Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60-75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the resistance of C. difficile to common decontamination methods. These results further demonstrate that the vegetative cells and spores of various C. difficile strains have different resistance properties that may impact decontamination of surfaces and hands.

At an altitude of 3,570 m, the volcanic lake Socompa in the Argentinean Andes is presently the highest site where actively forming stromatolite-like structures have been reported. Interestingly, pigment and microsensor analyses performed through the different layers of the stromatolites (50 mm-deep) showed steep vertical gradients of light and oxygen, hydrogen sulfide and pH in the porewater. Given the relatively good characterization of these physico-chemical gradients, the aim of this follow-up work was to specifically address how the bacterial diversity stratified along the top six layers of the stromatolites which seems the most metabolically important and diversified zone of the whole microbial community. We herein discussed how, in only 7 mm, a drastic succession of metabolic adaptations occurred: i.e., microbial communities shift from a UV-high/oxic world to an IR-low/anoxic/high H2S environment which force stratification and metabolic specialization of the bacterial community, thus, modulating the chemical faces of the Socompa stromatolites. The oxic zone was dominated by Deinococcus sp. at top surface (0.3 mm), followed by a second layer of Coleofasciculus sp. (0.3 to ∼2 mm). Sequences from anoxygenic phototrophic Alphaproteobacteria, along with an increasing diversity of phyla including Bacteroidetes, Spirochaetes were found at middle layers 3 and 4. Deeper layers (5-7 mm) were mostly occupied by sulfate reducers of Deltaproteobacteria, Bacteroidetes and Firmicutes, next to a high diversity and equitable community of rare, unclassified and candidate phyla. This analysis showed how microbial communities stratified in a physicochemical vertical profile and according to the light source. It also gives an insight of which bacterial metabolic capabilities might operate and produce a microbial cooperative strategy to thrive in one of the most extreme environments on Earth.

Diatoms (Bacillariophyceae) are aquatic photosynthetic microalgae with an ecological role as primary producers in the aquatic food web. They account substantially for global carbon, nitrogen, and silicon cycling. Elucidating the chemical space of diatoms is crucial to understanding their physiology and ecology. To expand the known chemical space of a cosmopolitan marine diatom, Skeletonema marinoi, we performed High-Resolution Liquid Chromatography-Tandem Mass Spectrometry (LC-MS2) for untargeted metabolomics data acquisition. The spectral data from LC-MS2 was used as input for the Metabolome Annotation Workflow (MAW) to obtain putative annotations for all measured features. A suspect list of metabolites previously identified in the Skeletonema spp. was generated to verify the results. These known metabolites were then added to the putative candidate list from LC-MS2 data to represent an expanded catalog of 1970 metabolites estimated to be produced by S. marinoi. The most prevalent chemical superclasses, based on the ChemONT ontology in this expanded dataset, were organic acids and derivatives, organoheterocyclic compounds, lipids and lipid-like molecules, and organic oxygen compounds. The metabolic profile from this study can aid the bioprospecting of marine microalgae for medicine, biofuel production, agriculture, and environmental conservation. The proposed analysis can be applicable for assessing the chemical space of other microalgae, which can also provide molecular insights into the interaction between marine organisms and their role in the functioning of ecosystems.

Local and regional associations between bacterial communities and nutrient and chemical concentrations were assessed in the Upper Mississippi River in Minnesota to determine if community structure was associated with discrete types of chemical inputs associated with different land cover. Bacterial communities were characterized by Illumina sequencing of the V6 region of 16S rDNA and compared to >40 chemical and nutrient concentrations. Local bacterial community structure was shaped primarily by associations among bacterial orders. However, order abundances were correlated regionally with nutrient and chemical concentrations, and were also related to major land coverage types. Total organic carbon and total dissolved solids were among the primary abiotic factors associated with local community composition and co-varied with land cover. Escherichia coli concentration was poorly related to community composition or nutrient concentrations. Abundances of 14 bacterial orders were related to land coverage type, and seven showed significant differences in abundance (P ≤ 0.046) between forested or anthropogenically-impacted sites. This study identifies specific bacterial orders that were associated with chemicals and nutrients derived from specific land cover types and may be useful in assessing water quality. Results of this study reveal the need to investigate community dynamics at both the local and regional scales and to identify shifts in taxonomic community structure that may be useful in determining sources of pollution in the Upper Mississippi River.

In agroecosystems, different cropping patterns cause changes in soil physicochemical properties and thus in microbial communities, which in turn affect crop yields. In this study, the yields of soybean continuous cropping for 5 years (C5), 10 years (C10), and 20 years (C20) and of soybean-corn rotational cropping (R) treatments were determined, and samples of the tillage layer soil were collected. High-throughput sequencing technology was used to analyze the diversity and composition of the soil bacterial and fungal communities. The factors influencing microbial communities, along with the effects of these communities and those of soil chemical indexes on yield, were further evaluated. The results showed that the community richness index of bacteria was higher in C20 than in R and that of fungi was highest in C5. The differences in the bacterial and fungal communities diversity indexes were not significant among the different continuous cropping treatments, respectively. The soil microbial community composition of all continuous cropping treatments differed significantly from R. The dominant bacterial phylum was Actinobacteriota and the dominant fungal phylum was Ascomycota. The relative abundance of Fusarium did not differ significantly among the continuous cropping treatments, while that of the plant pathogen fungi Lectera sp., Plectosphaerella sp., and Volutella sp. increased with continuous cropping years. Soil pH, SOM, N, and TP had significant effects on both bacterial and fungal communities, and TK and C/N had highly significant effects on fungal communities. The yield of C5 was significantly lower than that of R, and the differences in yield between C10, C20, and R were not significant. TN, TP, and pH had significant effects on yield, and fungal community abundance had a greater negative effect on yield than bacterial community abundance.

The dried tuber of Alisma orientale (Sam.) Juzep. (AOJ) is a traditional Chinese medicine with high medicinal value. The endophytic fungi of medicinal plants are a treasure house of natural compounds. However, there is a lack of research on the diversity and biological activity of endophytic fungi of AOJ. In this study, high-throughput sequencing technology was used to study the diversity of endophytic fungi in the roots and stems of AOJ, and endophytic fungi with a high output of phenols and flavonoids were screened by chromogenic reaction, and the antioxidant and antibacterial activities and chemical constituents of crude extracts of their fermentation broth were studied. A total of 3,426 amplicon sequence variants (ASVs) belonging to 9 phyla, 27 classes, 64 orders, 152 families, and 277 genera were identified from AOJ. There were significant differences in the endophytic fungal communities of AOJ roots and stems, as well as in the endophytic fungal communities of triangular AOJ and circular AOJ. In addition, 31 strains of endophytic fungi were isolated from AOJ, of which 6 strains had good antioxidant and antibacterial activities. The crude extract of YG-2 had the strongest free radical scavenging ability and bacteriostatic ability, and its IC50 DPPH, IC50 ABTS, and IC50⋅OH values were 0.009 ± 0.000 mg/mL, 0.023 ± 0.002 mg/mL, and 0.081 ± 0.006 mg/mL, respectively. The results of LC-MS showed that the main component of the crude extract of YG-2 was caffeic acid (10.12 μmol/g). Overall, the results of this study preliminarily elucidated the diversity and community composition of endophytic fungi of AOJ, indicating that AOJ endophytic fungi have abundant secondary metabolites and good antioxidant and antibacterial activities. This study provides an important reference for further research, development and utilization of AOJ endophytic fungi and a theoretical basis for the further development of the endophytic fungus YG-2 (Chaetomium globosum) as a source of antioxidants.

Traditional Chinese fermented vegetables are a type of brine-salted fermented vegetable product. During the spontaneous fermentation, various compounds are produced, degraded, and converted, influencing the quality of the fermented pickle. To ascertain the effect of different containers on the fermentation process of the pickles, this study investigated the bacterial diversity and the chemical composition characteristics of the pickle (radish) fermented in commonly used containers including glass jars (GL), porcelain jars (PO), and plastic jars (PL). The correlation between chemical compounds and microbial community was further analyzed. The changes in pH values suggested that PL may facilitate the quickest fermentation of the pickles, while the process in PO progressed at the lowest rate. The PL brine samples contained higher levels of lactic acid and threonine, while more abundant volatile chemical compounds were evident in PO. The container materials had no significant influence on the microbial structure, wherein Lactobacillus was the absolute dominant genus in all containers. But container material did have an effect on the abundance of specific genus, such as Lactococcus and Pediococcus. The correlation between these major genera was also analyzed and gene function prediction indicated that the top three pathways were: carbohydrate metabolism, amino acid metabolism, and energy metabolism. Lactobacillus negatively correlated with methionine, tyrosine, lysine, and arginine, but positively correlated with ammonia, and lactic acid and acetic acid both just correlated with Pediococcus. This study provides new insights into the microbiota succession and chemical compounds involved in the vegetable fermentation.

Chlordecone (CLD) is a very persistent synthetic organochlorine pesticide found in the French West Indies. Recently published work has demonstrated the potential of zero-valent iron to dechlorinate CLD by in situ chemical reduction (ISCR) in soils under water-saturated conditions, forming mono- to penta-dechlorinated CLD transformation products. These transformation products are more mobile than CLD and less toxic; however, nothing is known about their further degradation, although increasing evidence of CLD biodegradation by bacteria is being found. The present study began with the enrichment from wastewater sludge of a CLD-transforming community which was then inoculated into fresh media in the presence of either CLD or two of the main ISCR transformation products, 10-monohydroCLD (-1Cl-CLD) and tri-hydroCLD (-3Cl-CLD). Carried out in triplicate batches and incubated at 38°C under anoxic conditions and in the dark, the cultures were sampled regularly during 3 months and analyzed for CLD, -1Cl-CLD, -3Cl-CLD, and possible transformation products by gas chromatography coupled to mass spectrometry. All batches showed a decrease in the amended substrates (CLD or hydroCLD). CLD degradation occurred with concomitant formation of a nine-carbon compound (pentachloroindene) and two sulfur-containing transformation products (chlordecthiol, CLD-SH; methyl chlordecsulfide, CLD-SCH3), demonstrating competing transformation pathways. In contrast, -1Cl-CLD and -3Cl-CLD only underwent a sequential reductive sulfidation/S-methylation process resulting in -1Cl-CLD-SH and -1Cl-CLD-SCH3 on the one hand, and -3Cl-CLD-SH, -3Cl-CLD-SCH3 on the other hand. Some sulfur-containing transformation products have been reported previously with single bacterial strains, but never in the presence of a complex microbial community. At the end of the experiment, bacterial and archaeal populations were investigated by 16S rRNA gene amplicon sequencing. The observed diversity was mostly similar in the CLD and -1Cl-CLD conditions to the inoculum with a dominant archaea genus, Methanobacterium, and four OTU affiliated to bacteria, identified at the family (Spirochaetaceae) or genus level (Desulfovibrio, Aminobacterium, and Soehngenia). On the other hand, in the -3Cl-CLD condition, although the same OTU were found, Clostridium sensu stricto 7, Candidatus Cloacimonas, and Proteiniphilum were also present at > 2% sequences. Presence of methanogens and sulfate-reducing bacteria could contribute to sulfidation and S-methylation biotransformations. Overall, these results contribute to increasing our knowledge on the biodegradability of CLD and its transformation products, helping to progress toward effective remediation solutions.

The soft coral-derived fungus Trichoderma harzianum (XS-20090075) was found to be a potential strain to produce substantial new compounds in our previous study. In order to explore its potential to produce more metabolites, chemical epigenetic manipulation was used on this fungus to wake its sleeping genes, leading to the significant changes of its secondary metabolites by using a histone deacetylase (HDAC) inhibitor. The most obvious difference was the original main products harziane diterpenoids were changed into cyclonerane sesquiterpenoids. Three new terpenoids were isolated from the fungal culture treated with 10 μM sodium butyrate, including cleistanthane diterpenoid, harzianolic acid A (1), harziane diterpenoid, harzianone E (2), and cyclonerane sesquiterpenoid, 3,7,11-trihydroxy-cycloneran (3), together with 11 known sesquiterpenoids (4-14). The absolute configurations of 1-3 were determined by single-crystal X-ray diffraction, ECD and OR calculations, and biogenetic considerations. This was the first time to obtain cleistanthane diterpenoid and africane sesquiterpenoid from genus Trichoderma, and this was the first chlorinated cleistanthane diterpenoid. These results demonstrated that the chemical epigenetic manipulation should be an efficient technique for the discovery of new secondary metabolites from marine-derived fungi.

Clubroot disease, caused by Plasmodiophora brassicae, is a serious soil-borne disease in Brassica crops worldwide. It seriously occurs in conducive soils of southern China, while never happens in some areas of northern China with suppressive soils. To understanding the differences, we measured the soil suppressiveness, chemical properties, and microbial communities in suppressive and conducive soils by bioassay and sequencing of 16S and 18S rRNA amplicons. The biological basis of clubroot suppressiveness was supported by the ability to remove it by pasteurization. The pH value and calcium content in the suppressive soils were higher than those in the conducive soils. Suppressive soils were associated with higher fungal diversity and bacterial abundance. The fungal phyla Chytridiomycota, Olpidiomycota, and Mucoromycota and the bacterial phyla Acidobacteriota and Gemmatimonadota were enriched in suppressive soils. More abundant beneficial microbes, including Chaetomium and Lysobacter, were found in the suppressive soils than in the conducive soils. Molecular ecological network analysis revealed that the fungal network of suppressive soils was more complex than that of conducive soils. Our results indicate that plant health is closely related to soil physicochemical and biological properties. This study is of great significance for developing strategies for clubtroot disease prevention and control.

This study was aimed to measure the impact of the application of a bio-fungicide against Botrytis cinerea on the microbiota involved in the alcoholic fermentation (AF) of Tempranillo Rioja wines. For this purpose, a bio-fungicide composed of the biological control bacterium Bacillus subtilis QST713 was applied to the vineyard. The microbial diversity was analyzed from grape biofilm to wine. Impact on microbial diversity was measured employing indexes assessed with the software PAST 3.10 P.D. Results were compared to non-treated samples and to samples treated with a chemical fungicide mainly composed by fenhexamid. Overall, the impact of the biological-fungicide (bio-fungicide) on the microbial diversity assessed for grape biofilm and for musts was not remarkable. Neither of the tested fungicides enhanced the growth of any species or acted against the development of any microbial groups. The bio-fungicide had no significant impact on the wine microbiota whereas the chemical fungicide caused a reduction of microbial community richness and diversity. Although environmental threats might generate a detriment of the microbial species richness, in this study the tested bio-fungicide did not modify the structure of the microbial community. Indeed, some of the Bacillus applied at the grape surface, were detected at the end of the AF showing its resilience to the harsh environment of the winemaking; in contrast, its impact on wine quality during aging is yet unknown.

Orfamide-type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudomonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudomonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P. protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens.

The chemical engineering of natural extracts has emerged as an effective strategy for the production of diverse libraries of chemicals, making it integral to drug discovery. A chemical engineering strategy based on the epoxidation and ring-opening reactions was used to prepare diversity-enhanced extracts of Chaetomium madrasense 375. Eleven unnatural cytochalasan derivatives (1-11) with unique functional groups, such as amine and isoxazole, were isolated and characterized from these chemically engineered extracts of C. madrasense 375. The identification of these new structures was accomplished through comprehensive spectroscopic analysis, supplemented by synthetic considerations. Notably, compounds 5 and 13-16 displayed potent phytotoxic effects on Arabidopsis thaliana, while compounds 1, 2, 5, 10, and 12 demonstrated inhibitory activities on LPS-induced NO production in RAW264.7 cells. Among them, compound 1 was found to be able to inhibit the upregulated expression of the inducible nitric oxide synthase (iNOS) protein induced by LPS, while also decreasing the production of pro-inflammatory cytokines (IL-6) and influencing the phosphorylation of p38, ERK1/2, and JNK at 100 μM. Our findings demonstrate that the chemical engineering of natural product extracts can be an efficient technique for the generation of novel bioactive molecules.

In this study, we conducted tests on the isolation, identification, characterization, and extraction of chemical molecules from Beauveria bassiana against Tuta absoluta larvae. The enzyme responses of T. absoluta to the crude extract were examined 24 h after treatment, and the number of dead larvae was calculated 24 and 48 h after treatment. Molecular docking studies were conducted to assess the interaction of important molecules with the acetylcholinesterase enzyme. The larvicidal activity of crude chemicals from fungi was high 24 h after treatment, with LC50 and LC90 values of 25.937 and 33.559 μg/mL, respectively. For a period of 48 h, the LC50 and LC90 values were 52.254 and 60.450 μg/mL, respectively. The levels of acetylcholinesterase, α-carboxylesterase, and β-carboxylesterase enzymes were lower in the treatment group after 24 h compared to the control group. The GC-MS test revealed that the crude extract consisted mainly of 9,10-octadecadienoic acid, which was the primary compound. Docking results indicated that 9,10-octadecadienoic acid showed a strong interaction with acetylcholinesterase (AChE). Our findings suggest that the chemical molecule 9,10-octadecadienoic acid derived from the entomopathogenic fungus B. bassiana is more toxic to T. absoluta larvae. We plan to conduct studies to test its effectiveness in semi-field conditions and to evaluate its stability in field conditions. We believe that this 9,10-octadecadienoic acid molecule could be used to control T. absoluta larvae in the near future without causing environmental pollution.

The application of biogas slurry and chemical fertilizer in paddy fields can be a practical method to reduce the environmental risk and utilize the nutrients of biogas slurry. The responses of bacterial and fungal communities to the application of biogas slurry and chemical fertilizer are important reflections of the quality of the ecological environment. In this study, based on a 3-year field experiment with different ratios of biogas slurry and chemical fertilizer (applying the same pure nitrogen amount), the Illumina MiSeq platform was used to investigate the bacterial and fungal community diversity and composition in paddy soil. Our results revealed that compared with the observations under regular chemical fertilization, on the basis of stable paddy yield, the application of biogas slurry combined with chemical fertilizer significantly enhanced the soil nutrient availability and bacterial community diversity and reduced the fungal community diversity. Dissolved organic carbon (DOC), DOC/SOC (soil organic carbon), available nitrogen (AN) and available phosphorus (AP) were positively correlated with the bacterial community diversity, but no soil property was significantly associated with the fungal community. The bacterial community was primarily driven by the application of biogas slurry combined with chemical fertilizer (40.78%), while the fungal community was almost equally affected by the addition of pure biogas slurry, chemical fertilizer and biogas slurry combined with chemical fertilizer (25.65-28.72%). Biogas slurry combined with chemical fertilizer significantly enriched Proteobacteria, Acidobacteria, Planctomycetes, Rokubacteria, and Ascomycota and depleted Chloroflexi, Bacteroidetes, Crenarchaeota, Basidiomycota, and Glomeromycota. The observation of the alteration of some bacteria- and fungus-specific taxa provides insights for the proper application of biogas slurry combined with chemical fertilizer, which has the potential to promote crop growth and inhibit pathogens.

The oomycete Phytophthora infestans is the causal agent of tomato and potato late blight, a disease that causes tremendous economic losses in the production of solanaceous crops. The similarities between oomycetes and the apicomplexa led us to hypothesize that dihydroorotate dehydrogenase (DHODH), the enzyme catalyzing the fourth step in pyrimidine biosynthetic pathway, and a validated drug target in treatment of malaria, could be a potential target for controlling P. infestans growth. In eukaryotes, class 2 DHODHs are mitochondrially associated ubiquinone-linked enzymes that catalyze the fourth, and only redox step of de novo pyrimidine biosynthesis. We characterized the enzymes from both the pathogen and a host, Solanum tuberosum. Plant DHODHs are known to be class 2 enzymes. Sequence analysis suggested that the pathogen enzyme (PiDHODHs) also belongs to this class. We confirmed the mitochondrial localization of GFP-PiDHODH showing colocalization with mCherry-labeled ATPase in a transgenic pathogen. N-terminally truncated versions of the two DHODHs were overproduced in E. coli, purified, and kinetically characterized. StDHODH exhibited a apparent specific activity of 41 ± 1 μmol min-1 mg-1, a kcat app of 30 ± 1 s-1, and a Km app of 20 ± 1 μM for L-dihydroorotate, and a Km app= 30 ± 3 μM for decylubiquinone (Qd). PiDHODH exhibited an apparent specific activity of 104 ± 1 μmol min-1 mg-1, a kcat app of 75 ± 1 s-1, and a Km app of 57 ± 3 μM for L-dihydroorotate, and a Km app of 15 ± 1 μM for Qd. The two enzymes exhibited different activities with different quinones and napthoquinone derivatives, and different sensitivities to compounds known to cause inhibition of DHODHs from other organisms. The IC50 for A77 1726, a nanomolar inhibitor of human DHODH, was 2.9 ± 0.6 mM for StDHODH, and 79 ± 1 μM for PiDHODH. In vivo, 0.5 mM A77 1726 decreased mycelial growth by approximately 50%, after 92 h. Collectively, our findings suggest that the PiDHODH could be a target for selective inhibitors and we provide a biochemical background for the development of compounds that could be helpful for the control of the pathogen, opening the way to protein crystallization.

QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, United States) and RBB + C (Yu and Morrison, 2004) methodologies are widely employed to extract microbial DNA from rumen samples and can exhibit different efficiencies of obtaining DNA yield, quality, and downstream amplicon sequence analysis. No study has conducted to investigate the contributions of chemical and mechanical lysis on DNA extraction, which included chemical lysis from QIAamp Fast DNA Stool Mini Kit (QIA) and RBB + C (YM), bead (BB), and sand beating (SB). Effects of chemical lysis and bead beating (BB) were investigated by conducting a 2 × 2 factorial-designed experiment with four methodologies, including QIA without (QIA-) and with BB (QIA + BB), and YM without (YM-) and with BB (YM + BB). Comparisons between bead and sand were conducted by comparing methodologies of YM + BB and YM + SB. Comparing with QIA, YM had lower (P ≤ 0.10) OD260 / 280 and diversity of ZOTUs and length polymorphism of protozoal amplicons but harvested greater (P ≤ 0.086) DNA from fibrolytic bacteria such as Ruminococcaceae lineages. Including BB increased (P = 0.001) total DNA yield without affecting (P ≥ 0.55) OD260 / 280 and richness of bacterial ZOTUs but decreased (P ≤ 0.08) richness of both ZOTUs and length polymorphism of protozoal amplicon. Bead beating and SB showed no difference (P ≥ 0.19) in DNA yield and quality and bacterial and protozoal community. In summary, chemical lysis provided by RBB + C and QIAamp Fast DNA Stool Mini Kit should be better to extract DNA for analyzing bacterial and protozoal community, respectively. Sand can be an alternative beater for DNA extraction, and mechanical lysis is not recommended for protozoal community analysis.

The use of additives in food products has become an important public health concern. In recent reports, dietary emulsifiers have been shown to affect the gut microbiota, contributing to a pro-inflammatory phenotype and metabolic syndrome. So far, it is not yet known whether similar microbiome shifts are observable for a more diverse set of emulsifier types and to what extent these effects vary with the unique features of an individual's microbiome. To bridge this gap, we investigated the effect of five dietary emulsifiers on the fecal microbiota from 10 human individuals upon a 48 h exposure. Community structure was assessed with quantitative microbial profiling, functionality was evaluated by measuring fermentation metabolites, and pro-inflammatory properties were assessed with the phylogenetic prediction algorithm PICRUSt, together with a TLR5 reporter cell assay for flagellin. A comparison was made between two mainstream chemical emulsifiers (carboxymethylcellulose and P80), a natural extract (soy lecithin), and biotechnological emulsifiers (sophorolipids and rhamnolipids). While fecal microbiota responded in a donor-dependent manner to the different emulsifiers, profound differences between emulsifiers were observed. Rhamnolipids, sophorolipids, and soy lecithin eliminated 91 ± 0, 89 ± 1, and 87 ± 1% of the viable bacterial population after 48 h, yet they all selectively increased the proportional abundance of putative pathogens. Moreover, profound shifts in butyrate (-96 ± 6, -73 ± 24, and -34 ± 25%) and propionate (+13 ± 24, +88 ± 50, and +29 ± 16%) production were observed for these emulsifiers. Phylogenetic prediction indicated higher motility, which was, however, not confirmed by increased flagellin levels using the TLR5 reporter cell assay. We conclude that dietary emulsifiers can severely impact the gut microbiota, and this seems to be proportional to their emulsifying strength, rather than emulsifier type or origin. As biotechnological emulsifiers were especially more impactful than chemical emulsifiers, caution is warranted when considering them as more natural alternatives for clean label strategies.

Streptomyces are Gram-positive bacteria that occupy diverse ecological niches including host-associations with animals and plants. Members of this genus are known for their overwhelming repertoire of natural products, which has been exploited for almost a century as a source of medicines and agrochemicals. Notwithstanding intense scientific and commercial interest in Streptomyces natural products, surprisingly little is known of the intra- and/or inter-species ecological roles played by these metabolites. In this report we describe the chemical structures, biological properties, and biosynthetic relationships between natural products produced by Streptomyces isolated from internal tissues of predatory Conus snails, collected from the Great Barrier Reef, Australia. Using chromatographic, spectroscopic and bioassays methodology, we demonstrate that Streptomyces isolated from five different Conus species produce identical chemical and antifungal profiles - comprising a suite of polycyclic tetramic acid macrolactams (PTMs). To investigate possible ecological (and evolutionary) relationships we used genome analyses to reveal a close taxonomic relationship with other sponge-derived and free-living PTM producing Streptomyces (i.e., Streptomyces albus). In-depth phylogenomic analysis of PTM biosynthetic gene clusters indicated PTM structure diversity was governed by a small repertoire of genetic elements, including discrete gene acquisition events involving dehydrogenases. Overall, our study shows a Streptomyces-Conus ecological relationship that is concomitant with specific PTM chemical profiles. We provide an evolutionary framework to explain this relationship, driven by anti-fungal properties that protect Conus snails from fungal pathogens.

Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.