Mapping Geological Events and Nitrogen Fixation Evolution Onto the Timetree of the Evolution of Nitrogen-Fixation Genes.

Nitrogen is essential for all organisms, but biological nitrogen fixation (BNF) occurs only in a small fraction of prokaryotes. Previous studies divided nitrogenase-gene-carrying prokaryotes into Groups I to IV and provided evidence that BNF first evolved in bacteria. This study constructed a timetree of the evolution of nitrogen-fixation genes and estimated that archaea evolved BNF much later than bacteria and that nitrogen-fixing cyanobacteria evolved later than 1,900 MYA, considerably younger than the previous estimate of 2,200 MYA. Moreover, Groups III and II/I diverged ∼2,280 MYA, after the Kenorland supercontinent breakup (∼2,500-2,100 MYA) and the Great Oxidation Event (∼2,400-2,100 MYA); Groups III and Vnf/Anf diverged ∼2,086 MYA, after the Yarrabubba impact (∼2,229 MYA); and Groups II and I diverged ∼1,920 MYA, after the Vredefort impact (∼2,023 MYA). In summary, this study provided a timescale of BNF events and discussed the possible effects of geological events on BNF evolution.

Origin and Evolution of Nitrogen Fixation in Prokaryotes.

The origin of nitrogen fixation is an important issue in evolutionary biology. While nitrogen is required by all living organisms, only a small fraction of bacteria and archaea can fix nitrogen. The prevailing view is that nitrogen fixation first evolved in archaea and was later transferred to bacteria. However, nitrogen-fixing (Nif) bacteria are far larger in number and far more diverse in ecological niches than Nif archaea. We, therefore, propose the bacteria-first hypothesis, which postulates that nitrogen fixation first evolved in bacteria and was later transferred to archaea. As >30,000 prokaryotic genomes have been sequenced, we conduct an in-depth comparison of the two hypotheses. We first identify the six genes involved in nitrogen fixation in all sequenced prokaryotic genomes and then reconstruct phylogenetic trees using the six Nif proteins individually or in combination. In each of these trees, the earliest lineages are bacterial Nif protein sequences and in the oldest clade (group) the archaeal sequences are all nested inside bacterial sequences, suggesting that the Nif proteins first evolved in bacteria. The bacteria-first hypothesis is further supported by the observation that the majority of Nif archaea carry the major bacterial Mo (molybdenum) transporter (ModABC) rather than the archaeal Mo transporter (WtpABC). Moreover, in our phylogeny of all available ModA and WtpA protein sequences, the earliest lineages are bacterial sequences while archaeal sequences are nested inside bacterial sequences. Furthermore, the bacteria-first hypothesis is supported by available isotopic data. In conclusion, our study strongly supports the bacteria-first hypothesis.

Retroconversion of estrogens into androgens by bacteria via a cobalamin-mediated methylation.

Steroid estrogens modulate physiology and development of vertebrates. Conversion of C19 androgens into C18 estrogens is thought to be an irreversible reaction. Here, we report a denitrifying Denitratisoma sp. strain DHT3 capable of catabolizing estrogens or androgens anaerobically. Strain DHT3 genome contains a polycistronic gene cluster, emtABCD, differentially transcribed under estrogen-fed conditions and predicted to encode a cobalamin-dependent methyltransferase system conserved among estrogen-utilizing anaerobes; an emtA-disrupted DHT3 derivative could catabolize androgens but not estrogens. These data, along with the observed androgen production in estrogen-fed strain DHT3 cultures, suggested the occurrence of a cobalamin-dependent estrogen methylation to form androgens. Consistently, the estrogen conversion into androgens in strain DHT3 cell extracts requires methylcobalamin and is inhibited by propyl iodide, a specific inhibitor of cobalamin-dependent enzymes. The identification of the cobalamin-dependent estrogen methylation thus represents an unprecedented metabolic link between cobalamin and steroid metabolism and suggests that retroconversion of estrogens into androgens occurs in the biosphere.

Vibrio salinus sp. nov., a marine nitrogen-fixing bacterium isolated from the lagoon sediment of an islet inside an atoll in the western Pacific Ocean.

A marine, facultatively anaerobic, nitrogen-fixing bacterium, designated strain DNF-1T, was isolated from the lagoon sediment of Dongsha Island, Taiwan. Cells grown in broth cultures were Gram-negative rods that were motile by means of monotrichous flagella. Cells grown on plate medium produced prosthecae and vesicle-like structures. NaCl was required and optimal growth occurred at about 2-3% NaCl, 25-30 °C and pH 7-8. The strain grew aerobically and was capable of anaerobic growth by fermenting D-glucose or other carbohydrates as substrate. Both the aerobic and anaerobic growth could be achieved with NH4Cl as a sole nitrogen source. When N2 served as the sole nitrogen source only anaerobic growth was observed. Major cellular fatty acids were C14:0, C16:0 and C16:1 ω7c, while major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was 42.2 mol% based on the genomic DNA data. Phylogenetic analyses based on 16S rRNA genes and the housekeeping genes, gapA, pyrH, recA and gyrB, revealed that the strain formed a distinct lineage at species level in the genus Vibrio of the family Vibrionaceae. These results and those from genomic, chemotaxonomic and physiological studies strongly support the assignment of a novel Vibrio species. The name Vibrio salinus sp. nov. is proposed for the novel species, with DNF-1T (= BCRC 81209T = JCM 33626T) as the type strain. This newly proposed species represents the second example of the genus Vibrio that has been demonstrated to be capable of anaerobic growth by fixing N2 as the sole nitrogen source.

Vibrio nitrifigilis sp. nov., a marine nitrogen-fixing bacterium isolated from the lagoon sediment of an islet inside an atoll.

A nitrogen-fixing isolate of facultatively anaerobic, marine bacterium, designated strain NFV-1T, was recovered from the lagoon sediment of Dongsha Island, Taiwan. It was a Gram-negative rod which exhibited motility with monotrichous flagellation in broth cultures. The strain required NaCl for growth and grew optimally at about 25-35 °C, 3% NaCl and pH 7-8. It grew aerobically and could achieve anaerobic growth by fermenting D-glucose or other carbohydrates as substrates. NH4Cl could serve as a sole nitrogen source for growth aerobically and anaerobically, whereas growth with N2 as the sole nitrogen source was observed only under anaerobic conditions. Cellular fatty acids were predominated by C16:1 ω7c, C16:0, and C18:1 ω7c. The major polar lipids consisted of phosphatidylethanolamine and phosphatidylserine. Strain NFV-1T had a DNA G + C content of 42.5 mol%, as evaluated according to the chromosomal DNA sequencing data. Analyses of sequence similarities and phylogeny based on the 16S rRNA genes, together with the housekeeping genes, gyrB, ftsZ, mreB, topA and gapA, indicated that the strain formed a distinct species-level lineage in the genus Vibrio of the family Vibrionaceae. These phylogenetic data and those from genomic and phenotypic characterisations support the establishment of a novel Vibrio species, for which the name Vibrio nitrifigilis sp. nov. (type strain NFV-1T = BCRC 81211T = JCM 33628T) is proposed.

Long-term warming destabilizes aquatic ecosystems through weakening biodiversity-mediated causal networks.

Understanding how ecosystems will respond to climate changes requires unravelling the network of functional responses and feedbacks among biodiversity, physicochemical environments, and productivity. These ecosystem components not only change over time but also interact with each other. Therefore, investigation of individual relationships may give limited insights into their interdependencies and limit ability to predict future ecosystem states. We address this problem by analyzing long-term (16-39 years) time series data from 10 aquatic ecosystems and using convergent cross mapping (CCM) to quantify the causal networks linking phytoplankton species richness, biomass, and physicochemical factors. We determined that individual quantities (e.g., total species richness or nutrients) were not significant predictors of ecosystem stability (quantified as long-term fluctuation of phytoplankton biomass); rather, the integrated causal pathway in the ecosystem network, composed of the interactions among species richness, nutrient cycling, and phytoplankton biomass, was the best predictor of stability. Furthermore, systems that experienced stronger warming over time had both weakened causal interactions and larger fluctuations. Thus, rather than thinking in terms of separate factors, a more holistic network view, that causally links species richness and the other ecosystem components, is required to understand and predict climate impacts on the temporal stability of aquatic ecosystems.

Comparative Genome Analysis Reveals Cyanidiococcus gen. nov., A New Extremophilic Red Algal Genus Sister to Cyanidioschyzon (Cyanidioschyzonaceae, Rhodophyta).

The taxonomic placement of strains belonging to the extremophilic red alga Galdieria maxima has been controversial due to the inconsistent phylogenetic position inferred from molecular phylogenetic analyses. Galdieria maxima nom. inval. was classified in this genus based on morphology and molecular data in the early work, but some subsequent molecular phylogenetic analyses have inferred strains of G. maxima to be closely related to the genus Cyanidioschyzon. To address this controversy, an isolated strain identified as G. maxima using the rbcL gene sequence as the genetic barcode was examined using a comprehensive analysis across morphological, physiological, and genomic traits. Herein are reported the chloroplast-, mitochondrion-, and chromosome-level nuclear genome assemblies. Comparative analysis of orthologous gene clusters and genome arrangements suggested that the genome structure of this strain was more similar to that of the generitype of Cyanidioschyzon, C. merolae than to the generitype of Galdieria, G. sulphuraria. While the ability to uptake various forms of organic carbon for growth is an important physiological trait of Galdieria, this strain was identified as an ecologically obligate photoautotroph (i.e., the inability to utilize the natural concentrations of organic carbons) and lacked various gene models predicted as sugar transporters. Based on the genomic, morphological, and physiological traits, we propose this strain to be a new genus and species, Cyanidiococcus yangmingshanensis. Re-evaluation of the 18S rRNA and rbcL gene sequences of the authentic strain of G. maxima, IPPAS-P507, with those of C. yangmingshanensis suggests that the rbcL sequences of "G. maxima" deposited in GenBank correspond to misidentified isolates.

Highly Oxygenated Constituents from a Marine Alga-Derived Fungus Aspergillus giganteus NTU967.

Agar-based disc diffusion antimicrobial assay has shown that the ethyl acetate extract of the fermented broth of Aspergillus giganteus NTU967 isolated from Ulva lactuca exhibited significant antimicrobial activity in our preliminary screening of bioactive fungal strains. Therefore, column chromatography of the active principles from liquid- and solid-state fermented products of the fungal strain was carried out, and which had led to isolation of eleven compounds. Their structures were determined by spectral analysis to be seven new highly oxygenated polyketides, namely aspergilsmins A-G (1-7), along with previously reported patulin, deoxytryptoquivaline, tryptoquivaline and quinadoline B. Among these, aspergilsmin C (3) and patulin displayed promising anticancer activities against human hepatocellular carcinoma SK-Hep-1 cells and prostate cancer PC-3 cells with IC50 values between 2.7-7.3 µM. Furthermore, aspergilsmin C (3) and patulin exhibited significant anti-angiogenic functions by impeding cell growth and tube formation of human endothelial progenitor cells without any cytotoxicity.

Metabolites Involved in Aerobic Degradation of the A and B Rings of Estrogen.

Various bacteria, mainly actinobacteria and proteobacteria, are capable of aerobic estrogen degradation. In a previous study, we used the obligate aerobic alphaproteobacterium Sphingomonas sp. strain KC8 as a model microorganism to identify the initial metabolites involved in the oxygenolytic cleavage of the estrogen A ring: 4-hydroxyestrone, a meta-cleavage product, and a dead-end product pyridinestrone acid. In this study, we identified the downstream metabolites of this aerobic degradation pathway using ultraperformance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). 4-Norestrogen-5(10)-en-3-oyl-coenzyme A and its closely related deconjugated (non-coenzyme A [non-CoA]) structure, 4-norestrogenic acid, were detected in the estrone-grown strain KC8 cultures. The structure of 4-norestrogenic acid was elucidated using nuclear magnetic resonance (NMR) spectroscopy. The extracellular distribution and the accumulation of 4-norestrogenic acid in the bacterial cultures indicate that the estrogen-degrading bacteria cannot degrade this deconjugated product. We also observed temporal accumulation and subsequent consumption of a common steroid metabolite, 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP), in the bacterial cultures. The metabolite profile and genomic analyses shed light on the biochemical mechanisms involved in the degradation of the A and B rings of natural estrogens. In this proposed aerobic pathway, C-4 of the meta-cleavage product is removed by a 2-oxoacid oxidoreductase through oxidative decarboxylation to produce the 4-norestrogen-5(10)-en-3-oyl-CoA. Subsequently, the B ring is cleaved by hydrolysis. The resulting A/B-ring-cleaved product is transformed into a common steroid metabolite HIP through ß-oxidation reactions. Accordingly, the A and B rings of different steroids are degraded through at least three peripheral pathways, which converge at HIP, and HIP is then degraded through a common central pathway.IMPORTANCE Estrogens, often detected in surface waters worldwide, have been classified as endocrine disrupting chemicals and carcinogens. Bacterial degradation is crucial for removing natural estrogens from natural and engineered ecosystems; however, current knowledge regarding the biochemical mechanisms and catabolic enzymes involved in estrogen biodegradation is very limited. Our estrogen metabolite profile and genomic analyses on estrone-degrading bacteria enabled us to characterize the aerobic estrogen degradation pathway. The results greatly expand our understanding of microbial steroid degradation. In addition, the characteristic metabolites, dead-end products, and degradation genes can be used as biomarkers to investigate the fate and biodegradation potential of estrogens in the environment.

Estrogen Degraders and Estrogen Degradation Pathway Identified in an Activated Sludge.

The environmental release and fate of estrogens are becoming an increasing public concern. Bacterial degradation has been considered the main process for eliminating estrogens from wastewater treatment plants. Various bacterial isolates are reportedly capable of aerobic estrogen degradation, and several estrogen degradation pathways have been proposed in proteobacteria and actinobacteria. However, the ecophysiological relevance of estrogen-degrading bacteria in the environment is unclear. In this study, we investigated the estrogen degradation pathway and corresponding degraders in activated sludge collected from the Dihua Sewage Treatment Plant, Taipei, Taiwan. Cultivation-dependent and cultivation-independent methods were used to assess estrogen biodegradation in the collected activated sludge. Estrogen metabolite profile analysis revealed the production of pyridinestrone acid and two A/B-ring cleavage products in activated sludge incubated with estrone (1 mM), which are characteristic of the 4,5-seco pathway. PCR-based functional assays detected sequences closely related to alphaproteobacterial oecC, a key gene of the 4,5-seco pathway. Metagenomic analysis suggested that Novosphingobium spp. are major estrogen degraders in estrone-amended activated sludge. Novosphingobium sp. strain SLCC, an estrone-degrading alphaproteobacterium, was isolated from the examined activated sludge. The general physiology and metabolism of this strain were characterized. Pyridinestrone acid and the A/B-ring cleavage products were detected in estrone-grown strain SLCC cultures. The production of pyridinestrone acid was also observed during the aerobic incubation of strain SLCC with 3.7 nM (1 µg/liter) estrone. This concentration is close to that detected in many natural and engineered aquatic ecosystems. The presented data suggest the ecophysiological relevance of Novosphingobium spp. in activated sludge.IMPORTANCE Estrogens, which persistently contaminate surface water worldwide, have been classified as endocrine disruptors and human carcinogens. We contribute new knowledge on the major estrogen biodegradation pathway and estrogen degraders in wastewater treatment plants. This study considerably advances the understanding of environmental estrogen biodegradation, which is instrumental for the efficient elimination of these hazardous pollutants. Moreover, this study substantially improves the understanding of microbial estrogen degradation in the environment.

Substrate uptake and subcellular compartmentation of anoxic cholesterol catabolism in Sterolibacterium denitrificans.

Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. (13)C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a ß-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.

Anoxic androgen degradation by the denitrifying bacterium Sterolibacterium denitrificans via the 2,3-seco pathway.

The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 androgens. By using a (13)C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (≤ 500 µM) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C19 androgens.

Diterpene glycosides and polyketides from Xylotumulus gibbisporus.

Four new tetracyclic diterpene glycosides, namely, sordarins C-F (1-4), and three new γ-lactone polyketides, namely, xylogiblactones A-C (5-7), along with sordarin were isolated from the ethyl acetate extracts of the fermented broths of Xylotumulus gibbisporus YMJ863. The structures of 1-7 were elucidated on the basis of spectroscopic data analyses. The configurations of 1-4 were deduced by NOESY, molecular modeling, and comparison with the literature. The relative configurations of 5-7 were deduced by X-ray crystallographic analysis of 5. Compounds 1-5 and sordarin were evaluated in an antifungal assay using Candida albicans ATCC 18804, C. albicans ATCC MYA-2876, and Saccharomyces cerevisiae ATCC 2345, and only sordarin exhibited significant antifungal activities against these fungal strains, with MIC values of 64.0, 32.0, and 32.0 µg/mL, respectively. The effect of compounds 1-7 and sordarin on the inhibition of NO production in lipopolysaccharide-activated murine macrophages was also evaluated. Compounds 2 and sordarin inhibited NO production with IC50 values of 327.2±46.6 and 157.1±24.1 µM, respectively.

Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans.

The aerobic degradation of steroids by bacteria has been studied in some detail. In contrast, only little is known about the anaerobic steroid catabolism. Steroidobacter denitrificans can utilize testosterone under both oxic and anoxic conditions. By conducting metabolomic investigations, we demonstrated that S. denitrificans adopts the 9,10-seco-pathway to degrade testosterone under oxic conditions. This pathway depends on the use of oxygenases for oxygenolytic ring fission. Conversely, the detected degradation intermediates under anoxic conditions suggest a novel, oxygenase-independent testosterone catabolic pathway, the 2,3-seco-pathway, which differs significantly from the aerobic route. In this anaerobic pathway, testosterone is first transformed to 1-dehydrotestosterone, which is then reduced to produce 1-testosterone followed by water addition to the C-1/C-2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione. The A-ring of this compound is cleaved by hydrolysis as evidenced by H2(18)O-incorporation experiments. Regardless of the growth conditions, testosterone is initially transformed to 1-dehydrotestosterone. This intermediate is a divergence point at which the downstream degradation pathway is governed by oxygen availability. Our results shed light into the previously unknown cleavage of the sterane ring structure without oxygen. We show that, under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring.

An in vitro study of the antimicrobial effects of indigo naturalis prepared from Strobilanthes formosanus Moore.

Indigo naturalis is effective in treating nail psoriasis coexisting with microorganism infections. This study examines the antimicrobial effects of indigo naturalis prepared from Strobilanthes formosanus Moore. Eight bacterial and seven fungal strains were assayed using the agar diffusion method to examine the effects of indigo naturalis and its bioactive compounds. The bioactive compounds of indigo naturalis were purified sequentially using GFC, TLC, and HPLC. Their structures were identified using mass spectrometry and NMR spectroscopy. UPLC-MS/MS was applied to compare the metabolome profiles of indigo naturalis ethyl-acetate (EA) extract and its source plant, Strobilanthes formosanus Moore. The results of in vitro antimicrobial assays showed that indigo naturalis EA-extract significantly (≥1 mg/disc) inhibits Gram-positive bacteria (Staphylococcus aureus, S. epidermis and methicillin-resistant S. aureus (MRSA)) and mildly inhibits non-dermatophytic onychomycosis pathogens (Aspergillus fumigates and Candida albicans), but has little effect on dermatophyes. Isatin and tryptanthrin were identified as the bioactive compounds of indigo naturalis using S. aureus and S. epidermis as the bioassay model. Both bioactive ingredients had no effect on all tested fungi. In summary, indigo naturalis prepared from Strobilanthes formosanus Moore exhibits antimicrobial effects on Staphylococcus and non-dermatophytic onychomycosis pathogens. Tryptanthrin and isatin may be its major bioactive ingredients against Staphylococcus and the inhibitory effect on MRSA may be due to other unidentified ingredients.

Valorization of soybean pulp for sustainable α-ketoisocaproate production using engineered Bacillus subtilis whole-cell biocatalyst.

The disposal of soybean pulp (okara) (∼14 M tons annually) represents a global concern. α-ketoisocaproate (KIC) is an intrinsic l-leucine metabolite boosting mammalian muscle growth and has great potential in animal husbandry. However, the use of pure l-leucine (5000 USD/kg) for KIC (22 USD/kg) bioproduction is cost-prohibitive in practice, while okara rich in l-leucine (10%) could serve as an economical alternative. Following the concept of a circular bioeconomy, we managed to develop a cost-efficient platform to valorize okara into KIC. In this study, proteolytic Bacillus subtilis strain 168 capable of utilizing okara as a comprehensive substrate was employed as the whole-cell biocatalyst for KIC bioproduction. First, we elucidated the function of genes involved in KIC downstream metabolism in strain 168, including those encoding 2-oxoisovalerate dehydrogenase (bkdAA), 2-oxoisovalerate decarboxylase (bkdAB), enoyl-CoA hydratase (fadB), and bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (fadN). Among those KIC downstream metabolizing mutants of strain 168, the 2-oxoisovalerate decarboxylase gene knockout strain (ΔbkdAB) was found to have a better accumulation of KIC. To further improve the KIC yield, a soluble l-amino acid deaminase (LAAD) from Proteus vulgaris was heterologously expressed in the ΔbkdAB strain and a ∼50% conversion of total l-leucine contained in okara was catalyzed into KIC, along with a ∼50% reduction of CO2 emission compared to the wild-type cultures. Altogether, this renovated biocatalytic system provides an alternative platform to valorize okara for producing value-added chemicals in an eco-friendly manner.

Ustilago maydis PR-1-like protein has evolved two distinct domains for dual virulence activities.

The diversification of effector function, driven by a co-evolutionary arms race, enables pathogens to establish compatible interactions with hosts. Structurally conserved plant pathogenesis-related PR-1 and PR-1-like (PR-1L) proteins are involved in plant defense and fungal virulence, respectively. It is unclear how fungal PR-1L counters plant defense. Here, we show that Ustilago maydis UmPR-1La and yeast ScPRY1, with conserved phenolic resistance functions, are Ser/Thr-rich region mediated cell-surface localization proteins. However, UmPR-1La has gained specialized activity in sensing phenolics and eliciting hyphal-like formation to guide fungal growth in plants. Additionally, U. maydis hijacks maize cathepsin B-like 3 (CatB3) to release functional CAPE-like peptides by cleaving UmPR-1La's conserved CNYD motif, subverting plant CAPE-primed immunity and promoting fungal virulence. Surprisingly, CatB3 avoids cleavage of plant PR-1s, despite the presence of the same conserved CNYD motif. Our work highlights that UmPR-1La has acquired additional dual roles to suppress plant defense and sustain the infection process of fungal pathogens.

Harnessing microbial phylum-specific molecular markers for assessment of environmental estrogen degradation.

Steroidal estrogens are ubiquitous contaminants that have garnered attention worldwide due to their endocrine-disrupting and carcinogenic activities at sub-nanomolar concentrations. Microbial degradation is one of the main mechanisms through which estrogens can be removed from the environment. Numerous bacteria have been isolated and identified as estrogen degraders; however, little is known about their contribution to environmental estrogen removal. Here, our global metagenomic analysis indicated that estrogen degradation genes are widely distributed among bacteria, especially among aquatic actinobacterial and proteobacterial species. Thus, by using the Rhodococcus sp. strain B50 as the model organism, we identified three actinobacteria-specific estrogen degradation genes, namely aedGHJ, by performing gene disruption experiments and metabolite profile analysis. Among these genes, the product of aedJ was discovered to mediate the conjugation of coenzyme A with a unique actinobacterial C17 estrogenic metabolite, 5-oxo-4-norestrogenic acid. However, proteobacteria were found to exclusively adopt an α-oxoacid ferredoxin oxidoreductase (i.e., the product of edcC) to degrade a proteobacterial C18 estrogenic metabolite, namely 3-oxo-4,5-seco-estrogenic acid. We employed actinobacterial aedJ and proteobacterial edcC as specific biomarkers for quantitative polymerase chain reaction (qPCR) to elucidate the potential of microbes for estrogen biodegradation in contaminated ecosystems. The results indicated that aedJ was more abundant than edcC in most environmental samples. Our results greatly expand the understanding of environmental estrogen degradation. Moreover, our study suggests that qPCR-based functional assays are a simple, cost-effective, and rapid approach for holistically evaluating estrogen biodegradation in the environment.

Circulating androgen regulation by androgen-catabolizing gut bacteria in male mouse gut.

Abnormally high circulating androgen levels have been considered a causative factor for benign prostatic hypertrophy and prostate cancer in men. Recent animal studies on gut microbiome suggested that gut bacteria are involved in sex steroid metabolism; however, the underlying mechanisms and bacterial taxa remain elusive. Denitrifying betaproteobacteria Thauera spp. are metabolically versatile and often distributed in the animal gut. Thauera sp. strain GDN1 is an unusual betaproteobacterium capable of catabolizing androgen under both aerobic and anaerobic conditions. We administered C57BL/6 mice (aged 7 weeks) with strain GDN1 through oral gavage. The strain GDN1 administration caused a minor increase in the relative abundance of Thauera (≤0.1%); however, it has profound effects on the host physiology and gut bacterial community. The results of our ELISA assay and metabolite profile analysis indicated an approximately 50% reduction in serum androgen levels in the strain GDN1-administered male mice. Moreover, androgenic ring-cleaved metabolites were detected in the fecal extracts of the strain GDN1-administered mice. Furthermore, our RT - qPCR results revealed the expression of the androgen catabolism genes in the gut of the strain GDN1-administered mice. We found that the administered strain GDN1 regulated mouse serum androgen levels, possibly because it blocked androgen recycling through enterohepatic circulation. This study discovered that sex steroids serve as a carbon source of gut bacteria; moreover, host circulating androgen levels may be regulated by androgen-catabolizing gut bacteria. Our data thus indicate the possible applicability of androgen-catabolic gut bacteria as potent probiotics in alternative therapy of hyperandrogenism.

Bioactive pulvinones from a marine algicolous fungus Aspergillus terreus NTU243.

Marine fungi are regarded as an under-explored source of structurally interesting and bioactive natural products with the potential to provide attractive lead compounds for drug discovery. In this study, several fungal strains were isolated from marine algae collected from the northeastern coast of Taiwan. In the preliminary antimicrobial screening against bacteria and fungi, the ethyl acetate extract of the fermented products of Aspergillus terreus NTU243 derived from a green alga Ulva lactuca was found to exhibit significant antimicrobial activities. Therefore, bioassay-guided separations of the active principle from liquid and solid fermented products of A. terreus NTU243 were undertaken, which resulted in the isolation and purification of 16 compounds. Their structures were elucidated by spectroscopic analysis to be four previously undescribed aspulvinones S-V as well as twelve known compounds. All the isolates were assessed for anti-inflammatory activity by measuring the amount of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 cells, and aspulvinone V, butyrolactone I, and (+)-terrein inhibited 45.0%, 34.5%, and 49.2% of NO production, respectively, at 10 µM concentration. Additionally, zymography showed that the conditioned medium of THP-1 cells post-LPS challenged significantly enhanced matrix metalloproteinase (MMP)-9-mediated gelatinolysis, and pretreatment with aspulvinones U and V significantly attenuated MMP-9-mediated gelatinolysis by 56.0% and 67.8%, separately.