Metabolic Profiling of Rhizobacteria Serratia plymuthica and Bacillus subtilis Revealed Intra- and Interspecific Differences and Elicitation of Plipastatins and Short Peptides Due to Co-cultivation.

Rhizobacteria live in diverse and dynamic communities having a high impact on plant growth and development. Due to the complexity of the microbial communities and the difficult accessibility of the rhizosphere, investigations of interactive processes within this bacterial network are challenging. In order to better understand causal relationships between individual members of the microbial community of plants, we started to investigate the inter- and intraspecific interaction potential of three rhizobacteria, the S. plymuthica isolates 4Rx13 and AS9 and B. subtilis B2g, using high resolution mass spectrometry based metabolic profiling of structured, low-diversity model communities. We found that by metabolic profiling we are able to detect metabolite changes during cultivation of all three isolates. The metabolic profile of S. plymuthica 4Rx13 differs interspecifically to B. subtilis B2g and surprisingly intraspecifically to S. plymuthica AS9. Thereby, the release of different secondary metabolites represents one contributing factor of inter- and intraspecific variations in metabolite profiles. Interspecific co-cultivation of S. plymuthica 4Rx13 and B. subtilis B2g showed consistently distinct metabolic profiles compared to mono-cultivated species. Thereby, putative known and new variants of the plipastatin family are increased in the co-cultivation of S. plymuthica 4Rx13 and B. subtilis B2g. Interestingly, intraspecific co-cultivation of S. plymuthica 4Rx13 and S. plymuthica AS9 revealed a distinct interaction zone and showed distinct metabolic profiles compared to mono-cultures. Thereby, several putative short proline-containing peptides are increased in co-cultivation of S. plymuthica 4Rx13 with S. plymuthica AS9 compared to mono-cultivated strains. Our results demonstrate that the release of metabolites by rhizobacteria alters due to growth and induced by social interactions between single members of the microbial community. These results form a basis to elucidate the functional role of such interaction-triggered compounds in establishment and maintenance of microbial communities and can be applied under natural and more realistic conditions, since rhizobacteria also interact with the plant itself and many other members of plant and soil microbiota.

Cobamide remodeling in the freshwater microalga Chlamydomonas reinhardtii.

Microalgae are not able to produce cobamides (Cbas, B12 vitamers) de novo. Hence, the production of catalytically active Cba-containing methionine synthase (MetH), which is present in selected representatives, is dependent on the availability of exogenous B12 vitamers. Preferences in the utilization of exogenous Cbas equipped with either adenine or 5,6-dimethylbenzimidazole as lower base have been reported for some microalgae. Here, we investigated the utilization of norcobamides (NorCbas) for growth by the Cba-dependent Chlamydomonas reinhardtii mutant strain (ΔmetE). The growth yields in the presence of NorCbas were lower in comparison to those achieved with Cbas. NorCbas lack a methyl group in the linker moiety of the nucleotide loop. C. reinhardtii was also tested for the remodeling of NorCbas (e.g. adeninyl-norcobamide) in the presence of different benzimidazoles. Extraction of the NorCbas from C. reinhardtii, their purification, and identification confirmed the exchange of the lower base of the vitamers. However, the linker moiety of the NorCbas nucleotide loop was not exchanged. This observation strongly indicates the presence of an alternative mode of Cba deconstruction in C. reinhardtii that differs from the amidohydrolase (CbiZ)-dependent pathway described in Cba-remodeling bacteria and archaea.

ReDU: a framework to find and reanalyze public mass spectrometry data.

We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

A suite of complementary biocontrol traits allows a native consortium of root-associated bacteria to protect their host plant from a fungal sudden-wilt disease.

The beneficial effects of plant--bacterial interactions in controlling plant pests have been extensively studied with single bacterial isolates. However, in nature, bacteria interact with plants in multitaxa consortia, systems which remain poorly understood. Previously, we demonstrated that a consortium of five native bacterial isolates protected their host plant Nicotiana attenuata from a sudden wilt disease. Here we explore the mechanisms behind the protection effect against the native pathosystem. Three members of the consortium, Pseudomonas azotoformans A70, P. frederiksbergensis A176 and Arthrobacter nitroguajacolicus E46, form biofilms when grown individually in vitro, and the amount of biofilm increased synergistically in the five-membered consortium, including two Bacillus species, B. megaterium and B. mojavensis. Fluorescence in situ hybridization and scanning electron microscopy in planta imaging techniques confirmed biofilm formation and revealed locally distinct distributions of the five bacterial strains colonizing different areas on the plant-root surface. One of the five isolates, K1 B. mojavensis produces the antifungal compound surfactin, under in vitro and in vivo conditions, clearly inhibiting fungal growth. Furthermore, isolates A70 and A176 produce siderophores under in vitro conditions. Based on these results we infer that the consortium of five bacterial isolates protects its host against fungal phytopathogens via complementary traits. The study should encourage researchers to create synthetic communities from native strains of different genera to improve bioprotection against wilting diseases.

Metabolomic and transcriptomic data on major metabolic/biosynthetic pathways in workers and soldiers of the termite Prorhinotermes simplex (Isoptera: Rhinotermitidae) and chemical synthesis of intermediates of defensive (E)-nitropentadec-1-ene biosynthesis.

Production of nitro compounds has only seldom been recorded in arthropods. The aliphatic nitroalkene (E)-nitropentadec-1-ene (NPD), identified in soldiers of the termite genus Prorhinotermes, was the first case documented in insects in early seventies. Yet, the biosynthetic origin of NPD has long remained unknown. We previously proposed that NPD arises through the condensation of amino acids glycine and/or l-serine with tetradecanoic acid along a biosynthetic pathway analogous to the formation of sphingolipids. Here, we provide a metabolomics and transcriptomic data of the Prorhinotermes simplex termite workers and soldiers. Data are related to NPD biosynthesis in P. simplex soldiers. Original metabolomics data were deposited in MetaboLights metabolomics database and are become publicly available after publishing the original article. Additionally, chemical synthesis of biosynthetic intermediates of NPD in nonlabeled and stable labeled forms are reported. Data extend our poor knowledge of arthropod metabolome and transcriptome and would be useful for comparative study in termites or other arthropods. The data were used for de-replication of NPD biosynthesis and published separately (Jirosová et al., 2017) [1].

Deciphering the genome and secondary metabolome of the plant pathogen Fusarium culmorum.

Fusarium culmorum is one of the most important fungal plant pathogens that causes diseases on a wide diversity of cereal and non-cereal crops. We report herein for the first time the genome sequence of F. culmorum strain PV and its associated secondary metabolome that plays a role in the interaction with other microorganisms and contributes to its pathogenicity on plants. The genome revealed the presence of two terpene synthases, trichodiene and longiborneol synthase, which generate an array of volatile terpenes. Furthermore, we identified two gene clusters, deoxynivalenol and zearalenone, which encode for the production of mycotoxins. Linking the production of mycotoxins with in vitro bioassays, we found high virulence of F. culmorum PV on maize, barley and wheat. By using ultra-performance liquid chromatography-mass spectrometry, we confirmed several compounds important for the behaviour and lifestyle of F. culmorum. This research sets the basis for future studies in microbe-plant interactions.

Acylated Quinic Acids Are the Main Salicortin Metabolites in the Lepidopteran Specialist Herbivore Cerura vinula.

Salicortin is a phenolic glucoside produced in Salicaceae as a chemical defense against herbivory. The specialist lepidopteran herbivorous larvae of Cerura vinula are able to overcome this defense. We examined the main frass constituents of C. vinula fed on Populus nigra leaves, and identified 11 quinic acid derivatives with benzoate and/or salicylate substitution. We asked whether the compounds are a result of salicortin breakdown and sought answers by carrying out feeding experiments with highly 13C-enriched salicortin. Using HRMS and NMR analyses, we were able to confirm that salicortin metabolism in C. vinula proceeds through deglucosylation and ester hydrolysis, after which saligenin is oxidatively transformed into salicylic acid and, eventually, conjugated to quinic acid. To the best of our knowledge, this is the first report of a detoxification pathway based on conjugation with quinic acid.

Selective Utilization of Benzimidazolyl-Norcobamides as Cofactors by the Tetrachloroethene Reductive Dehalogenase of Sulfurospirillum multivorans.

The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

Co-option of the sphingolipid metabolism for the production of nitroalkene defensive chemicals in termite soldiers.

The aliphatic nitroalkene (E)-1-nitropentadec-1-ene (NPD), reported in early seventies in soldiers of the termite genus Prorhinotermes, was the first documented nitro compound produced by insects. Yet, its biosynthetic origin has long remained unknown. Here, we investigated in detail the biosynthesis of NPD in P. simplex soldiers. First, we track the dynamics in major metabolic pathways during soldier ontogeny, with emphasis on likely NPD precursors and intermediates. Second, we propose a hypothesis of NPD formation and verify its individual steps using in vivo incubations of putative precursors and intermediates. Third, we use a de novo assembled RNA-Seq profiles of workers and soldiers to identify putative enzymes underlying NPD formation. And fourth, we describe the caste- and age-specific expression dynamics of candidate initial genes of the proposed biosynthetic pathway. Our observations provide a strong support to the following biosynthetic scenario of NPD formation, representing an analogy of the sphingolipid pathway starting with the condensation of tetradecanoic acid with l-serine and leading to the formation of a C16 sphinganine. The C16 sphinganine is then oxidized at the terminal carbon to give rise to 2-amino-3-hydroxyhexadecanoic acid, further oxidized to 2-amino-3-oxohexadecanoic acid. Subsequent decarboxylation yields 1-aminopentadecan-2-one, which then proceeds through six-electron oxidation of the amino moiety to give rise to 1-nitropentadecan-2-one. Keto group reduction and hydroxyl moiety elimination lead to NPD. The proposed biosynthetic sequence has been constructed from age-related quantitative dynamics of individual intermediates and confirmed by the detection of labeled products downstream of the administered labeled intermediates. Comparative RNA-Seq analyses followed by qRT-PCR validation identified orthologs of serine palmitoyltransferase and 3-ketodihydrosphingosine reductase genes as highly expressed in the NPD production site, i.e. the frontal gland of soldiers. A dramatic onset of expression of the two genes in the first days of soldier's life coincides with the start of NPD biosynthesis, giving further support to the proposed biosynthetic hypothesis.

Local phytochemical response of Musa acuminata × balbisiana Colla cv. 'Bluggoe' (ABB) to colonization by Sternorrhyncha.

The interaction of two Sternorrhyncha species, the banana aphid (Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae, Aphidinae)), vector of the banana bunchy top virus (BBTV), and the latania scale (Hemiberlesia lataniae Signoret (Hemiptera: Diaspididae, Diaspidinae)) with Musa acuminata × balbisiana Colla (ABB Group) 'Bluggoe' (Musaceae) was investigated by a combination of conventional and spatially resolved analytical techniques, 1H NMR, UHPLC-MS, and matrix-free UV-laser desorption/ionization MS imaging. After infestation, the feeding sites of P. nigronervosa on the pseudostem and the exocarp of banana fruit developed a red tinge, in which tissue-specific accumulations of phenylphenalenones were discovered. Phenylphenalenones were also detected in the black mats of sooty molds growing on the banana aphid exudates and in the dorsal scales of H. lataniae. This suggests that although these secondary metabolites play a role in the reaction of banana plants towards attack by sucking insects, an aphid and an armored scale have established mechanisms to exude these metabolites before they deploy their deleterious effect.

Cultured roots of Xiphidium caeruleum: Phenylphenalenones and their biosynthetic and extractant-dependent conversion.

Phytochemical investigation of root cultures of Xiphidium caeruleum (Haemodoraceae) resulted in the structure elucidation of five previously undescribed phenylphenalenone-type compounds, structure revision of a phenylphenalenone glucoside, and identification of nine additional constituents previously reported from other Haemodoraceae and Musaceae plants. The observed extractant-dependent metabolic profiles indicated that phenylphenalenones had been converted hydrolytically and oxidatively. Stable isotope labeling experiments extended the understanding of the phenylphenalenone pathway in plants and provided evidence for a network of biosynthetic and spontaneous conversions linking phenylphenalenones and their derivatives detected in extracts of cultured roots of this plant.

Glaciations, gradients, and geography: multiple drivers of diversification of bush frogs in the Western Ghats Escarpment.

The historical processes underlying high diversity in tropical biodiversity hotspots like the Western Ghats of Peninsular India remain poorly understood. We sampled bush frogs on 13 massifs across the Western Ghats Escarpment and examined the relative influence of Quaternary glaciations, ecological gradients and geological processes on the spatial patterns of lineage and clade diversification. The results reveal a large in situ radiation (more than 60 lineages), exhibiting geographical structure and clade-level endemism, with two deeply divergent sister clades, North and South, highlighting the biogeographic significance of an ancient valley, the Palghat Gap. A majority of the bush frog sister lineages were isolated on adjacent massifs, and signatures of range stasis provide support for the dominance of geological processes in allopatric speciation. In situ diversification events within the montane zones (more than 1800 m) of the two highest massifs suggest a role for climate-mediated forest-grassland persistence. Independent transitions along elevational gradients among sub-clades during the Miocene point to diversification along the elevational gradient. The study highlights the evolutionary significance of massifs in the Western Ghats with the high elevations acting as centres of lineage diversification and the low- and mid-elevations of the southern regions, with deeply divergent lineages, serving as museums.

Phenylbenzoisoquinolindione alkaloids accumulate in stamens of Xiphidium caeruleum Aubl. flowers.

Xiphidium caeruleum (Haemodoraceae) flower organs such as carpels, pedicels, petals, and stamens were separately investigated for their phytochemical profile. The stamens appeared to be a rich source of previously undescribed phenylbenzoisoquinolindiones, a group of phenylphenalenone-derived alkaloids, also named aza-phenylphenalenones. Nine previously undescribed compounds with an identical aza-phenylphenalenone core structure but different amino acid-derived side chains at position 2 were isolated and their structures elucidated by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). In addition, some previously reported phenylbenzoisoquinolindiones, phenylbenzoisochromenones and flavonoids were found in stamens and other parts of the flowers. The specific occurrence of heterocyclic phenylphenalenone-type compounds in X. caeruleum suggests these are involved in physiological or ecological processes.

Phenylphenalenones protect banana plants from infection by Mycosphaerella fijiensis and are deactivated by metabolic conversion.

Phenylphenalenones, polycyclic aromatic natural products from some monocotyledonous plants, are known as phytoalexins in banana (Musa spp.). In this study, (1) H nuclear magnetic resonance (NMR)-based metabolomics along with liquid chromatography and mass spectrometry were used to explore the chemical responses of the susceptible 'Williams' and the resistant 'Khai Thong Ruang' Musa varieties to the ascomycete fungus Mycosphaerella fijiensis, the agent of the black leaf Sigatoka disease. Principal component analysis discriminated strongly between infected and non-infected plant tissue, mainly because of specialized metabolism induced in response to the fungus. Phenylphenalenones are among the major induced compounds, and the resistance level of the plants was correlated with the progress of the disease. However, a virulent strain of M. fijiensis was able to overcome plant resistance by converting phenylphenalenones to sulfate conjugates. Here, we report the first metabolic detoxification of fungitoxic phenylphenalenones to evade the chemical defence of Musa plants.

Chemical Composition and Antimicrobial Activity of Populus nigra Shoot Resin.

The chemical composition of Populus nigra shoot resin has been investigated by chromatographic and spectroscopic methods. The analyses resulted in identification of 19 known compounds. The resin exhibited low activity against selected microorganisms.

High resolution mass spectrometry imaging reveals the occurrence of phenylphenalenone-type compounds in red paracytic stomata and red epidermis tissue of Musa acuminata ssp. zebrina cv. 'Rowe Red'.

The banana epidermis and in particular their stomata are conducive sites for the penetration of pathogenic fungi which can severely limit global banana production. The red pseudostem of the ornamental banana Musa acuminata ssp. zebrina cv. 'Rowe Red' was used to study the chemical constituents of the epidermal cell layer using matrix-free laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometric imaging (LDI-FT-ICR-MSI). The high resolution of this technique allowed phenylphenalenone-type compounds to be located in single plant cells. Some of these secondary metabolites were identified as constitutive compounds and found in specialized epidermal cells in banana pseudostem tissue. Especially the red paracytic stomata revealed higher signal intensities of certain phenylphenalenones than normal epidermis cells. The ease of detection of polycyclic aromatic compounds on the cellular level is discussed with regard to future investigations of plant-pathogen interactions.

Monitoring metabolites from Schizophyllum commune interacting with Hypholoma fasciculare combining LESA-HR mass spectrometry and Raman microscopy.

Microbial competition for territory and resources is inevitable in habitats with overlap between niches of different species or strains. In fungi, competition is brought about by antagonistic mycelial interactions which alter mycelial morphology, metabolic processes, secondary metabolite release, and extracellular enzyme patterns. Until now, we were not able study in vivo chemical interactions of different colonies growing on the same plate. In this report, we developed a fast and least invasive approach to identify, quantify, and visualize co culture-induced metabolites and their location of release within Schizophyllum commune. The pigments indigo, indirubin, and isatin were used as examples to show secondary metabolite production in the interaction zone with Hypholoma fasciculare. Using a combinatory approach of Raman spectroscopy imaging, liquid extraction surface analysis (LESA), and high-resolution mass spectrometry, we identified, quantified, and visualized the presence of indigo and indirubin in the interaction zone. This approach allows the investigation of metabolite patterns between wood degrading species in competition to gain insight in community interactions, but could also be applied to other microorganisms. This method advances analysis of living, still developing colonies and are in part not destructive as Raman spectroscopy imaging is implemented.