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.

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

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.

Frictional characteristics of nano-confined water mediated hole-doped single-layer graphene on silica surface.

We have investigated the frictional properties of single-layer graphene (SLG) coated rough silica substrate under the influence of nano-confined hydration layer underneath SLG. Through the friction and surface potential measurements by atomic force microscopy (AFM), we found polygonal features in AFM images of SLG-protected silica surface that exhibit simultaneously larger friction and higher surface potential as compared to their surrounding areas due to water layers confined under SLG. Nano-confined water layers at the SLG-silica interface can induce the hole-doping effect in SLG, resulting in a more positively-charged and hydrophilic surface that favors adsorption of ambient water molecules. Therefore, during friction measurements, nanoscale capillary bridges can form within the interstices of AFM probe-SLG contact, leading to larger adhesion and friction. The friction forces were found to respectively have negative and positive dependence on the sliding velocity inside and outside the polygonal regions due to different surface wettability. Hence, it is possible to manipulate the frictional properties of SLG-coated silica by the amount of hydration layer confined underneath SLG. Our results may find applications in friction control for future nano-devices.

Sustained Dechlorination of Vinyl Chloride to Ethene in Dehalococcoides-Enriched Cultures Grown without Addition of Exogenous Vitamins and at Low pH.

Trichloroethene (TCE) bioremediation has been demonstrated at field sites using microbial cultures harboring TCE-respiring Dehalococcoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In these cultures, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are dominant acetogens. At neutral pH, Acetobacterium supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in cobalamin (∼20-fold). Sustained dechlorination of VC to ethene was achieved at pH as low as 5.5. Below pH 5.0, dechlorination was not stimulated by DMB supplementation but was restored by raising pH to neutral. Cell-extract assays revealed that vinyl chloride reductase activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the importance of cobamide-producing populations and pH in microbial dechlorinating communities for successful bioremediation at field sites.

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.

Specific and efficient knockdown of intracellular miRNA using partially neutralized phosphate-methylated DNA oligonucleic acid-loaded mesoporous silica nanoparticles.

Antisense oligonucleotides (ASOs) are molecules used to regulate RNA expression by targeting specific RNA sequences. One specific type of ASO, known as neutralized DNA (nDNA), contains site-specific methyl phosphotriester (MPTE) linkages on the phosphate backbone, changing the negatively charged DNA phosphodiester into a neutralized MPTE with designed locations. While nDNA has previously been employed as a sensitive nucleotide sequencing probe for the PCR, the potential of nDNA in intracellular RNA regulation and gene therapy remains underexplored. Our study aims to evaluate the regulatory capacity of nDNA as an ASO probe in cellular gene expression. We demonstrated that by tuning MPTE locations, partially and intermediately methylated nDNA loaded onto mesoporous silica nanoparticles (MSNs) can effectively knock down the intracellular miRNA, subsequently resulting in downstream mRNA regulation in colorectal cancer cell HCT116. Additionally, the nDNA ASO-loaded MSNs exhibit superior efficacy in reducing miR-21 levels over 72 hours compared to the efficacy of canonical DNA ASO-loaded MSNs. The reduction in the miR-21 level subsequently resulted in the enhanced mRNA levels of tumour-suppressing genes PTEN and PDCD4. Our findings underscore the potential of nDNA in gene therapies, especially in cancer treatment via a fine-tuned methylation location.

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.

One-Pot De Novo Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions.

Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.

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.

A Fluorescent Vector of Carbon Dot to Deliver Rab13 and Rab14 Plasmids for Promoting Neurite Outgrowth.

The complex processes of neuron differentiation and neuron repair are critical for treating nervous system injuries and neurodegenerative diseases. Neurite outgrowth plays a crucial role in these processes by enabling the formation of connections between neurons and the generation of neuroplasticity to restore the function of the nervous system. In this study, we fabricated functionalized carbon dots (CDs) with distinctive photoluminescence and low cytotoxicity for use as fluorescence imaging probes and nanocarriers to deliver plasmid DNAs to neurons effectively for inducing neurite outgrowth. CDs were prepared through a reflux process in nitric acid solution, and their surface was then modified using polyethylenimine (PEI) to obtain positively charged CDs for increasing the absorption of plasmid DNAs and the efficiency of cell uptake. Experimental results indicated that the fabricated CDs maintained a low cytotoxicity and exhibited a high neuron uptake of up to 97%. An improvement in the plasmid DNA ingestion of neurons resulted in enhanced expression of Rab13-Q67L and Rab14 proteins, which considerably promoted neurite sprouting and elongation. After the fabricated PEI-modified CDs were used to deliver the Rab13-Q67L and Rab14 plasmids, more than 56% of the neurons had a neurite length that was greater than twice the size of their soma. Thus, DNA delivery through functionalized CDs has a high potential for use in gene therapy for neuronal injuries and diseases.

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.

Omics and mechanistic insights into di-(2-ethylhexyl) phthalate degradation in the O2-fluctuating estuarine sediments.

Di-(2-ethylhexyl) phthalate (DEHP) represents the most used phthalate plasticizer with an annual production above the millions of tons worldwide. Due to its inadequate disposal, outstanding chemical stability, and extremely low solubility (3 mg/L), endocrine-disrupting DEHP often accumulates in urban estuarine sediments at concentrations above the predicted no-effect concentration (20-100 mg/kg). Our previous study suggested that microbial DEHP degradation in estuarine sediments proceeds synergistically where DEHP side-chain hydrolysis to form phthalic acid represents a bottleneck. Here, we resolved this bottleneck and deconstructed the microbial synergy in O2-fluctuating estuarine sediments. Metagenomic analysis and RNA sequencing suggested that orthologous genes encoding extracellular DEHP hydrolase NCU65476 in Acidovorax sp. strain 210-6 are often flanked by the co-expressed composite transposon and are widespread in aquatic environments worldwide. Therefore, we developed a turbidity-based microplate assay to characterize NCU65476. The optimized assay conditions (with 1 mM Ca2+ and pH 6.0) increased the DEHP hydrolysis rate by a factor of 10. Next, we isolated phthalic acid-degrading Hydrogenophaga spp. and Thauera chlorobenzoica from Guandu estuarine sediment to study the effect of O2(aq) on their metabolic synergy with strain 210-6. The results of co-culture experiments suggested that after DEHP side-chain hydrolysis by strain 210-6, phthalic acid can be degraded by Hydrogenophaga sp. when O2(aq) is above 1 mg/L or degraded by Thauera chlorobenzoica anaerobically. Altogether, our data demonstrates that DEHP could be degraded synergistically in estuarine sediments via divergent pathways responding to O2 availability. The optimized conditions for NCU65476 could facilitate the practice of DEHP bioremediation in estuarine sediments.

Identification of essential ß-oxidation genes and corresponding metabolites for oestrogen degradation by actinobacteria.

Steroidal oestrogens (C18 ) are contaminants receiving increasing attention due to their endocrine-disrupting activities at sub-nanomolar concentrations. Although oestrogens can be eliminated through photodegradation, microbial function is critical for removing oestrogens from ecosystems devoid of sunlight exposure including activated sludge, soils and aquatic sediments. Actinobacteria were found to be key oestrogen degraders in manure-contaminated soils and estuarine sediments. Previously, we used the actinobacterium Rhodococcus sp. strain B50 as a model microorganism to identify two oxygenase genes, aedA and aedB, involved in the activation and subsequent cleavage of the estrogenic A-ring respectively. However, genes responsible for the downstream degradation of oestrogen A/B-rings remained completely unknown. In this study, we employed tiered comparative transcriptomics, gene disruption experiments and mass spectrometry-based metabolite profile analysis to identify oestrogen catabolic genes. We observed the up-regulation of thiolase-encoding aedF and aedK in the transcriptome of strain B50 grown with oestrone. Consistently, two downstream oestrogenic metabolites, 5-oxo-4-norestrogenic acid (C17 ) and 2,3,4-trinorestrogenic acid (C15 ), were accumulated in aedF- and aedK-disrupted strain B50 cultures. Disruption of fadD3 [3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP)-coenzyme A-ligase gene] in strain B50 resulted in apparent HIP accumulation in oestrone-fed cultures, indicating the essential role of fadD3 in actinobacterial oestrogen degradation. In addition, we detected a unique meta-cleavage product, 4,5-seco-estrogenic acid (C18 ), during actinobacterial oestrogen degradation. Differentiating the oestrogenic metabolite profile and degradation genes of actinobacteria and proteobacteria enables the cost-effective and time-saving identification of potential oestrogen degraders in various ecosystems through liquid chromatography-mass spectrometry analysis and polymerase chain reaction-based functional assays.

A novel testosterone catabolic pathway in bacteria.

Forty years ago, Coulter and Talalay (A. W. Coulter and P. Talalay, J. Biol. Chem. 243:3238-3247, 1968) established the oxygenase-dependent pathway for the degradation of testosterone by aerobes. The oxic testosterone catabolic pathway involves several oxygen-dependent reactions and is not available for anaerobes. Since then, a variety of anaerobic bacteria have been described for the ability to degrade testosterone in the absence of oxygen. Here, a novel, oxygenase-independent testosterone catabolic pathway in such organisms is described. Steroidobacter denitrificans DSMZ18526 was shown to be capable of degrading testosterone in the absence of oxygen and was selected as the model organism in this study. In a previous investigation, we identified the initial intermediates involved in an anoxic testosterone catabolic pathway, most of which are identical to those of the oxic pathway demonstrated in Comamonas testosteroni. In this study, five additional intermediates of the anoxic pathway were identified. We demonstrated that subsequent steps of the anoxic pathway greatly differ from those of the established oxic pathway, which suggests that a novel pathway for testosterone catabolism is present. In the proposed anoxic pathway, a reduction reaction occurs at C-4 and C-5 of androsta-1,4-diene-3,17-dione, the last common intermediate of both the oxic and anoxic pathways. After that, a novel hydration reaction occurs and a hydroxyl group is thus introduced to the C-1α position of C(19)steroid substrates. To our knowledge, an enzymatic hydration reaction occurring at the A ring of steroid compounds has not been reported before.

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering.

One aspect of the study of the origins of life focuses on how primitive chemistries assembled into the first cells on Earth and how these primitive cells evolved into modern cells. Membraneless droplets generated from liquid-liquid phase separation (LLPS) are one potential primitive cell-like compartment; current research in origins of life includes study of the structure, function, and evolution of such systems. However, the goal of primitive LLPS research is not simply curiosity or striving to understand one of life's biggest unanswered questions, but also the possibility to discover functions or structures useful for application in the modern day. Many applicational fields, including biotechnology, synthetic biology, and engineering, utilize similar phaseseparated structures to accomplish specific functions afforded by LLPS. Here, we briefly review LLPS applied to primitive compartment research and then present some examples of LLPS applied to biomolecule purification, drug delivery, artificial cell construction, waste and pollution management, and flavor encapsulation. Due to a significant focus on similar functions and structures, there appears to be much for origins of life researchers to learn from those working on LLPS in applicational fields, and vice versa, and we hope that such researchers can start meaningful cross-disciplinary collaborations in the future.

Mechanistic and phylogenetic insights into actinobacteria-mediated oestrogen biodegradation in urban estuarine sediments.

Steroidal oestrogens are often accumulated in urban estuarine sediments worldwide at microgram per gram levels. These aromatic steroids have been classified as endocrine disruptors and group 1 carcinogens. Microbial degradation is a naturally occurring mechanism that mineralizes oestrogens in the biosphere; however, the corresponding genes in oestrogen-degrading actinobacteria remain unidentified. In this study, we identified a gene cluster encoding several putative oestrogen-degrading genes (aed; actinobacterial oestrogen degradation) in actinobacterium Rhodococcus sp. strain B50. Among them, the aedA and aedB genes involved in oestrogenic A-ring cleavage were identified through gene-disruption experiments. We demonstrated that actinobacterial oestrone 4-hydroxylase (AedA) is a cytochrome P450-type monooxygenase. We also detected the accumulation of two extracellular oestrogenic metabolites, including pyridinestrone acid (PEA) and 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP), in the oestrone-fed strain B50 cultures. Since actinobacterial aedB and proteobacterial edcB shared < 40% sequence identity, 4-hydroxyestrone 4,5-dioxygenase genes (namely aedB and edcB) could serve as a specific biomarker to differentiate the contribution of actinobacteria and proteobacteria in environmental oestrogen degradation. Therefore, 4-hydroxyestrone 4,5-dioxygenase genes and the extracellular metabolites PEA and HIP were used as biomarkers to investigate oestrogen biodegradation in an urban estuarine sediment. Interestingly, our data suggested that actinobacteria are active oestrogen degraders in the urban estuarine sediment.