was developed to recover these antibiotic-producing strains within 20 CF sputum. Using this approach, we have isolated an unusual Gram-positive bacterium identified as Paenibacillus alvei by Api galleries and 16S rRNA gene sequence analysis. This strain has inhibited the growth of P. aeruginosa, S. aureus and Klebsiella pneumoniae, in co-cultures. A liquid mineral medium named MODT50 was designed and optimised for the production and the recovery of the antimicrobial compounds. The supernatant has inhibited the growth of all Gram-positive strains tested, even Methicillin-resistant S. aureus. One antimicrobial compound with a peptide structure (mainly active against S. aureus, Micrococcus luteus, and Pseudomonas stutzeri) has been purified and characterised by liquid chromatography-mass spectrometry. The new active molecule (m/z 786.6) named depsipeptide L possesses a 15-guanidino-3-hydroxypentadecanoic acid side chain (m/z 298) linked on a cyclic part of four amino acids residues (Ser, two Leu/Ile, Arg). This work reports for the first time the production of such a molecule by a P. alvei strain in a mineral medium. The CF lung microflora might represent a valuable source for the discovery of new antimicrobial-producing strains.

Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.

A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.