Unveiling the fungal diversity and associated secondary metabolism on black apples.

Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena.IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

Fungal-Associated Molecules Induce Key Genes Involved in the Biosynthesis of the Antifungal Secondary Metabolites Nunamycin and Nunapeptin in the Biocontrol Strain Pseudomonas fluorescens In5.

Pseudomonas fluorescens In5 synthesizes the antifungal cyclic lipopeptides (CLPs) nunamycin and nunapeptin, which are similar in structure and genetic organization to the pseudomonas-derived phytotoxins syringomycin and syringopeptin. Regulation of syringomycin and syringopeptin is dependent on the two-component global regulatory system GacS-GacA and the SalA, SyrF, and SyrG transcription factors, which activate syringomycin synthesis in response to plant signal molecules. Previously, we demonstrated that a specific transcription factor, NunF, positively regulates the synthesis of nunamycin and nunapeptin in P. fluorescens In5 and that the nunF gene is upregulated by fungal-associated molecules. This study focused on further unravelling the complex regulation governing CLP synthesis in P. fluorescens In5. Promoter fusions were used to show that the specific activator NunF is dependent on the global regulator of secondary metabolism GacA and is regulated by fungal-associated molecules and low temperatures. In contrast, GacA is stimulated by plant signal molecules leading to the hypothesis that P. fluorescens is a hyphosphere-associated bacterium carrying transcription factor genes that respond to signals indicating the presence of fungi and oomycetes. Based on these findings, we present a model for how synthesis of nunamycin and nunapeptin is regulated by fungal- and oomycete-associated molecules.IMPORTANCE Cyclic lipopeptide (CLP) synthesis gene clusters in pseudomonads display a high degree of synteny, and the structures of the peptides synthesized are very similar. Accordingly, the genomic island encoding the synthesis of syringomycin and syringopeptin in P. syringae pv. syringae closely resembles that of P. fluorescens In5, which contains genes coding for synthesis of the antifungal and anti-oomycete peptides nunamycin and nunapeptin, respectively. However, the regulation of syringomycin and syringopeptin synthesis is different from that of nunamycin and nunapeptin synthesis. While CLP synthesis in the plant pathogen P. syringae pv. syringae is induced by plant signal molecules, such compounds do not significantly influence synthesis of nunamycin and nunapeptin in P. fluorescens In5. Instead, fungal-associated molecules positively regulate antifungal peptide synthesis in P. fluorescens In5, while the synthesis of the global regulator GacA in P. fluorescens In5 is positively regulated by plant signal molecules but not fungal-associated molecules.

Isolation of Methyl Troposulfenin from Phaeobacter inhibens.

An S-methylated analogue of tropodithietic acid (TDA, 1), methyl troposulfenin (2), was isolated from the marine alphaproteobacterium Phaeobacter inhibens. The structure was elucidated by NMR and HRMS. Its inhibitory effect against the fish pathogen Vibrio anguillarum was 4-fold to 100-fold lower than that of the known antibacterial compound TDA. Methyl troposulfenin lacks the acidic proton of TDA, indicating that the methylation turns the potent antibacterial TDA into an inactive compound, and thereby, this analysis supports the proposed mode of action of TDA.

The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production.

Aspergillus niger secretes proteins throughout the colony except for the zone that forms asexual spores called conidia. Inactivation of flbA that encodes a regulator of G-protein signaling results in colonies that are unable to reproduce asexually and that secrete proteins throughout the mycelium. In addition, the ΔflbA strain shows cell lysis and has thinner cell walls. Expression analysis showed that 38 predicted transcription factor genes are differentially expressed in strain ΔflbA. Here, the most down-regulated predicted transcription factor gene, called fum21, was inactivated. Growth, conidiation, and protein secretion were not affected in strain Δfum21. Whole genome expression analysis revealed that 63 and 11 genes were down- and up-regulated in Δfum21, respectively, when compared to the wild-type strain. Notably, 24 genes predicted to be involved in secondary metabolism were down-regulated in Δfum21, including 10 out of 12 genes of the fumonisin cluster. This was accompanied by absence of fumonisin production in the deletion strain and a 25% reduction in production of pyranonigrin A. Together, these results link FlbA-mediated sporulation-inhibited secretion with mycotoxin production.

Phaeobacter piscinae sp. nov., a species of the Roseobacter group and potential aquaculture probiont.

Four heterotrophic, antimicrobial, motile, marine bacterial strains, 27-4T, 8-1, M6-4.2 and S26, were isolated from aquaculture units in Spain, Denmark and Greece. All four strains produced the antibiotic compound tropodithietic acid, which is a key molecule in their antagonism against fish pathogenic bacteria. Cells of the strains were Gram-reaction-negative, rod-shaped and formed star-shaped aggregates in liquid culture and brown-coloured colonies on marine agar. The predominant cellular fatty acids were C18 : 1ω7c, C16 : 0, C11 methyl C18 : 1ω7c and C16 : 0 2-OH, and the polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an aminolipid, a phospholipid and an unidentified lipid. The strains grew optimally at 31-33 °C. Growth was observed at a salt concentration between 0.5 and 5-6 % NaCl with an optimum at 2-3 %. The pH range for growth of the strains was from pH 6 to 8-8.5 with an optimum at pH 7. Based on 16S rRNA gene sequence analysis, the strains are affiliated with the genus Phaeobacter. The genome sequences of the strains have a DNA G+C content of 60.1 % and share an average nucleotide identity (ANI) of more than 95 %. The four strains are distinct from the type strains of the closely related species Phaeobactergallaeciensis and Phaeobacterinhibens based on an ANI of 90.5-91.7 and 89.6-90.4 %, respectively, and an in silico DNA-DNA hybridization relatedness of 43.9-46.9 and 39.8-41.9 %, respectively. On the basis of phylogenetic analyses as well as phenotypic and chemotaxonomic properties, the isolates are considered to represent a novel species, for which the name Phaeobacter piscinae sp. nov. is proposed. The type strain is 27-4T (=DSM 103509T=LMG 29708T).

Influence of Niche-Specific Nutrients on Secondary Metabolism in Vibrionaceae.

UNLABELLED: Many factors, such as the substrate and the growth phase, influence biosynthesis of secondary metabolites in microorganisms. Therefore, it is crucial to consider these factors when establishing a bioprospecting strategy. Mimicking the conditions of the natural environment has been suggested as a means of inducing or influencing microbial secondary metabolite production. The purpose of the present study was to determine how the bioactivity of Vibrionaceae was influenced by carbon sources typical of their natural environment. We determined how mannose and chitin, compared to glucose, influenced the antibacterial activity of a collection of Vibrionaceae strains isolated because of their ability to produce antibacterial compounds but that in subsequent screenings seemed to have lost this ability. The numbers of bioactive isolates were 2- and 3.5-fold higher when strains were grown on mannose and chitin, respectively, than on glucose. As secondary metabolites are typically produced during late growth, potential producers were also allowed 1 to 2 days of growth before exposure to the pathogen. This strategy led to a 3-fold increase in the number of bioactive strains on glucose and an 8-fold increase on both chitin and mannose. We selected two bioactive strains belonging to species for which antibacterial activity had not previously been identified. Using ultrahigh-performance liquid chromatography-high-resolution mass spectrometry and bioassay-guided fractionation, we found that the siderophore fluvibactin was responsible for the antibacterial activity of Vibrio furnissii and Vibrio fluvialis These results suggest a role of chitin in the regulation of secondary metabolism in vibrios and demonstrate that considering bacterial ecophysiology during development of screening strategies will facilitate bioprospecting. IMPORTANCE: A challenge in microbial natural product discovery is the elicitation of the biosynthetic gene clusters that are silent when microorganisms are grown under standard laboratory conditions. We hypothesized that, since the clusters are not lost during proliferation in the natural niche of the microorganisms, they must, under such conditions, be functional. Here, we demonstrate that an ecology-based approach in which the producer organism is allowed a temporal advantage and where growth conditions are mimicking the natural niche remarkably increases the number of Vibrionaceae strains producing antibacterial compounds.

Influence of Iron on Production of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in Phaeobacter inhibens.

Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography-high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe(3+) coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe(III)(TDA)2]x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.

Stachybotrys mycotoxins: from culture extracts to dust samples.

The filamentous fungus Stachybotrys chartarum is known for its toxic metabolites and has been associated with serious health problems, including mycotoxicosis, among occupants of contaminated buildings. Here, we present results from a case study, where an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for known and tentatively identified compounds characterized via UHPLC-quadruple time-of-flight (QTOF) screening of fungal culture extracts, wall scrapings and reference standards. The UHPLC-MS/MS method was able to identify 12 Stachybotrys metabolites, of which four could be quantified based on authentic standards and a further six estimated based on similarity to authentic standards. Samples collected from walls contaminated by S. chartarum in a water-damaged building showed that the two known chemotypes, S and A, coexisted. More importantly, a link between mycotoxin concentrations found on contaminated surfaces and in settled dust was made. One dust sample, collected from a water-damaged room, contained 10 pg/cm(2) macrocyclic trichothecenes (roridin E). For the first time, more than one spirocyclic drimane was detected in dust. Spirocyclic drimanes were detected in all 11 analysed dust samples and in total amounted to 600 pg/cm(2) in the water-damaged room and 340 pg/cm(2) in rooms adjacent to the water-damaged area. Their wide distribution in detectable amounts in dust suggested they could be good candidates for exposure biomarkers. Graphical abstract Stachybotrys growing on a gypsum board, and some of the compounds it produces.

Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

Fungal and chemical diversity in hay and wrapped haylage for equine feed.

The presence of fungi and mycotoxins in silage (fermented maize) for cattle and other ruminants have been studied extensively compared to wrapped haylage (fermented grass) for horses and other monogastric animals. The purpose of this work was to examine the fungal diversity of wrapped haylage and conventional hay and to analyse the forage sample for fungal metabolites. Faeces samples were also analysed to study the fate of fungi and metabolites. Fungal diversity of the samples was determined by direct plating on DG18, V8 and MEA and chemical analyses were done using LC-MS/MS. The results show that Sordaria fimicola was common in both hay and haylage, while Penicillium spp. was prevalent in haylage and Aspergillus spp. in hay. Communiols were found in all types of samples together with gliocladic acid. Roquefortines and fumigaclavines were found in haylage with no visible fungal growth, but not in hay. In haylage hot spot samples, a series of Penicillium metabolites were detected: Andrastins, fumigaclavines, isofumigaclavines, marcfortines, mycophenolic acid, PR toxins, and roquefortines. Penicillium solitum was found repeatedly in haylage and haylage hot spot samples and viridicatols were detected in a hot spot sample, which has not been reported before. Even haylage with no visible fungal growth contained more metabolites than hay. Individually, the metabolites detected in haylage may, in high doses, be mutagenic, neurotoxic or immunosuppressive; but the synergistic effect of small doses may also have other or greater negative health effects on equines than on ruminants.

Phylogenetic distribution of roseobacticides in the Roseobacter group and their effect on microalgae.

The Roseobacter-group species Phaeobacter inhibens produces the antibacterial tropodithietic acid (TDA) and the algaecidal roseobacticides with both compound classes sharing part of the same biosynthetic pathway. The purpose of this study was to investigate the production of roseobacticides more broadly in TDA-producing roseobacters and to compare the effect of producers and non-producers on microalgae. Of 33 roseobacters analyzed, roseobacticide production was a unique feature of TDA-producing P. inhibens, P. gallaeciensis and P. piscinae strains. One TDA-producing Phaeobacter, 27-4, did not produce roseobacticides, possibly due to a transposable element. TDA-producing Ruegeria and Pseudovibrio did not produce roseobacticides. Addition of roseobacticide-containing bacterial extracts affected the growth of the microalgae Rhodomonas salina, Thalassiosira pseudonana and Emiliania huxleyi, while growth of Tetraselmis suecica was unaffected. During co-cultivation, growth of E. huxleyi was initially stimulated by the roseobacticide producer DSM 17395, while the subsequent decline in algal cell numbers during senescence was enhanced. Strain 27-4 that does not produce roseobacticides had no effect on algal growth. Both bacterial strains, DSM 17395 and 27-4, grew during co-cultivation presumably utilizing algal exudates. Furthermore, TDA-producing roseobacters have potential as probiotics in marine larviculture and it is promising that the live feed Tetraselmis was unaffected by roseobacticides-containing extracts.

Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation.

Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/α-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/α-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the α-ketoglutarate-binding ability.

Growth on Chitin Impacts the Transcriptome and Metabolite Profiles of Antibiotic-Producing Vibrio coralliilyticus S2052 and Photobacterium galatheae S2753.

Members of the Vibrionaceae family are often associated with chitin-containing organisms, and they are thought to play a major role in chitin degradation. The purpose of the present study was to determine how chitin affects the transcriptome and metabolome of two bioactive Vibrionaceae strains, Vibrio coralliilyticus and Photobacterium galatheae. We focused on chitin degradation genes and secondary metabolites based on the assumption that these molecules in nature confer an advantage to the producer. Growth on chitin caused upregulation of genes related to chitin metabolism and of genes potentially involved in host colonization and/or infection. The expression of genes involved in secondary metabolism was also significantly affected by growth on chitin, in one case being 34-fold upregulated. This was reflected in the metabolome, where the antibiotics andrimid and holomycin were produced in larger amounts on chitin. Other polyketide synthase/ nonribosomal peptide synthetase (PKS-NRPS) clusters in P. galatheae were also strongly upregulated on chitin. Collectively, this suggests that both V. coralliilyticus and P. galatheae have a specific lifestyle for growth on chitin and that their secondary metabolites likely play a crucial role during chitin colonization. IMPORTANCE The bacterial family Vibrionaceae (vibrios) is considered a major player in the degradation of chitin, the most abundant polymer in the marine environment; however, the majority of studies on the topic have focused on a small number of Vibrio species. In this study, we analyzed the genomes of two vibrios to assess their genetic potential for the degradation of chitin. We then used transcriptomics and metabolomics to demonstrate that chitin strongly affects these vibrios at both the transcriptional and metabolic levels. We observed a strong increase in production of secondary metabolites, suggesting an ecological role for these molecules during chitin colonization in the marine environment.

Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF.

Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun-nup regulon. Organization of the regulon is similar to clusters found in other CLP-producing pseudomonads except for the border regions where putative LuxR-type regulators are located. This study focuses on understanding the regulatory role of the LuxR-type-encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real-time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun-nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.

Occurrence and fumonisin B2 producing potential of Aspergillus section Nigri in Brazil nuts.

Bertholletia excelsa is the tree that produces Brazil nuts which have vast economic importance in the Amazon region and as an export commodity. The aim of this study was to assess the presence of Aspergillus section Nigri in Brazil nut samples at different stages of its production chain and to verify the toxigenic potential for fumonisin B2 (FB2) production of these isolates along with the presence of this mycotoxin in Brazil nut samples. The fungal infection ranged from 0 to 80% at the different stages of the harvest and processing chain and the water activity of the nuts from 0.273 to 0.994. A total of 1052 A. section Nigri strains were isolated from Brazil nuts and 200 strains were tested for their ability to produce FB2: 41 strains (20.5%) were FB2 producers with concentrations ranging from 0.09 to 37.25 mg/kg; 2 strains (1%) showed traces of FB2, less than the detection limit (0.08 mg/kg); and 157 (78.5%) were not FB2 producers. Although several samples showed high contamination by A. section Nigri, no sample was contaminated by FB2.

"On-water" conjugate additions of anilines.

The conjugate addition of anilines onto unsaturated ketones, esters and N-acylpyrroles was investigated. Based on a recently proposed explanation for the phenomenon of on-water catalysis, operationally simple and mild reaction conditions for effecting these addition reactions have been developed. The success of these additions provides further support for the acid-catalysed nature of on-water chemistry.