Combination of S1-N-Terminal and S1-C-Terminal Domain Antigens Targeting Double Receptor-Binding Domains Bolsters Protective Immunity of a Nanoparticle Vaccine against Porcine Epidemic Diarrhea Virus.

Variants of coronavirus porcine epidemic diarrhea virus (PEDV) frequently emerge, causing an incomplete match between the vaccine and variant strains, which affects vaccine efficacy. Designing vaccines with rapidly replaceable antigens and high efficacy is a promising strategy for the prevention of infection with PEDV variant strains. In our study, three different types of self-assembled nanoparticles (nps) targeting receptor-binding N-terminal domain (NTD) and C-terminal domain (CTD) of S1 protein, named NTDnps, CTDnps, and NTD/CTDnps, were constructed and evaluated as vaccine candidates against PEDV. NTDnps and CTDnps vaccines mediated significantly higher neutralizing antibody (NAb) titers than NTD and CTD recombinant proteins in mice. The NTD/CTDnps in varying ratios elicited significantly higher NAb titers when compared with NTDnps and CTDnps alone. The NTD/CTDnps (3:1) elicited NAb with titers up to 92.92% of those induced by the commercial vaccine. Piglets immunized with NTD/CTDnps (3:1) achieved a passive immune protection rate of 83.33% of that induced by the commercial vaccine. NTD/CTDnps (3:1) enhanced the capacity of mononuclear macrophages and dendritic cells to take up and present antigens by activating major histocompatibility complex I and II molecules to stimulate humoral and cellular immunity. These data reveal that a combination of S1-NTD and S1-CTD antigens targeting double receptor-binding domains strengthens the protective immunity of nanoparticle vaccines against PEDV. Our findings will provide a promising vaccine candidate against PEDV.

Construction and immune effect evaluation of the S protein heptad repeat-based nanoparticle vaccine against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV), a highly virulent enteropathogenic coronavirus, is a significant threat to the pig industry. High frequency mutations in the PEDV genome have limited the effectiveness of current vaccines in providing immune protection. Developing efficient vaccines that can quickly adapt to mutant strains is a challenging but crucial task. In this study, we chose the pivotal protein heptad repeat (HR) responsible for coronavirus entry into host cells, as the vaccine antigen. HR-Fer nanoparticles prepared using ferritin were evaluated them as PEDV vaccine candidates. Nanoparticle vaccines elicited stronger neutralizing antibody responses in mice compared to monomer vaccines. Additionally, HR protein delivered via nanoparticles increased antigen uptake by antigen-presenting cells in vitro by 2.75-fold. The collective results suggest that HR can be used as antigens for vaccines, and the HR vaccine based on ferritin nanoparticles significantly enhances immunogenicity.

A Mini-Review on the Common Antiviral Drug Targets of Coronavirus.

Coronaviruses in general are a zoonotic pathogen with significant cross-species transmission. They are widely distributed in nature and have recently become a major threat to global public health. Vaccines are the preferred strategy for the prevention of coronaviruses. However, the rapid rate of virus mutation, large number of prevalent strains, and lag in vaccine development contribute to the continuing frequent occurrence of coronavirus diseases. There is an urgent need for new antiviral strategies to address coronavirus infections effectively. Antiviral drugs are important in the prevention and control of viral diseases. Members of the genus coronavirus are highly similar in life-cycle processes such as viral invasion and replication. These, together with the high degree of similarity in the protein sequences and structures of viruses in the same genus, provide common targets for antiviral drug screening of coronaviruses and have led to important advances in recent years. In this review, we summarize the pathogenic mechanisms of coronavirus, common drugs targeting coronavirus entry into host cells, and common drug targets against coronaviruses based on biosynthesis and on viral assembly and release. We also describe the common targets of antiviral drugs against coronaviruses and the progress of antiviral drug research. Our aim is to provide a theoretical basis for the development of antiviral drugs and to accelerate the development and utilization of commonly used antiviral drugs in China.

Natural hyperoside extracted from hawthorn exhibits antiviral activity against porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and death in piglets, resulting in significant economic losses for the pork industry. There is an urgent need for new treatment strategies. Here, we focused on optimizing the process of purifying natural hyperoside (nHYP) from hawthorn and evaluating its effectiveness against PEDV both in vitro and in vivo. Our findings demonstrated that nHYP with a purity >98% was successfully isolated from hawthorn with an extraction rate of 0.42 mg/g. Furthermore, nHYP exhibited strong inhibitory effects on PEDV replication in cells, with a selection index of 9.72. nHYP significantly reduced the viral load in the intestines of piglets and protected three of four piglets from death caused by PEDV infection. Mechanistically, nHYP could intervene in the interaction of PEDV N protein and p53. The findings implicate nHYP as having promising therapeutic potential for combating PEDV infections.

Licorice extract inhibits porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and even death in piglets, resulting in significant economic losses to the pig industry. Because of the ongoing mutation of PEDV, there might be variations between the vaccine strain and the prevailing strain, causing the vaccine to not offer full protection against different PEDV variant strains. Therefore, it is necessary to develop anti-PEDV drugs to compensate for vaccines. This study confirmed the anti-PEDV effect of licorice extract (Le) in vitro and in vivo. Le inhibited PEDV replication in a dose-dependent manner in vitro. By exploring the effect of Le on the life cycle of PEDV, we found that Le inhibited the attachment, internalization, and replication stages of the virus. In vivo, all five piglets in the PEDV-infected group died within 72 h. In comparison, the Le-treated group had a survival rate of 80 % at the same time, with significant relief of clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Our results suggested that Le can exert anti-PEDV effects in vitro and in vivo. Le is effective and inexpensive; therefore it has the potential to be developed as a new anti-PEDV drug.

A survey of fecal virome and bacterial community of the diarrhea-affected cattle in northeast China reveals novel disease-associated ecological risk factors.

Limited information on the virome and bacterial community hampers our ability to discern systemic ecological risk factors that cause cattle diarrhea, which has become a pressing issue in the control of disease. A total of 110 viruses, 1,011 bacterial genera, and 322 complete viral genomes were identified from 70 sequencing samples mixed with 1,120 fecal samples from 58 farms in northeast China. For the diarrheic samples, the identified virome and bacterial community varied in terms of composition, abundance, diversity, and geographic distribution in relation to different disease-associated ecological factors; the abundance of identified viruses and bacteria was significantly correlated with the host factors of clinical status, cattle type, and age, and with environmental factors such as aquaculture model and geographical location (P < 0.05); a significant interaction occurred between viruses and viruses, bacteria and bacteria, as well as between bacteria and viruses (P < 0.05). The abundance of SMB53, Butyrivibrio, Facklamia, Trichococcus, and Turicibacter was significantly correlated with the health status of cattle (P < 0.05). The proportion of BRV, BCoV, BKV, BToV, BoNoV, BoNeV, BoAstV, BEV, BoPV, and BVDV in 1,120 fecal samples varied from 1.61% to 12.05%. A series of significant correlations were observed between the prevalence of individual viruses and the disease-associated ecological factors. A genome-based phylogenetic analysis revealed high variability of 10 bovine enteric viruses. The bovine hungarovirus was initially identified in both dairy and beef cattle in China. This study elucidates the fecal virome and bacterial community signatures of cattle affected by diarrhea, and reveals novel disease-associated ecological risk factors, including cattle type, cattle age, aquaculture model, and geographical location.IMPORTANCEThe lack of data on the virome and bacterial community restricts our capability to recognize ecological risk factors for bovine diarrhea disease, thereby hindering our overall comprehension of the disease's cause. In this study, we found that, for the diarrheal samples, the identified virome and bacterial community varied in terms of composition, abundance, diversity, configuration, and geographic distribution in relation to different disease-associated ecological factors. A series of significant correlations were observed between the prevalence of individual viruses and the disease-associated ecological factors. Our study aims to uncover novel ecological risk factors of bovine diarrheal disease by examining the pathogenic microorganism-host-environment disease ecology, thereby providing a new perspective on the control of bovine diarrheal diseases.

Soil drying legacy does not affect phenanthrene fate in soil but modifies bacterial community response.

Alteration of the structure of soil microbial communities following the elimination of hydrophobic organic pollutants (e.g., polycyclic aromatic hydrocarbons, PAHs) is generally assessed using DNA-based techniques, and soil is often required to dry prior to pollutant addition, to facilitate a better mix when establishing microcosms. However, the drying practice may have a legacy effect on soil microbial community structure, which would in turn influence the biodegradation process. Here, we used 14C-labeled phenanthrene to examine the potential side effects of precedent short-term drought events. The results indicate that the drying practice had legacy effects on soil microbial community structure, illustrated by irreversible shifts in the communities. The legacy effects had no significant impact on phenanthrene mineralization and non-extractable residue formation. However, they altered the response of bacterial communities to PAH degradation, leading to a decrease in the abundance of potential PAH degradation genes plausibly attributed to moderately abundant taxa. Based on a comparison of the varied effects of different drying intensity levels, an accurate description of microbial responses to phenanthrene degradation strongly relies on the establishment of stable microbial communities before PAH amendment. Concurrent alterations in the communities resulting from environmental perturbation could greatly mask minor alterations from the degradation of recalcitrant hydrophobic PAH. In practice, to minimize the legacy effects, a soil equilibration step with a reduced drying intensity is indispensable.

A Catalog of over 5,000 Metagenome-Assembled Microbial Genomes from the Caprinae Gut Microbiota.

Most microbiome studies regarding the ruminant digestive tract have focused on the rumen microbiota, whereas only a few studies were performed on investigating the gut microbiota of ruminants, which limits our understanding of this important component. Herein, the gut microbiota of 30 Caprinae animals (sheep and goats) from six provinces in China was characterized using ultradeep (>100 Gbp per sample) metagenome shotgun sequencing. An inventory of Caprinae gut microbial species containing 5,046 metagenomic assembly genomes (MAGs) was constructed. Particularly, 2,530 of the genomes belonged to uncultured candidate species. These genomes largely expanded the genomic repository of the current microbes in the Caprinae gut. Several enzymes and biosynthetic gene clusters encoded by these Caprinae gut species were identified. In summary, our study extends the gut microbiota characteristics of Caprinae and provides a basis for future studies on animal production and animal health. IMPORTANCE We constructed a microbiota catalog containing 5,046 MAGs from Caprinae gut from six regions of China. Most of the MAGs do not overlap known databases and appear to be potentially new species. We also characterized the functional spectrum of these MAGs and analyzed the differences between different regions. Our study enriches the understanding of taxonomic, functional, and metabolic diversity of Caprinae gut microbiota. We are confident that the manuscript will be of utmost interest to a wide range of readers and be widely applied in future research.

Up-regulated 60S ribosomal protein L18 in PEDV N protein-induced S-phase arrested host cells promotes viral replication.

Coronavirus subverts the host cell cycle to create a favorable cellular environment that enhances viral replication in host cells. Previous studies have revealed that nucleocapsid (N) protein of the coronavirus porcine epidemic diarrhea virus (PEDV) interacts with p53 to induce cell cycle arrest in S-phase and promotes viral replication. However, the mechanism by which viral replication is increased in the PEDV N protein-induced S-phase arrested cells remains unknown. In the current study, the protein expression profiles of PEDV N protein-induced S-phase arrested Vero E6 cells and thymidine-induced S-phase arrested Vero E6 cells were characterized by tandem mass tag-labeled quantitative proteomic technology. The effect of differentially expressed proteins (DEPs) on PEDV replication was investigated. The results indicated that a total of 5709 proteins, including 20,560 peptides, were identified, of which 58 and 26 DEPs were identified in the PEDV N group and thymidine group, respectively (P < 0.05; ratio ≥ 1.2 or ≤ 0.8). The unique DEPs identified in the PEDV N group were mainly involved in DNA replication, transcription, and protein synthesis, of which 60S ribosomal protein L18 (RPL18) exhibited significantly up-regulated expression in the PEDV N protein-induced S-phase arrested Vero E6/IPEC-J2 cells and PEDV-infected IPEC-J2 cells (P < 0.05). Further studies revealed that the RPL18 protein could significantly enhance PEDV replication (P < 0.05). Our findings reveal a mechanism regarding increased viral replication when the PEDV N protein-induced host cells are in S-phase arrest. These data also provide evidence that PEDV maintains its own replication by utilizing protein synthesis-associated ribosomal proteins.

Isolation, identification, and pathogenicity of a goose astrovirus causing fatal gout in goslings.

Since November 2016, severe infectious diseases characterized by gout and kidney swelling and caused by goose astrovirus (GoAstV) have affected goslings in major goose-producing areas in China. In 2021, a similar serious infectious disease broke out in commercial goose farms in Heilongjiang Province, China. In this study, strain HLJ2021 was successfully isolated from goose embryos. Electron microscopy showed that the viral particles are spherical, with a diameter of about 28 nm. The complete genomic length of strain HLJ2021 is 7210 nt, and it encodes three viral proteins. A phylogenetic analysis showed that strain HLJ2021 belongs to GoAstV-2 (G2). Compared with the two original GoAstV strains, amino acid site 540Q of the strain HLJ2021 spike domain has a mutation that affects the protein structure. One potential recombination event occurred between strains HLJ2021 and AstV/HB01/Goose/0123/19, which led to the generation of recombinant strain AstV/HN03/Goose/0402/19. Strain HLJ2021 also showed strong pathogenicity in goslings. Goslings infected with GoAstV began to die at 48 h post-infection (hpi), with a mortality rate of 83.3% at 240 hpi. At autopsy, visceral urate deposits, severe renal hemorrhage and swelling, and urate in the ureter were observed in the dead goslings. These findings extend our understanding of the evolution of GoAstV, which causes gout. The isolated GoAstV strain HLJ2021 provides a potential resource for the development of biological products for the prevention of goose gout.

Goose Astrovirus in China: A Comprehensive Review.

Goose astroviruses (GoAstVs) are small non-enveloped viruses with a genome consisting of a single-stranded positive-sense RNA molecule. A novel GoAstV was identified in Shandong in 2016 and quickly spread to other provinces in China, causing gout in goslings, with a mortality rate of approximately 50%. GoAstV can also cause gout in chickens and ducks, indicating its ability to cross the species barrier. GoAstV has only been reported in China, where it has caused serious losses to the goose-breeding industry. However, in view of its cross-species transmission ability and pathogenicity in chickens and ducks, GoAstV should be a concern to poultry breeding globally. As an emerging virus, there are few research reports concerning GoAstV. This review summarizes the current state of knowledge about GoAstV, including the epidemiology, evolution analysis, detection methods, pathogenicity, pathogenesis, and potential for cross-species transmission. We also discuss future outlooks and provide recommendations. This review can serve as a valuable reference for further research on GoAstV.

Occurrence, distribution, and input pathways of per- and polyfluoroalkyl substances in soils near different sources in Shanghai.

Per- and polyfluoroalkyl substances (PFAS) are complex emerging pollutants that are widely distributed in soils. The compositions of PFAS vary according to the emission sources. However, the soil distributions of PFAS from different sources are still poorly understood. In this study, the concentrations and compositions of 18 PFAS in soils close to potential sources (industrial areas, airports, landfills, fire stations and agricultural areas) were investigated in Shanghai. The total PFAS concentrations varied from 0.64 to 294 µg kg-1d.w.. Among the sites, the highest PFAS concentration was found near the fire station (average = 57.9 µg kg-1d.w.), followed by the industrial area (average = 8.53 µg kg-1d.w.). The detection frequencies of the 18 PFAS ranged from 47.5% to 100%. Perfluorooctanoic acid (PFOA) and perfluoroheptanoic acid (PFHpA) were detected in all samples. The detection frequencies of PFAS near the fire station were higher than those near other sources. The PFAS in soils were mainly composed of short-chain perfluoroalkyl carboxylic acids (C ≤ 8). Elevated concentrations of long-chain perfluoroalkyl carboxylic acids (C > 12) were found in industrial area. Principal component analysis revealed that long-chain PFAS had different factor loadings compared to short-chain PFAS. With the exception of agricultural soils, the correlations between individual PFAS were more positive than negative. Strong positive correlations were found within three groups of perfluoroalkyl carboxylic acids (C5-C7, C9-C12, and C14-C18), suggesting their similar inputs and transportation pathways. The PFAS in soils around the fire station were likely directly emitted from a point source. In contrast, the PFAS in soils near the other sites had multiple input pathways, including both direct emission and precursor degradation.

Bovine Astrovirus-A Comprehensive Review.

Bovine astrovirus (BoAstV) is a small non-enveloped virus with a single-stranded positive-sense RNA. In 1978, BoAstV was first found in calf diarrhea fecal samples in the United Kingdom and since then it has been reported in many other countries. It has wide tissue tropism and can infect multiple organs, including the intestine, nerves and respiratory tract. Since BoAstV is prevalent in healthy as well as clinically infected bovines, and is mostly associated with co-infection with other viruses, the pathogenic nature of BoAstV is still unclear. At present, there are no stable passage cell lines available for the study of BoAstV and animal model experiments have not been described. In addition, it has been reported that BoAstV may have the possibility of cross-species transmission. This review summarizes the current state of knowledge about BoAstV, including the epidemiology, evolution analysis, detection methods, pathogenesis and potential cross species transmission, to provide reference for further research of BoAstV.

Advances in Bovine Coronavirus Epidemiology.

Bovine coronavirus (BCoV) is a causative agent of enteric and respiratory disease in cattle. BCoV has also been reported to cause a variety of animal diseases and is closely related to human coronaviruses, which has attracted extensive attention from both cattle farmers and researchers. However, there are few comprehensive epidemiological reviews, and key information regarding the effect of S-gene differences on tissue tendency and potential cross-species transmission remain unclear. In this review, we summarize BCoV epidemiology, including the transmission, infection-associated factors, co-infection, pathogenicity, genetic evolution, and potential cross-species transmission. Furthermore, the potential two-receptor binding motif system for BCoV entry and the association between BCoV and SARS-CoV-2 are also discussed in this review. Our aim is to provide valuable information for the prevention and treatment of BCoV infection throughout the world.

Epidemiological survey and genetic diversity of bovine coronavirus in Northeast China.

In 2020, to trace the prevalence and evolution of bovine coronavirus (BCoV) in China, a total of 1383 samples (1016 fecal samples and 367 nasal swab samples) were collected from 1016 cattle exhibiting diarrhea symptoms on dairy farms and beef cattle farms in Heilongjiang Province, Northeast China. All samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) detection of the BCoV N gene, followed by an analysis of its epidemiology and genetic evolution. The results indicated that of the 1016 diarrhea-affected cattle, 15.45% (157/1016) were positive for BCoV, in which positive rates of the fecal and nasal swab samples were 12.20% (124/1016) and 21.53% (79/367), respectively. Of the 367 cattle whose nasal swab samples were collected, the BCoV positive rate of the corresponding fecal samples was 15.26% (56/367). BCoV infection was significantly associated with age, farming pattern, cattle type, farm latitude, sample type, and clinical symptom (p < 0.05). Of the 203 BCoV-positive samples, 20 spike (S) genes were successfully sequenced. The 20 identified BCoV strains shared nucleotide homologies of 97.7-100.0%, and their N-terminal domain of S1 subunit (S1-NTD: residues 15-298) differed genetically from the reference strains of South Korea and Europe. The 20 identified BCoV strains were clustered in the Asia-North America group (GII group) in the global strain-based phylogenetic tree and formed three clades in the Chinese strain-based phylogenetic tree. The HLJ/HH-10/2020 strain was clustered into the Europe group (GI group) in the S1-NTD-based phylogenetic tree, exhibiting N146/I, D148/G, and L154/F mutations that affect the S protein structure. Of the identified BCoV strains, one potential recombination event occurred between the HLJ/HH-20/2020 and HLJ/HH-10/2020 strains, which led to the generation of the recombinant BCV-AKS-01 strain. A selective pressure analysis on the S protein revealed one positively selected site (Asn509) among the 20 identified BCoV strains located inside the putative receptor binding domain (residues 326-540). These data provide a greater understanding of the epidemiology and evolution of BCoV in China.

Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.

Isolation of diverse pyrene-degrading bacteria via introducing readily utilized phenanthrene.

Bacteria able to degrade pyrene play a critical role in the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). However, the traditional isolation procedure only obtains strains related to the genus Mycobacterium. The aim of the present study was to develop a modified method to isolate taxonomically distinct pyrene-degrading strains. The results indicated that replacing pyrene with phenanthrene in the isolation step improved the isolation efficiency. Using the modified method, six PAH degraders belonging to the genera Bosea, Arthrobacter, Paenibacillus, Bacillus, and Rhodococcus were obtained. They were capable of effectively utilizing pyrene (∼100%) as their sole carbon source, and could co-metabolize the degradation of benzo [a]pyrene (26.9-71.5%). In contrast, a small amount of pyrene (5.6%) and benzo [a]pyrene (8.6%) were degraded by a phenanthrene-degrading Sphingobium sp. NS7 under the same conditions. The difference in PAHs degradation between agar plate culture and liquid culture may lead to the low isolation efficiency in the traditional procedure. Hereditary stability analysis showed that PAH degradation capability of the Bosea, Paenibacillus, and Rhodococcus strains were easily lost without PAH pressure, which may partly explain why those strains were difficult to obtain using the traditional method.

Influence of bacterial community composition and soil factors on the fate of phenanthrene and benzo[a]pyrene in three contrasting farmland soils.

The fate of polycyclic aromatic hydrocarbons (PAHs) determines their potential risk in soil, which may be directly affected by abiotic conditions and indirectly through the changes in decomposer communities. In comparison, the indirect effects on the fate remain largely elusive. In this study, the fate of phenanthrene and benzo[a]pyrene and the corresponding bacterial changes were investigated in three contaminated farmland soils using a 14C tracer method and Miseq sequencing. The results showed that most benzo[a]pyrene was consistently extractable with dichloromethane (DCM) after the 60-day incubation (60.4%-78.2%), while phenanthrene was mainly mineralized to CO2 during the 30-day incubation (40.4%-58.7%). Soils from Guangzhou (GZ) showed a different distribution pattern of 14C-PAHs exemplified by low mineralization and disparate bound residue formation. The PAH fate in the Shenyang (SY) and Nanjing (NJ) soils were similar to each other than to that in the GZ soil. The fate in the GZ soil seemed to be linked to the distinct edaphic properties, such as organic matter content, however soil microbial community could have influenced the distribution pattern of PAHs. This potential role of microorganisms was reflected by the unique changes in the copy numbers of Gram positive RHDα gene, and by the distinct shifts in bacterial community composition during the incubation. A quite different shift in bacterial communities was found in the GZ microcosms which may influence PAH mineralization and non-extractable residue (NER) formation.

Oxidation of benzo[a]pyrene by laccase in soil enhances bound residue formation and reduces disturbance to soil bacterial community composition.

Laccases are capable of rapidly oxidizing benzo[a]pyrene. It is thought that the metabolites with an increase in water solubility caused by the oxidation of benzo[a]pyrene may stimulate the subsequent mineralization. However, to date, there has been no experimental evidence to support this. In this study, the fate of benzo[a]pyrene in soil affected by laccase amendment and the resulting soil bacterial responses were investigated. Laccase amendment promoted benzo[a]pyrene dissipation (15.6%) from soil, accompanied by trace mineralization (<0.58 ±â€¯0.02%) and substantial bound residue formation (∼80%). An increase of ∼15% in the bound residue fraction was observed by laccase amendment, which mainly resulted from covalent binding of the residues to humin fraction. During the incubation, the abundance of bacterial 16S rRNA and polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes did not change markedly. In contrast, benzo[a]pyrene treated with laccase resulted in a smaller shift in the bacterial community composition, indicating a reduced disturbance to the soil microbial communities. These results here suggest that benzo[a]pyrene contaminated soil can be detoxified by laccase amendment mainly due to the enhanced bound residue formation to soil organic matter via covalent binding.

Characterization of a polycyclic aromatic ring-hydroxylation dioxygenase from Mycobacterium sp. NJS-P.

Ring-hydroxylating dioxygenases (RHDs) play a critical role in the biodegradation of polycyclic aromatic hydrocarbons (PAHs). In this study, genes pdoAB encoding a dioxygenase capable of oxidizing various PAHs with up to five-ring benzo[a]pyrene were cloned from Mycobacterium sp. NJS-P. The α-subunit of the PdoAB showed 99% and 93% identity to that from Mycobacterium sp. S65 and Mycobacterium sp. py136, respectively. An Escherichia coli expression experiment revealed that the enzyme is able to oxidize anthracene, phenanthrene, pyrene and benzo[a]pyrene, but not to fluoranthene and benzo[a]anthracene. Furthermore, the results of in silico analysis showed that PdoAB has a large substrate-binding pocket satisfying for accommodation of HMW PAHs, and suggested that the binding energy of intermolecular interaction may predict the substrate conversion of RHDs towards HMW PAHs, especially those may have steric constraints on the substrate-binding pocket, such as benzo[a]pyrene and benzo[a]anthracene.