Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Visualization and mapping of literature on the scientific analysis of wall paintings: a bibliometric analysis from 2011 to 2021.

As non-renewable cultural heritage, wall paintings play an important role in society. To reveal the trends in the scientific analysis of mural paintings, 845 relevant research articles published from 2011 to 2021 were collected from the Web of Science database and analyzed. The VOSviewer software was adopted to map the network data of scientific publications, so that relationships among authors, countries, institutions can be displayed, and the co-occurrence of keywords and co-citation can be analyzed. The results revealed close and strong interconnections between the top authors, suggesting a considerable strong research link in this field. The cooperation between research institutions was relatively close. The most productive country of relevant publications was Italy. The leading journals for the scientific analysis of wall paintings were Journal of Raman Spectroscopy and Journal of Cultural Heritage. At present, the hotspots of scientific analysis and research on wall painting are revealing the composition, distribution, origin, and deterioration mechanism of pigments, alongside with evaluating the effects and mechanism of conservation materials and techniques. On the one hand, a possible development direction in this field is introducing more cutting-edge analysis and data processing methods. On the other hand, scientific analysis is increasingly adopted to guide the research and development of mural conservation materials.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

Physiology, Taxonomy, and Sulfur Metabolism of the Sulfolobales, an Order of Thermoacidophilic Archaea.

The order Sulfolobales (phylum Crenarchaeota) is a group of thermoacidophilic archaea. The first member of the Sulfolobales was discovered in 1972, and current 23 species are validly named under the International Code of Nomenclature of Prokaryotes. The majority of members of the Sulfolobales is obligately or facultatively chemolithoautotrophic. When they grow autotrophically, elemental sulfur or reduced inorganic sulfur compounds are their energy sources. Therefore, sulfur metabolism is the most important physiological characteristic of the Sulfolobales. The functions of some enzymes and proteins involved in sulfur reduction, sulfur oxidation, sulfide oxidation, thiosulfate oxidation, sulfite oxidation, tetrathionate hydrolysis, and sulfur trafficking have been determined. In this review, we describe current knowledge about the physiology, taxonomy, and sulfur metabolism of the Sulfolobales, and note future challenges in this field.

[Advances in bacterial Rieske non-heme iron ring-hydroxylating dioxygenases that initiate polycyclic aromatic hydrocarbons degradation].

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants, which have received widespread attentions due to their carcinogenic and mutagenic toxicity. The microbial degradation of PAHs are usually started from the hydroxylation, followed by dehydrogenation, ring cleavage and step-by-step removal of branched chains, and finally mineralized by the tricarboxylic acid cycle. Rieske type non-heme iron aromatic ring-hydroxylating dioxygenases (RHOs) or cytochrome P450 oxidases are responsible for the conversion of hydrophobic PAHs into hydrophilic derivatives by the ring hydroxylation. The ring hydroxylation is the first step of PAHs degradation and also one of the rate-limiting steps. Here, we review the distribution, substrate specificity, and substrate recognition mechanisms of RHOs, along with some techniques and methods used for the research of RHOs and PAHs.

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

Reply to the 'Comment on "Design and circuit simulation of nanoscale vacuum channel transistors"' by R. Forbes, Nanoscale Adv., 2020, 2, DOI: 10.1039/D0NA00687D.

Prof. Forbes takes a critical view of our paper on nanoscale vacuum channel transistors (NVCTs), arguing mainly about the weaknesses in the theory of field electron emission. On the one hand, we agree with the theoretical derivation details given by Prof. Forbes, and we would correct the "simplified formula" accordingly (eqn (2)-(5)). On the other hand, the main part of our work focuses on the simulation results of structural parameters and circuit behavior, which is not involved with eqn (2)-(5). Thus, the reported results and conclusions remain reliable.

Low-fouling CNT-PEG-hydrogel coated quartz crystal microbalance sensor for saliva glucose detection.

Saliva glucose detection based on a quartz crystal microbalance (QCM) sensor has emerged as a promising tool and a non-invasive diagnostic technique for diabetes. However, the low glucose concentration and strong protein interference in the saliva hinder the QCM sensors from practical applications. In this study, we present a robust and simple anti-fouling CNT-PEG-hydrogel film-coated QCM sensor for the detection of saliva glucose with high sensitivity. The CNT-PEG-hydrogel film consists of two layers; the bottom base PBA-hydrogel film is designed to recognize the glucose while the top CNT-PEG layer is used to restrict protein adsorption and improve the biocompatibility. Our results show that this CNT-PEG-hydrogel film exhibited a 10-fold enhancement on the detection limit compared to the PBA-hydrogel. Meanwhile, the adsorption of proteins on the surface of the CNT-PEG-hydrogel film, including bovine serum albumin (BSA), mucin (MUC), and fibrinogen (FIB), were reduced by 99.1%, 77.8%, and 83.7%, respectively. The CNT-PEG-hydrogel film could detect the typical saliva glucose level (0-50 mg L-1) in 10% saliva with a good responsivity. To sum up, this new tool with low-fouling film featuring high stability, specificity, and selectivity holds great potential for non-invasive monitoring of saliva glucose in human physiological levels.

Design and circuit simulation of nanoscale vacuum channel transistors.

Nanoscale vacuum channel transistors (NVCTs) are promising candidates in electronics due to their high frequency, fast response and high reliability, and have attracted considerable attention for structural design and optimization. However, conventional modeling for vacuum devices tends to focus on the work function or electric field distribution for an individual structure. Therefore, it is desirable for a new simulation method to explore the function circuits of NVCTs, e.g. high-speed logic circuits. In this study, a complete simulation of the fabrication, structure design and circuit simulation of NVCTs is demonstrated. First, the fabrication process was designed to be compatible with current semiconductor technology. Then, the "fabricated" structure was directly employed to investigate the influence of the structure parameters on the electrical performance. Furthermore, we explore the possibility of implementing an invert circuit with a single optimal NVCT. To the best of our knowledge, this is the first demonstration of a vacuum-state invertor with a circuit-simulation module in which NVCT functions as a conventional triode or FET. These simulation results illustrate the feasibility of integrating NVCTs into functional circuits and provide a theoretical method for future on-chip vacuum transistors applied in logic or radio-frequency (RF) devices.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

[Microbial community structure and function in acid mine drainage from Mengzi, Yunnan Province].

In order to explore the microbial communities and functions in distinct environments of acid mine drainage (AMD) ecosystem and fully comprehend the patterns of AMD formation and development, high-throughput sequencing technology was used to study the prokaryotic community composition in AMD puddles and surrounding rills in a mining area in Mengzi, Yunnan Province, China. By combining with physicochemical properties of the samples, we distinguished the key factors affecting the community structure and analyzed the environmental functions of the microflora. We discovered that the main phyla in AMD puddles were Euryarchaeota, Proteobacteria (including the class α-, γ- and δ-Proteobacteria), Nitrospirae, Firmicutes, Actinobacteria, and Acidobacteria. Community structure of the AMD puddles apparently differed from that of the surrounding rills. Microbial diversity was significantly positively correlated with pH, whereas the relative abundance of Thermoplasmata was negatively correlated with pH and this class might play a predominant role in community structure. There was high relative abundance of the genus Ferroplasma (6.60%-86.34%) in different samples of AMD puddles. Acidithiobacillus spp. were the major iron- and/or sulfur-oxidizing bacteria in AMD solutions and sediments, whereas relative abundance of the obligate iron-oxidizer Leptospirillum spp. was lower, and Ferrovum spp. were almost only present in the AMD solutions. In addition, acidophilic or acid-tolerant heterotrophic bacteria were widely distributed in the AMD solutions and sediments, which might promote the growth of iron- and/or sulfur-oxidizers and catalyze the oxidative/reductive dissolution of metal ores. Our results suggested that pH significantly impacted the microbial community structure of AMD environment by affecting the microbial diversity and microflora distribution.