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.

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Biosynthetic Basis for Structural Diversity of Aminophenylpyrrole-Derived Alkaloids.

Bacterial aminophenylpyrrole-derived alkaloids (APPAs) represent high value lead compounds. Pyrrolnitrin, which was developed into globally important fungicides, is the only reported APPA produced by Proteobacteria. Recently, various APPAs showing diverse bioactivities were discovered from Bacteroidetes. Here, a bioinformatics and phylogenetic approach enabled the elucidation of the biosynthesis of the highly diverse APPAs in Cytophagales bacteria and their chemical diversification strategy. The biosynthetic gene clusters were identified in producer strains, and the biosynthesis was experimentally validated by heterologous expression experiments in E. coli. First, one enzyme-dependent biosynthetic step yields the tryptophan-derived precursor 3-(2'-aminophenyl)-pyrrole. Second, a spontaneous Pictet-Spengler-like coupling reaction enables the bacterial producer strains to create a library of tricyclic alkaloids, since several aldehydes can be applied as substrates. The diversity of this natural products class is further enlarged by the catalytic action of a methyltransferase, which adds one or more methyl groups to the aminophenyl intermediate.

Correction to: Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway.

There is an error in the original publication of this paper. Figures 1-6 were shown in the wrong version, thus corrected figures provided were in this article.

Soil Bacteria Isolated From Tunisian Arid Areas Show Promising Antimicrobial Activities Against Gram-Negatives.

Arid regions show relatively fewer species in comparison to better-watered biomes, but the competition for the few nutrients is very distinct. Here, in total 373 bacterial strains were isolated from rhizospheric soils obtained from three different sampling sites in Tunisia. Their potential for the production of antimicrobial compounds was evaluated. Bacterial strains, showing antibacterial activity against pathogenic bacteria, were isolated from all three sites, one strain from the Bou-Hedma national park, 15 strains from Chott-Djerid, and 13 strains from Matmata, respectively. The dominant genus was Bacillus, with 27 out of 29 strains. Most interestingly, 93% of the isolates showed activity against Gram-positive and Gram-negative test bacteria. Strain Bacillus sp. M21, harboring high inhibitory potential, even against clinical isolates of Gram-negative bacteria, was analyzed in detail to enable purification and identification of the bioactive compound responsible for its bioactivity. Subsequent HPLC-MS and NMR analyses resulted in the identification of 1-acetyl-ß-carboline as active component. Furthermore, fungicides of the bacillomycin and fengycin group, which in addition show antibiotic effects, were identified. This work highlights the high potential of the arid-adapted strains for the biosynthesis of specialized metabolites and suggest further investigation of extreme environments, since they constitute a promising bioresource of biologically active compounds.

Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway.

Sulforaphane (SFN) has been considered as an indirect antioxidant and potential inducer of the Nrf2-ARE pathway. This study was conducted to investigate the protective role of SFN against oxidative stress in bovine granulosa cells (GCs). GCs were collected from antral follicles (4-8 mm) and cultured according to the experimental design where group 1 = control, group 2 = treated with SFN, group 3 = treated with hydrogen peroxide (H2O2), group 4 = pretreated with SFN and then with H2O2 (protective) and group 5 = treated with H2O2 followed by SFN treatment (rescuing). Results showed that SFN pretreatment significantly increases cell viability and reduces cytotoxicity in GCs under oxidative stress. Following H2O2 exposure, expression of NRF2 was found to be significantly increased (p < 0.05) in SFN-pretreated cells, while no significant differences were observed between group 3 and group 5, although the expression was significantly increased compared to the control group. Moreover, the relative abundance of the NRF2 downstream target antioxidant genes (CAT, PRDX1, SOD1 and TXN1) were higher (fold change ranged from 7 to 14, p < 0.05) in sulforaphane pretreated GCs. Low level of ROS and lipid accumulation and higher mitochondrial activity were observed in GCs pretreated with SFN, whereas no such changes were observed in GCs treated with SFN after exposure to oxidative stress (group 5). Thus, we suggest that SFN pretreatment effectively protects GCs against oxidative damage through the activation of the NRF2-ARE pathway, whereas addition of SFN during oxidative insult failed to rescue GCs.

Diversity and Antimicrobial Potential of Predatory Bacteria from the Peruvian Coastline.

The microbiome of three different sites at the Peruvian Pacific coast was analyzed, revealing a lower bacterial biodiversity at Isla Foca than at Paracas and Manglares, with 89 bacterial genera identified, as compared to 195 and 173 genera, respectively. Only 47 of the bacterial genera identified were common to all three sites. In order to obtain promising strains for the putative production of novel antimicrobials, predatory bacteria were isolated from these sampling sites, using two different bait organisms. Even though the proportion of predatory bacteria was only around 0.5% in the here investigated environmental microbiomes, by this approach in total 138 bacterial strains were isolated as axenic culture. 25% of strains showed antibacterial activity, thereby nine revealed activity against clinically relevant methicillin resistant Staphylococcus aureus (MRSA) and three against enterohemorrhagic Escherichia coli (EHEC) strains. Phylogeny and physiological characteristics of the active strains were investigated. First insights into the chemical basis of the antibacterial activity indicated the biosynthetic production of the known compounds ariakemicin, kocurin, naphthyridinomycin, pumilacidins, resistomycin, and surfactin. However, most compounds remained elusive until now. Hence, the obtained results implicate that the microbiome present at the various habitats at the Peruvian coastline is a promising source for heterotrophic bacterial strains showing high potential for the biotechnological production of antibiotics.

Identification and heterologous expression of the kocurin biosynthetic gene cluster.

The antibiotically bioactive thiopeptide compound kocurin was identified in extracts from a newly isolated Kocuria rosea strain. The axenic strain was retrieved from a soil sample of the intertidal area at the Paracas National Park, Peru. The genetic basis of this promising natural product with activity against methicillin-resistant Staphylococcus aureus (MRSA) strains was revealed by comparative genome analysis of this new isolate and other reported thiopeptide producer strains. The functionality of the predicted gene locus was experimentally proven by heterologous expression in Streptomyces coelicolor M1146. Expression of the gene cluster under the control of a constitutive promoter enabled the transgenic strain to produce kocurin in selected media. The kocurin biosynthetic gene cluster comprises nine open reading frames and spans around 12 kbp of the genome.