Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).

The complete genome sequence of Sedimentibacter sp. strain MB35-C1, isolated from the sewage sludge.

Here, we report the complete genome sequence of Sedimentibacter sp. strain MB35-C1, which was isolated from sewage sludge at the Wastewater Treatment Plant of Sanming Steel Co. Ltd. in Fujian, China. The resulting genome of strain MB35-C1 is a single contig of 3,621,605 bp.

The complete genome sequence of Paludicola sp. MB14-C6, isolated from the lake waters of Donghu, Wuhan City, China.

Here, we report the complete genome sequence of Paludicola sp. strain MB14-C6, which was isolated from the lake waters of Donghu, situated at Wuhan City, Hubei Province, China. The genome of strain MB14-C6 was chosen for further species delineation and comparative genomic analysis.

Complete genome sequence of Anaerotignum sp. strain MB30-C6, isolated from sewage sludge of the wastewater treatment plant at a steel factory.

We report the complete genome sequence of Anaerotignum sp. strain MB30-C6, which was isolated from the dehydrated sludge collected at the wastewater treatment plant of Sanming Steel Co. Ltd. in Fujian, China. The resulting genome of strain MB30-C6 is a single contig of 3,104,838 bp with 39.49% GC content.

Whole-genome sequencing of Aminobacterium sp. strain MB27-C1, isolated from the wastewater treatment plant of a steel mill.

Here, we report the complete genome sequence of Aminobacterium sp. strain MB27-C1, which was isolated from sewage sludge collected at the wastewater treatment plant of Sanming Steel Co. Ltd. in Fujian, China. The resulting genome of strain MB27-C1 is a single contig of 2,427,830 bp with 41.58% GC content.

The complete genome sequence of Kineothrix sp. MB12-C1, isolated from the feces of black soldier fly larvae.

Here, we present the complete genome sequence of Kineothrix sp. MB12-C1 (= BCRC 81406), isolated from the feces of black soldier fly (Hermetia illucens) larvae. The genome of strain MB12-C1 was chosen for further species classification and comparative genomic analysis.

Complete genome sequence of Proteiniborus sp. MB09-C3, isolated from the feces of the black soldier fly larvae.

Here, we report the complete genome sequence of Proteiniborus sp. MB09-C3 (= BCRC 81405), isolated from the feces of black soldier fly (Hermetia illucens) larvae. The genome of strain MB09-C3 was selected for further species delineation and comparative genomic analysis.

Complete genome sequence of Proteiniclasticum sp. QWL-01, isolated from sewage sludge.

Here, we report the complete genome sequence of Proteiniclasticum sp. QWL-01 (= BCRC 81396), isolated from sewage sludge of the Wastewater Treatment Plant of Sanming Steel Co. Ltd., Fujian, China. The genome of strain QWL-01 was selected for further species delineation and comparative genomic analysis.

Complete Genome Sequence of Tissierella sp. Strain Yu-01, Isolated from the Feces of the Black Soldier Fly.

We report the complete genome sequence of Tissierella sp. strain Yu-01 (=BCRC 81391), isolated from the feces of black soldier fly (Hermetia illucens) larvae. This fly has increasingly been gaining attention because of its usefulness for recycling organic waste. The genome of strain Yu-01 was selected for further species delineation.

Phocaeicola oris sp. nov., an anaerobic bacterium isolated from the saliva of a patient with oral squamous cell carcinoma.

A Gram-stain-negative or -positive, strictly anaerobic, non-spore-forming and pleomorphic bacterium (designated 14-104T) was isolated from the saliva sample of a patient with oral squamous cell carcinoma. It was an acid-tolerant neutralophilic mesophile, growing at between 20 and 40 °C (with optimum growth at 30 °C) and pH between pH 3.0 and 7.0 (with optimum growth at pH 6.0-7.0). It contained anteiso-C15 : 0 and C15 : 0 as the major fatty acids. The genome size of strain 14-104T was 2.98 Mbp, and the G+C content was 39.6 mol%. It shared <87 % 16S rRNA sequence similarity, <71 % orthologous average nucleotide identity, <76 % average amino acid identity and <68 %% of conserved proteins with its closest relative, Phocaeicola abscessus CCUG 55929T. Reconstruction of phylogenetic and phylogenomic trees revealed that strain 14-104T and P. abscessus CCUG 55929T were clustered as a distinct clade without any other terminal node. The phylogenetic and phylogenomic analyses along with physiological and chemotaxonomic data indicated that strain 14-104T represents a novel species in the genus Phocaeicola, for which the name Phocaeicola oris sp. nov. is proposed. The type strain is 14-104T (=BCRC 81305T= NBRC 115041T).

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.

Complete Genome Sequence of Methanofollis aquaemaris BCRC 16166T, Isolated from a Marine Aquaculture Fishpond.

The hydrogenotrophic methanogen Methanofollis aquaemaris BCRC 16166T (= N2F9704T = DSM 14661T) was isolated from a marine aquaculture fishpond near Wang-gong (Taiwan, Republic of China). The genome of strain BCRC 16166T was selected for sequencing in order to provide further information about the species delineation and its infected virus.

Draft Genomes of Methanocalculus taiwanensis P2F9704aT and Methanocalculus chunghsingensis K1F9705bT, Hydrogenotrophic Methanogens Belonging to the Family Methanocalculaceae.

The family Methanocalculaceae comprises hydrogen- and formate-utilizing methanogens. Here, we report two additional draft genome sequences of Methanocalculaceae, those of Methanocalculus taiwanensis P2F9704aT (equivalent to BCRC 16182T and DSM 14663T) and Methanocalculus chunghsingensis K1F9705bT (equivalent to DSM 14646T and OCM 772T), which were selected for further species delineation and comparative genomic analyses.

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.

Potential syntrophic relationship between coral-associated Prosthecochloris and its companion sulfate-reducing bacterium unveiled by genomic analysis.

Endolithic microbial symbionts in the coral skeleton may play a pivotal role in maintaining coral health. However, compared to aerobic micro-organisms, research on the roles of endolithic anaerobic micro-organisms and microbe-microbe interactions in the coral skeleton are still in their infancy. In our previous study, we showed that a group of coral-associated Prosthecochloris (CAP), a genus of anaerobic green sulphur bacteria, was dominant in the skeleton of the coral Isopora palifera. Though CAP is diverse, the 16S rRNA phylogeny presents it as a distinct clade separate from other free-living Prosthecochloris. In this study, we build on previous research and further characterize the genomic and metabolic traits of CAP by recovering two new high-quality CAP genomes - Candidatus Prosthecochloris isoporae and Candidatus Prosthecochloris sp. N1 - from the coral I. palifera endolithic cultures. Genomic analysis revealed that these two CAP genomes have high genomic similarities compared with other Prosthecochloris and harbour several CAP-unique genes. Interestingly, different CAP species harbour various pigment synthesis and sulphur metabolism genes, indicating that individual CAPs can adapt to a diversity of coral microenvironments. A novel high-quality genome of sulfate-reducing bacterium (SRB)- Candidatus Halodesulfovibrio lyudaonia - was also recovered from the same culture. The fact that CAP and various SRB co-exist in coral endolithic cultures and coral skeleton highlights the importance of SRB in the coral endolithic community. Based on functional genomic analysis of Ca. P. sp. N1, Ca. P. isoporae and Ca. H. lyudaonia, we also propose a syntrophic relationship between the SRB and CAP in the coral skeleton.

Metagenomic, phylogenetic, and functional characterization of predominant endolithic green sulfur bacteria in the coral Isopora palifera.

BACKGROUND: Endolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown. RESULTS: This study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB's abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP. CONCLUSION: We suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons.

Exploration of Fungal Metabolic Interactions Using Imaging Mass Spectrometry on Nanostructured Silicon.

Application of matrix-assisted laser desorption/ionization imaging mass spectrometry to microbiology and natural product research has opened the door to the exploration of microbial interactions and the consequent discovery of new natural products and their functions in the interactions. However, several drawbacks of matrix-assisted laser desorption/ionization imaging mass spectrometry have limited its application especially to complicated and uneven microbial samples. Here, we applied nanostructured silicon as a substrate for surface-assisted laser desorption/ionization mass spectrometry for microbial imaging mass spectrometry to explore fungal metabolic interactions. We chose Phellinus noxius and Aspergillus strains to evaluate the potential of microbial imaging mass spectrometry on nanostructured silicon because both fungi produce a dense mass of aerial mycelia, which is known to complicate the collection of high-quality imaging mass spectrometry data. Our simple and straightforward sample imprinting method and low background interference resulted in an efficient analysis of small metabolites from the complex microbial interaction samples.

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.

Biochemical Mechanisms and Microorganisms Involved in Anaerobic Testosterone Metabolism in Estuarine Sediments.

Current knowledge on the biochemical mechanisms underlying microbial steroid metabolism in anaerobic ecosystems is extremely limited. Sulfate, nitrate, and iron [Fe (III)] are common electron acceptors for anaerobes in estuarine sediments. Here, we investigated anaerobic testosterone metabolism in anaerobic sediments collected from the estuary of Tamsui River, Taiwan. The anaerobic sediment samples were spiked with testosterone (1 mM) and individual electron acceptors (10 mM), including nitrate, Fe3+, and sulfate. The analysis of androgen metabolites indicated that testosterone biodegradation under denitrifying conditions proceeds through the 2,3-seco pathway, whereas testosterone biodegradation under iron-reducing conditions may proceed through an unidentified alternative pathway. Metagenomic analysis and PCR-based functional assays suggested that Thauera spp. were the major testosterone degraders in estuarine sediment samples incubated with testosterone and nitrate. Thauera sp. strain GDN1, a testosterone-degrading betaproteobacterium, was isolated from the denitrifying sediment sample. This strain tolerates a broad range of salinity (0-30 ppt). Although testosterone biodegradation did not occur under sulfate-reducing conditions, we observed the anaerobic biotransformation of testosterone to estrogens in some testosterone-spiked sediment samples. This is unprecedented since biotransformation of androgens to estrogens is known to occur only under oxic conditions. Our metagenomic analysis suggested that Clostridium spp. might play a role in this anaerobic biotransformation. These results expand our understanding of microbial metabolism of steroids under strictly anoxic conditions.

Biochemical Mechanisms and Catabolic Enzymes Involved in Bacterial Estrogen Degradation Pathways.

Estrogens have been classified as group 1 carcinogens by the World Health Organization and represent a significant concern given that they are found in surface waters worldwide, and long-term exposure to estrogen-contaminated water can disrupt sexual development in animals. To date, the estrogen catabolic enzymes and genes remain unknown. Using a tiered functional genomics approach, we identified three estrogen catabolic gene clusters in Sphingomonas sp. strain KC8. We identified several estrone-derived compounds, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. We characterized 17ß-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase, responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A ring, respectively. The characteristic pyridinestrone acid was detected in estrone-spiked samples collected from two wastewater treatment plants and two suburban rivers in Taiwan. The results significantly expand our understanding of microbial degradation of aromatic steroids at molecular level.