Abstract:Host defense peptides are a class of small molecular peptides ubiquitous in vertebrates, with a broad spectrum of antimicrobial activities and immunomodulatory functions such as anti-inflammation, regulation of cell chemotaxis, promotion of angiogenesis, and repair of injury. The previous studies about host defense peptides mainly focused on the resistance against bacterial and fungal infections. The recent studies have demonstrated that host defense peptides have a wide range of antiviral activities and thus have potential application prospects in the prevention and treatment of viral diseases. This paper reviews the molecular mechanisms of host defense peptides against viral infections, which include direct killing of viruses, regulation of viral infection, and participation in host immune regulation against viral infections, which will provide a reference for the research on host defense peptides against viral infections and the development of host defense peptides as new antiviral biological agents.

Abstract:RecJ proteins, the members of aspartate-histidine-histidine (DHH) superfamily of phosphoesterases, are ubiquitous in bacteria, eukaryotes, and archaea. In bacteria, the archetypal RecJ protein is a 5′→3′ ssDNA exonuclease that functions in biological processes such as mismatch repair, homologous recombination, and base excision repair. Cell division cycle 45 (Cdc45) protein, a homologue of bacterial RecJ nuclease, is essential in eukaryotes, but has no nuclease activity. Cdc45 protein forms Cdc45-MCM-GINS (CMG) complex with minichromosome maintenance (MCM) and Go-Ichi-Ni-Sa (GINS), and the complex plays a key role in DNA replication in eukaryotes. Almost all archaea whose genomes have been sequenced encode one or more RecJ protein homologues. Unlike bacterial RecJ nuclease, archaeal RecJ protein has diverse nuclease activities and can form a complex with MCM and GINS which shows functions similar to eukaryotic CMG complex. Thus, archaeal RecJ protein is an important component that is involved in archaeal DNA replication, repair and recombination. Based on current reports on archaeal RecJ protein, this study reviewed the activities, structures, and functions of archaeal RecJ protein, focusing on similarities and differences between different archaeal RecJ proteins and between archaeal RecJ proteins and bacterial RecJ nucleases/eukaryotic RecJ homologues. In addition, we summarized the research trends in archaeal RecJ protein.

Abstract:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a global COVID-19 pandemic threatening human health and safety. However, it is urgent to find effective therapeutic agents and targets in response to the emergence of novel variants. Interferon-stimulated genes (ISGs), a class of genes upregulated by interferons (IFNs), play a crucial role in host resistance against viral infection. Studies have demonstrated that ISGs are able to target different stages of viral replication cycle to exert the effect against viral infection, whereas SARS-CoV-2 has evolved strategies to interfere with or evade host innate immune response. Comprehensively understanding the interactions between SARS-CoV-2 and ISGs is critical for the design of antiviral therapeutics. This review aims to briefly introduce the mechanisms of different ISGs against SARS-CoV-2, which provides ideas and theoretical basis for the development of novel antiviral agents.

Abstract:The dissimilatory reduction of nitrate and nitrite to produce ammonium is an accessory pathway of nitrogen transformation, which provides a basis for the reuse of nitrogen in the ecosystem and has become a research hotspot in recent years. According to the available reports, the occurrence mechanism and intensity of ammonium production by dissimilatory reduction of different nitrogen sources are different, which determines the efficiency of microbial ammonium production. Therefore, it is necessary to clarify the metabolic mechanism of dissimilatory reduction of different nitrogen sources. This paper introduces the species of microorganisms involved in the dissimilatory reduction of nitrate and nitrite and elaborates on the ammonium production pathways and mechanisms. Further, we analyzed the effects of single and mixed nitrogen sources on microbial ammonium production, summarized the advantages of ammonium production by actinomycetes compared with other microorganisms, and prospected the future research directions. This review aims to provide a theoretical basis for the application of microbial dissimilatory nitrate and nitrite reduction to ammonium.

Abstract:The tripartite motif (TRIM) protein family is composed of a variety of proteins with E3 ubiquitin ligase activity. These proteins are involved in different cell processes and play a role in the host immune response to viral infection. The proteins of TRIM family can exert antiviral activity by enhancing host innate immune response or directly degrading viral proteins. In some cases, some viruses can use TRIM family proteins to regulate cytokine expression to promote their infections. This article reviews the research progress in the role of TRIM family proteins in viral replication and the related mechanisms, aiming to provide a reference for studying the mechanism of viral infection.

Abstract:Seed, the specific reproductive organ of spermatophyte, is recognized as a potent reservoir and vector for beneficial and pathogenic microorganisms which play important roles in the growth, development, health, quality, and yield of plants. Plant microbiome, especially rhizosphere microbiome and phyllosphere microbiome, has attracted wide attention and flourished with the development of microbial ecology and related technology. However, seed microbiome is still a virgin field absent from enough concern. Bacteria are well known as the dominant microorganisms in seed endophytes. In this review we summarized the progress in the composition, biological function, transmission, and core microbiome of seed bacteriome. We further expounded the definition and significance of core microbiome in seed endophytes and proposed the questions to be solved, aiming to provide guidance for the future research in the leading-edge area.

Abstract:The metabolic diversity of anaerobic ammonia-oxidizing bacteria (AnAOB) allows them to widely distribute in different natural ecosystems such as oceans, wetlands, and land, which are even detected in some extremely hot and cold environments. In this review, the discovery, distribution, and denitrification contribution of anaerobic ammonia-oxidizing bacteria in different ecosystems were reviewed and summarized, and the main environmental factors affecting their distribution were analyzed as well. This review may help us understand the real role and function of anaerobic ammonia-oxidizing bacteria better in the global nitrogen cycle. Based on the anaerobic ammonia oxidation (anammox) process, this paper explored novel biological nitrogen removal processes that could cooperate with it, to provide an ecological basis and new ideas for the promotion and development of the processes, thereby realizing the technological transformation of denitrification process.

Abstract:OxyR, an oxidative stress regulator of the LysR family, is essential for bacteria to survive when confronting the threat of hydrogen peroxide (H₂O₂). OxyR regulates the expression of multiple genes such as those encoding catalase and peroxidase to scavenge H₂O₂, participates in the iron metabolism to control the generation of peroxides, and repairs biomacromolecule damage. Functioning as a two-state redox switch depending on the formation of intracellular disulfide bond in the oxidizing and reducing environments, OxyR could recognize specific binding motif of target genes and activate/repress their expression. Here, we review the latest research progress in the OxyR-mediated regulation of gene expression in bacteria, which will help to further understand the role of OxyR in resisting oxidative stress and provide a molecular basis for preventing and controlling related pathogens.

Abstract:Cytolethal distending toxin (CDT) is an AB₂ toxin produced by a variety of Gram-negative bacteria. It was the first bacterial genotoxin described, encoding three polypeptides: CDTA, CDTB, and CDTC. CdtB is the active moiety that has the ability to damage multiple cell types. CDT has a new molecular mode of action, which can interfere with the progress of eukaryotic cell cycle, resulting in G2/M arrest and apoptosis. Such mechanism of action targets cells, and the available studies of CDT concentrate on the cellular level. However, CDT, as a virulence factor, ultimately causes pathogenic damage to the host, and the molecular mechanism of CDT-host interaction remains unclear. This paper comprehensively expounded the damage of CDT as a virulence factor to the host defense mechanism from three aspects, including damage to the epithelial barrier, acquired immunity, and promotion of inflammatory response, aiming to reveal the pathogenic mechanism of CDT and provide a theoretical basis and new ideas for clinical treatment.

Abstract:Ulcerative colitis (UC) is a life-long refractory disease with high incidence worldwide. Akkermansia muciniphila (A. muciniphila) and the metabolites short chain fatty acids (SCFA) have been found to protect intestinal mucosal barrier in UC, but the specific mechanisms fail to be summarized. Therefore, we analyze the research on the mechanical, chemical, immune, and biological barriers of intestinal mucosa and discuss the mechanisms of A. muciniphila and SCFA on intestinal mucosal barrier, hoping to provide a new perspective and mindset for the study of the pathogenesis and therapy of UC.

Abstract:[Objective] To evaluate the inhibitory and killing effects of two epigallocatechin gallate (EGCG) derivatives and compound potassium peroxymonosulfate powder (KMPS) on grass carp reovirus genotype I (GCRV) by in vitro cell experiments. [Methods] The safe concentrations of epigallocatechin gallate palmitate (EGCG-P), peracetylated epigallocatechin gallate (AcEGCG), and KMPS were assessed by MUSE and CCK-8 assays. Different concentrations of EGCG-P, AcEGCG, and KMPS were used to treat the cells infected with GCRV-JX01, and the inhibitory and killing effects of the three substances on the virus were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, qRT-PCR was employed to determine the expression levels of the genes involved in host immune responses in the cells treated with the three substances and then infected with the virus. [Results] EGCG-P and AcEGCG suppressed the viral proliferation in a concentration- dependent manner, and EGCG-P outperformed AcEGCG. The EGCG-P of 10 μg/mL and 40 μg/mL decreased the titer of GCRV-JX01 by 10² copies/μL and 10³ copies/μL, respectively, compared with the control group, while AcEGCG achieved the same inhibitory effect at the concentrations of 60 μg/mL and 120 μg/mL, respectively. EGCG-P, AcEGCG, and KMPS all had killing effect on GCRV-JX01, and the lowest dose for effective killing followed the trend of KMPS (5 μg/mL)<EGCG-P (120 μg/mL)<AcEGCG (180 μg/mL). Moreover, EGCG-P, AcEGCG, and KMPS up-regulated the expression of interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and myeloid differentiation factor (MyD88) compared with the control group. In particular, the up-regulation was more significant after the treatment with EGCG-P (50 μg/mL) for 8 h and AcEGCG (10 μg/mL) for 24 h. [Conclusion] EGCG-P, AcEGCG, and KMPS have good inhibitory and killing effects on GCRV-I. EGCG-P and KMPS at a concentration of 40 μg/mL can significantly inhibit GCRV replication without evoking an inflammatory response in 24 h. This study provides a reference for the development of environmentally friendly disinfection products for GCRV.

Abstract:[Objective] The research and development and application of eco-friendly biopesticides for the sustainable agricultural development has received widespread concern. Microbial metabolite pesticide (MMP) is one of the most popular biopesticides in China and also one of the most important green pesticides to be developed. [Methods] The nutrient agar (NA) containing Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A was used to screen the anti-Xoo Streptomyces strain HSW2009. High performance liquid chromatography (HPLC) and mass chromatography (MS) were employed to characterize the chemical structure of the anti-Xoo metabolite produced by HSW2009. Xoo was inoculated into adult rice plants (Oryza sativa L. subsp. japonica) by leaf-clipping, followed by spraying with the piericidin solution (0.1 g/L). The length of spot was measured 2 weeks after inoculation. Response surface methodology was employed to optimize the fermentation medium. PacBio SMRT together with Illumina HiSeq X Ten was applied for genome sequencing. Average nucleotide identity (ANI) was compared to analyze the relationship between HSW2009 and other Streptomyces strains. [Results] A Streptomyces strain HSW2009 was detected to have strong inhibitory effect on Xoo growth and it produced the active metabolite piericidin A1 (PIE). PIE application mitigated Xoo infection on rice leaves. An optimized MFM medium was developed for PIE production. In MFM, HSW2009 grew well, with the highest fermentation titer of PIE reaching 0.72 g/L. HSW2009 was different from the known PIE-producing Streptomyces strains in colony phenotype, PIE biosynthetic gene cluster, whole genome sequence, and ANI. [Conclusion] HSW2009 is a representative of a candidate novel species of Streptomyces producing high level of PIE and is named as Streptomyces shaowuensis sp. nov. It has potential to be developed as an industrial strain for piericidin production.

Abstract:[Objective] To express the truncated fragments of enhancin gene from Pseudaletia unipuncta granulovirus-Ps (PuGV-Ps) in Bacillus thuringiensis (Bt) and provide a theoretical basis for the construction of Bt engineering bacteria. [Methods] The codon of the truncated fragments of enhancin gene was optimized for the construction of the expression vectors of enhancin and the expression of fusion proteins. Then, the expression levels of enhancin under the guidance of two promoters were analyzed, and the synergistic activity of enhancin on Bt was determined. [Results] The expression vectors pHTP_cry1AcCoEn81, pHTRHCoEn81, and pHTNCCoEn81 were constructed in the study. SDS-PAGE showed that pHTP_cry1AcCoEn81 and pHTNCCoEn81 produced recombinant proteins of 81 kDa and 134 kDa, respectively. There was no significant difference in the expression level of enhancin or the yield of recombinant protein under the guidance of promoters P_cry1Ac and P_cry8E. Furthermore, the recombinant enhancin significantly increased the insecticidal activity of Bt against Plutella xylostella. [Conclusion] The codon-optimized enhancin of PuGV-Ps can be expressed in Bt and has significant synergistic activity, which provides theoretical guidance for the construction and application of Bt engineering bacteria with high efficiency.

Abstract:[Objective] To investigate the diversity and potential value of the algae-associated symbiotic or epiphytic actinomycete resources from South China Sea, we isolated and identified actinomycetes from different algae samples collected from Xisha Islands and evaluated their antibacterial activities. [Methods] We used two types of media for the isolation and purification of actinomycetes and the isolates were identified according to their 16S rRNA gene sequences and the phylogenetic tree. The activities of their fermentation products against 10 pathogenic bacterial strains including Streptococcus agalactiae were tested with the punching method. The whole genome of the target active strain was sequenced and the biosynthetic gene clusters of secondary metabolites were predicted and analyzed by AntiSMASH. [Results] A total of 36 strains of actinomycetes were isolated from six kinds of algae. Based on 16S rRNA gene sequence and phylogeny analysis, they belonged to Streptomyces (2 strains), Rhodococcus (2 strains), Nocardia (3 strains), Micromonospora (5 strains), and Salinispora (24 strains), respectively. The antibacterial tests showed that the fermentation products of the 36 strains displayed inhibitory effect against at least one of ten sensitive bacteria. The antibacterial activities of Salinispora spp. were stronger than those of other actinomycetes. AntiSMASH analysis showed that more than 22.28% of the gene sequences in HZ014 genome were related to the biosynthesis of secondary metabolites, indicating their great potential in natural product biosynthesis. [Conclusion] The algae of Xisha Islands are home to abundant rare culturable actinomycetes. In this study, marine obligate rare actinomycetes associated with marine algae, Salinispora spp., were reported for the first time. The fermentation products of 36 strains of actinomycetes displayed antibacterial activity, indicating the potential value in the prevention of fish diseases. These rare actinomycetes are potential resources for the development of marine drugs or biocontrol agents.

Abstract:[Objective] Cronobacter, a foodborne pathogen transmitted mainly by powdered infant formula, can cause necrotizing enterocolitis, bacteraemia, meningitis and other diseases. We analyzed the composition of capsular polysaccharides from four K:CA types of Cronobacter to explore the correlation between different capsular types and monosaccharide composition of capsular polysaccharides. [Methods] We measured the yields of capsular polysaccharides from 28 Cronobacter strains (4 K:CA types) by phenol-sulfuric acid method and determined the monosaccharide composition of the capsular polysaccharides by ¹H NMR. [Results] The capsular polysaccharide yield of Cronobacter was the best when the bacteria were cultured in milk agar for 48.0 h, while the monosaccharide composition did not change under different culture conditions. The yield of capsular polysaccharides varied among the four K:CA types of Cronobacter, of which the K2:CA2 type had the highest average yield. Further, we identified that two strains C. sakazakii ATCC 12868 and C. sakazakii ATCC 29004 with high yields of capsular polysaccharides (19.6% and 28.4%, respectively) were of K2:CA2 type. The capsular polysaccharides of 28 Cronobacter isolates contained 8 monosaccharides, among which β-ManpNAc was only in C. malonaticus cro1754B2 and C. sakazakii cro1573B3, and β-Ribp only in C. sakazakii cro771A2. In addition, α-Rhap, β-Glcp, and α-Glcp were the dominant monosaccharide components of capsular polysaccharide in strains of K1:CA1 type, K1:CA2 and K2:CA1 types, and K2:CA2 type, respectively. [Conclusion] This study preliminarily revealed the polysaccharide yields of four K:CA capsular types of Cronobacter and found that K2:CA2 type had the highest average yield of capsular polysaccharide. We confirmed the correlation between the monosaccharide composition of capsular polysaccharide and the capsular types of Cronobacter, which provided a theoretical and technical foundation for further research on the capsular properties of Cronobacter.

Abstract:[Objective] This paper aims to explore the growth and toxigenicity of Shiga toxin-producing Escherichia coli (STEC) under different stress conditions, and to provide basic data reference for the study of the stress response mechanism of STEC strains under stress. [Methods] Three STEC strains were selected for adaptive subculture at different pH values, different salt concentrations, and different temperatures, and their growth heterogeneity under stress was analyzed via the Gompertz model. The toxigenicity of the strains under stress was analyzed by the Vero cytotoxicity assay. [Results] Except the ST462 strain at pH 5.0, other STEC strains showed decrease in the maximum specific growth rate (μ_max) and extension of the lag period (λ) under stress conditions (pH 5.0 or 9.0, 1.5% NaCl or 2.5% NaCl, 10 ℃) (P<0.01). The viability of the cells treated with the toxins extracted from STEC strains under different stress conditions was lower than that under the optimal control conditions, while the opposite trend was observed at pH 5.0. [Conclusion] STEC strains activated the stress response mechanism under alkaline stress, higher salt concentrations, and low temperatures. Although the growth was inhibited, the toxins produced by the strains exposed to stress aggravated the damage to Vero cells, demonstrating increased toxicity. By analyzing the heterogeneity of the growth and toxigenicity in a stressful environment, this study helps to comprehensively understand the biological characteristics of STEC strains under stress and has guiding significance for improving the detection, sterilization, and prevention measures during food processing to ensure food safety.

Abstract:[Objective] To investigate the effects of bio-fermented rice straw on intestinal epithelial morphology and microflora of Hu sheep by microscopic observation and 16S rRNA gene high-throughput sequencing technology. [Methods] Twenty-one male lambs aged 70 d with similar body weight (25.15±0.47) kg were randomly divided into three groups based on the composition of dietary roughage, namely, the rice straw group, the bio-fermented rice straw group, and the alfalfa hay group, with dietary concentrate to forage ratio of 6:4. The experiment lasted for 7 weeks, including 3 weeks of the adaptation period and 4 weeks of the experimental period. At the end of the experiment, all animals were sacrificed to collect samples. Epithelial tissues of the rumen, jejunum, and colon were collected for morphological observation. The content of corresponding intestinal segments was collected for the analysis of microbiota and metabolites. [Results] Compared with the rice straw group, the bio-fermented rice straw group significantly increased the relative abundance of Fibrobacteres and the content of volatile fatty acids (VFA) in the rumen of Hu sheep, and promoted the development of the rumen epithelium. The bio-fermented rice straw significantly increased the relative abundance of Firmicutes and Verrucomicrobia in the jejunum of Hu sheep. It changed the structure of the jejunal microbial community and promoted the development of jejunal epithelial tissues. Moreover, the colonic microbial structure of Hu sheep was also changed by the bio-fermented rice straw. [Conclusion] Feeding bio-fermented rice straw is beneficial to the development of intestinal epithelium and increases the microbial diversity in the digestive tract of Hu sheep.

Abstract:[Objective] To isolate the microorganisms that can remove ammonia and hydrogen sulfide from biogas residue and pyrite soil and use them for composting, so as to reduce the odor release in the process of manure treatment and improve the working environment. [Methods] The microorganisms capable of removing ammonia and hydrogen sulfide were screened out by the culture method and identified by 16S rRNA gene sequencing. The strains with good removal performance were selected to prepare the microbial deodorants. The deodorants were then applied to the composting of manure, and their deodorization effects were preliminarily evaluated based on the concentrations of ammonia and hydrogen sulfide. [Results] A total of 12 ammonia-removing strains and 5 sulfur-removing strains were isolated, from which 5 strains with good removal performance were screened out and labeled as N-2, N-5, N-6, N-11, and S-3, respectively. The microbial deodorant composed of strains N-5, N-6, N-11, and S-3 had the best deodorization effect, with the ammonia- and sulfide-removing rates of 82.46% and 84.84%, respectively. The composting test proved that the microbial deodorant had deodorization effect. Especially in the early stage of composting, the microbial deodorant group reduced the release of ammonia and hydrogen sulfide in the process of turning on day 7 by 62.84% and 53.12%, respectively, compared with the control group. At the end of composting, the ammonia nitrogen content of the microbial deodorant group was 33.62% lower than that of the control group. [Conclusion] The microbial deodorant produced in this study can effectively reduce the release of odorant gas in the process of manure composting and thus demonstrates great application potential in improving the environment of livestock waste treatment.

Abstract:[Objective] To investigate the antifungal activity and mechanism of berberine against Cryptococcus neoformans. [Methods] The minimum inhibitory concentration (MIC) of berberine against the standard strain and clinical isolates of C. neoformans was determined by micro-broth dilution method. The synergistic effect of berberine with the marketed antifungal drugs (fluconazole and amphotericin B) was determined by checkerboard method. Further, we determined the effects of berberine on the expression of key virulence factors of Cryptococcus and on the macrophage-Cryptococcus interaction. The in vivo fungicidal activity of berberine was examined with the Galleria mellonella model of Cryptococcus infection. [Results] Berberine was a fungicidal compound with a MIC range of 8−16 µg/mL against the tested strains. Berberine at the sublethal concentration inhibited capsule size, melanin-producing ability, and sexual reproduction and enhanced the fungicidal ability of macrophages. The zinc-finger transcription factor Nrg1 mediated these important processes. The experiment with the G. mellonella model of cryptococcal infection showed that berberine prolonged the survival time of infected G. mellonella. [Conclusion] Berberine exhibits excellent anti-cryptococcal activity in vitro and in vivo and is expected to be a promising starting compound for the development of anti-cryptococcal agents.

Abstract:[Objective] The microbial fermentation of plant can increase the yield of polysaccharide and transform the original plant polysaccharide into a new fermentative polysaccharide with higher activity. According to the viable count, yield of Saussurea involucrata polysaccharide, and skincare efficacy, we conducted strain screening, aiming to obtain a strain suitable for the production of S. involucrata polysaccharide. [Methods] Different strains were used to produce S. involucrata polysaccharide. The viable count was determined by plate colony counting method, and the content of polysaccharide in the fermentation broth was determined by anthrone colorimetry. The cell models of barrier damage and inflammation were used to evaluate the skincare efficacy of the polysaccharide in cells, with the cell viability measured by MTT method and NO content measured by Griess method. The mouse model of atopic dermatitis was used to evaluate the efficacy of the polysaccharide in animals in terms of skin appearance, trans-epidermal water loss, skin pathology, changes in epidermal thickness, and the barrier protein filaggrin of skin tissue. [Results] There were significant differences in the viable count and yield of crude polysaccharide after fermentation by different strains. The viable counts of Bacillus subtilis CCFM1162, B. subtilis 165-M1, Lactobacillus casei CCFM1073, L. reuteri CCFM8631, and L. sakei GD17-9 were no less than 2.0×10⁸ CFU/mL and higher than those of the other strains. However, Saccharomyces cerevisiae HN7-A5, L. casei CCFM1073, L. reuteri CCFM8631, and L. sakei GD17-9 produced more than 1.37 g/L polysaccharide. On the basis of the polysaccharide yield and the viable count, the skincare efficacy of crude polysaccharide from S. involucrata fermented by L. casei CCFM1073, L. reuteri CCFM8631, L. sakei GD17-9, and S. cerevisiae HN7-A5 was evaluated with cell models. The results showed that the polysaccharides produced by the selected strains had little effect on the survival rate of HaCaT cells induced by sodium lauryl sulfate, while the polysaccharides decreased the NO content in the RAW264.7 cells exposed to lipopolysaccharide (LPS). The polysaccharides produced by L. sakei GD17-9 and L. reuteri CCFM8631 had better effect, reducing the NO content by 81% and 71%, respectively. According to the viable count, polysaccharide yield, and the skincare efficacy in cell models, we selected the crude polysaccharides from S. involucrata fermented by L. sakei GD17-9 and L. reuteri CCFM8631 to validate the skincare efficacy in the animal model. The results showed that the crude polysaccharides from S. involucrata fermented by L. reuteri CCFM8631 had better skincare efficacy. [Conclusion] L. reuteri CCFM8631 was the most suitable strain for the production of crude polysaccharide from S. involucrata, considering the viable count, the polysaccharide yield, and the skincare efficacy in cell and animal models.

Abstract:[Objective] To investigate the regulatory effect of the heavy metal-resistant bacteria on lead (Pb) and cadmium (Cd) resistance and flavonoid biosynthesis in chicory (Cichorium intybus L.) seedlings under Pb and Cd stress. [Methods] After inoculation of Ensifer adhaerens JB19, the growth indicators, the content of Pb and Cd, the activities of antioxidant enzymes, the content of total flavonoids, and the expression of genes related to flavonoid biosynthesis in the chicory seedlings exposed to different concentrations of Pb and Cd ((200+20) mg/kg, (400+40) mg/kg, (800+80) mg/kg) were determined. [Results] Strain JB19 significantly increased the biomass and chlorophyll content of chicory seedlings exposed to different concentrations of Pb and Cd. Under the Pb+Cd treatment of (200+20) mg/kg, the inoculation of strain JB19 decreased the content of H₂O₂, MDA, and Pb and Cd in the shoot by 25.7%, 26.1%, 53.2%, and 54.1%, respectively, compared with the control. Moreover, the strain strengthened the antioxidant enzyme system and up-regulated expression of the genes involved in flavonoid biosynthesis of chicory seedlings. Under the Pb+Cd treatment of (400+40) mg/kg, the strain increased the flavonoid content by 105.2% and up-regulated the expression of chalcone isomerase gene to reach 458.9%. [Conclusion] E. adhaerens JB19 can reduce the accumulation of heavy metals and improve the Pb and Cd resistance of chicory seedlings by increasing plant biomass, reducing active oxygen, inhibiting membrane lipid peroxidation, enhancing the activities of antioxidant enzymes, and regulating the levels of flavonoids.

Abstract:[Objective] To screen out the strong promoters suitable for the expression of alkaline pectinase in Bacillus amyloliquefaciens based on the transcription level and expression of the target gene and further analyze the selected strong promoters. [Methods] The promoter fragments were predicted and screened out by bioinformatics tools on the basis of the relative fluorescence intensity and enzyme activity. Further, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was carried out to determine the transcription levels of different promoters. [Results] The activities of PrapA, PmetE-1, and Phin-1 expressing alkaline pectinase were 9.8, 4.8, and 3.0 times that of P43 promoter, respectively. The three strong promoters screened out laid a foundation for the expression of other heterologous genes in B. amyloliquefaciens. [Conclusion] The strong promoters PrapA, PmetE-1, and Phin-1 screened out in this study can effectively improve the expression of alkaline pectinase.

Abstract:[Objective] To investigate the effects of sacbrood virus (SBV) infection on the genes associated with the immunity, metabolism, resistance to virus, and cell growth and metabolism of Apis cerana cerana and Apis mellifera ligustica. [Methods] The 2-day-old larvae were collected from the colonies of A. c. cerana and A. m. ligustica and reared in an incubator at 34 ℃ and RH 85%. The larvae were infected with SBV at 3 days old, and the dead larvae were then recorded every day. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to measure the expression levels of gene SBV, as well as the genes associated with immunity (Apidaecin, Abaecin, Hymenoptaecin, Defensin, Lys-1, Pgrp-lc, Kenny, and Domeless), metabolism (Ilp1, Hex110, and Vg), resistance to viruses (Dis3, Dicer, and Ago1), and cell development and metabolism (Vhdl, Co-1-iv, and Mta1), in the 4- and 7-day-old larvae. [Results] After infection with SBV at the same dose, all the larvae of A. c. cerana died at 8 days old, while some larvae of A. m. ligustica emerged. The relative expression of SBV in A. c. cerana was significantly higher than that in A. m. ligustica of the same days old. Compared with the control group, SBV infection significantly up-regulated the expression of Abaecin, Apidaecin, Hex110, Dicer, and Vhdl in the 4-day old larvae of A. c. cerana, and it up-regulated the expression of Hymenoptaecin and Ago1 and down-regulated that of Apidaecin, Abaecin, Vg, and Vhdl in the 4-day-old larvae of A. m. ligustica. In addition, SBV infection down-regulated the expression levels of Hex110, Dis3, and Ago1 in the 7-day-old larvae of A. c. cerana and Ilp1, Dicer, and Co-1-iv in the 7-day-old larvae of A. m. ligustica. [Conclusion] The larvae of A. c. cerana were more susceptible to SBV infection than those of A. m. ligustica. The significant differences in the expression levels of the genes involved in immunity and development between A. c. cerana and A. m. ligustica may be associated with the differences in the defense mechanism against diseases and pests, the regulation of nutrient metabolism, and the virus-caused siRNA response.

Abstract:[Objective] To explore the effects of retrogressive succession of the alpine wetland of Bayanbulak Swan Lake in Xinjiang on soil properties and microbial community structure by relying on the typical transect of retrogressive succession (swamp-swamped meadow-meadow). [Methods] The soil microbial community structure was analyzed by high-throughput sequencing. [Results] The retrogressive succession of the alpine wetland changed the species composition of soil microorganisms at the operational taxonomic unit (OTU) level. The ACE, Chao1, Simpson, and Shannon diversity indexes of soil microorganisms in the meadow area were lower than those in the swamp area and the swamped meadow area (P<0.05). With the occurrence of succession, the relative abundance of Proteobacteria, Acidobacteria, Bacteroidetes, and Ascomycota decreased, while that of Actinobacteria, Gemmatimonadetes, Basidiomycota, and Mortierellomycota increased. Principal coordinates analysis showed that soil microbial communities presented different degrees of dispersion at each stage of retrogressive succession. The species dispersion in the meadow area was larger than that in the swamped meadow and swamp areas where a certain degree of aggregation appeared. Further, the redundancy analysis indicated that soil organic carbon, soil water content, soil bulk density, microbial biomass nitrogen, and microbial biomass carbon were the key factors affecting the dominant microbial phyla and genera. [Conclusion] The retrogressive succession of the alpine wetland decreased the soil microbial community diversity and led to the transition from eutrophic to oligotrophic community.

Abstract:[Objective] To characterize the function of the transporter encoded by ydgF1 in Bacillus licheniformis 9945a. [Methods] The strain 9945a/pHY300-Shu-ydgF1 with ydgF1 being overexpressed and the strain 9945a ΔydgF1 with ydgF1 knocked out were constructed. Phosphate d-alanine (PDA) medium with d-alanine as the sole nitrogen source was designed to observe the growth ability of strains, and cell assimilating experiments were implemented. Real-time quantitative polymerase chain reaction (RT-qPCR) evaluated the relative expression of ydgF1 in 9945a and 9945a ΔydgF1 at different growth phases in the LB medium. Colony forming units (CFU) of 9945a and 9945a ΔydgF1 at the anaphase in the LB medium were determined by viable plate counting. [Results] In the PDA medium, the specific growth rate of 9945a ΔydgF1 was always lower than that of 9945a, while the maximum specific growth rate of 9945a/pHY300-Shu-ydgF1 was 0.336 h⁻¹, which was 1.98 times that of 9945a/pHY300-Shu. OD₆₀₀ of 9945a/pHY300-Shu-ydgF1 was 3.04 after culturing for 15 h, which was 1.73 times that of 9945a/pHY300-Shu. In cell assimilating experiments, the concentration of d-alanine assimilated by 9945a ΔydgF1 was (0.509±0.055) g/L, which was significantly lower than (0.759±0.038) g/L assimilated by 9945a. 9945a/pHY300-Shu-ydgF1 assimilated (0.821± 0.021) g/L, slightly higher than 9945a. The results of RT-qPCR showed the relative expression of ydgF1 in 9945a increased gradually at the transition and stationary phases, and the relative expression of ydgF1 in 9945a ΔydgF1 was almost zero at all different growth phases. CFU of 9945a and 9945a ΔydgF1 at the anaphase suggested that the knockout of ydgF1 weakened the viability of B. licheniformis 9945a in the anaphase. [Conclusion] YdgF1 assimilates d-alanine in B. licheniformis 9945a, which is beneficial to the long-term survival of B. licheniformis 9945a. In addition, it may also assimilate l-asparagine.

Abstract:[Objective] Lactobacillus paragasseri as a closely related species of L. gasseri, is one of the important bacteria in the intestinal and reproductive tract. Our research team previously found that L. paragasseri IMAU FB017 had good tolerance to stomach acid and bile salt. Therefore, we intended to analyze the genetic background of IMAU FB017 from the genome level, explore its functional gene characteristics, and dig into its potential probiotic genes, thereby laying a genetic foundation for its development and utilization. [Methods] In this study, the whole genome of IMAU FB017 was sequenced and assembled using Nanopore and Illumina sequencing technologies, and the genomic sequences of 18 L. paragasseri strains published by National Center for Biotechnology Information (NCBI) were combined for comparative genomic analysis. Roary software was used to identify core gene sets and pan gene sets. The Rapid Annotation using Subsystem Technology website was used for functional annotation of the genome to explore the genomic characteristics of IMAU FB017. [Results] The results showed that IMAU FB017 genome contained a ring chromosome and a plasmid, and the size of the ring chromosome was 1 880 023 bp, with 34.90% of the guanine/cytosine (G/C) content, containing 1 851 protein-coding sequences. The size of the plasmid was 43 639 bp, and the G/C content was 38.00%. A phylogenetic tree was constructed based on 903 core genes identified from 18 L. paragasseri genome sequences. It was found that L. paragasseri population formed three clades. Strain IMAU FB017 belonged to clades Ⅰ, which contained the most strains (13 strains, about 68%), and the strains from the same source had no significant aggregation trend. Functional annotation analysis found that the IMAU FB017 genome encoded genes related to N-acetylgalactosaamine and galactosaamine utilization, as well as probiotic genes related to oxalate catabolism, and contained a complete exopolysaccharide (EPS) gene cluster and three acid and bile salt tolerance gene clusters corresponding to the phenotype. [Conclusion] In this paper, the genetic background of IMAU FB017 has been analyzed at the genomic level, and the potential probiotic genes have been mined to provide references for its development and utilization.

Abstract:[Objective] To study the synergistic remediation efficiency of polycyclic aromatic hydrocarbons (PAHs) by mixed functional bacteria constructed by Pseudomonas putida B6-2 and Klebsiella sp. CW-D3T, and explore the effect of non-ionic surfactant Tween-80 on the degradation of PAHs by mixed bacteria, to provide the potentials and insights for the biodegradation of aromatic compounds. [Methods] In this study, we measured the bacteria-growth curve and the determined proportion of mixed bacteria by standard plate count method, thereby exploring the feasibility of mixed bacteria degradation system. We investigated the degradation efficiencies of PAHs by pure cultures and bacterial mixed cultures at different concentrations of Tween-80 using high-performance liquid chromatography. Finally, we conducted the cell surface hydrophobicity assay by an alkane adsorption method to explore the mechanism of Tween-80 in affecting the degradation of PAHs by mixed functional bacteria. [Results] The growth state of the two strains mixed in equal proportion was better than that of the pure culture system. After 7 days, the degradation rates of mixed PAHs (phenanthrene, fluoranthene, and pyrene) were 33.4%, 30.1%, and 28.6%, respectively, which were 1.31 times, 1.46 times and 1.42 times higher than those of strain CW-D3T. The degradation rates of phenanthrene, fluoranthene, and pyrene with the addition of 500 mg/L Tween-80 to the mixed bacterial culture were 47.7%, 43.2%, and 38.8%, respectively, which were 1.55 times, 1.38 times, and 1.31 times higher than those in the control group. However, higher concentrations of Tween-80 did not significantly promote or slightly inhibit the degradation of PAHs. The addition of Tween-80 increased the surface hydrophobicity of strain CW-D3T and mixed bacteria while decreasing that of strain B6-2. Combined with the number of bacterial colonies results, the higher concentration of Tween-80 produced certain toxicity to strain B6-2 and inhibited its growth, thereby affecting the degradation efficiency of mixed bacteria. [Conclusion] The composite functional bacteria constructed by P. putida B6-2 and Klebsiella sp. CW-D3T has a good efficiency in degrading aromatic compounds. A low concentration of Tween-80 can be utilized as a carbon source and change the hydrophobicity of the cell surface, thus significantly improving the removal efficiency of phenanthrene, fluoranthene and pyrene.

Abstract:[Objective] To develop an efficient method for isolating and cultivating zooxanthellae from reef-building corals and lay a foundation for the research on germplasm banking and physiological functions of zooxanthellae species associated with corals. [Methods] The zooxanthellae were isolated and enriched from reef-building coral tissues by micro-strainer filtration and density gradient centrifugation, and the cells were cultured in 96-well plates with the modified L1 medium. Single clones of zooxanthellae were obtained by single cell isolation, culture, and/or plate streaking. The species and phylogenetic relationship were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in combination with internal transcribed spacer 2 (ITS2) and large subunit (LSU) sequences. [Results] Three zooxanthellae strains were isolated from Acropora pruinose of Weizhou Island, Galaxea fascicularis and Acropora tenuis of Xisha Islands and numbered AP21C1, GF21D1, and AT21A113, respectively. The three strains showed the ITS2 genotypes of C1, D1, and A113 and the sequences basically consistent with Cladocopium goreaui, Durusdinium trenchii, and Symbiodinium natans, respectively. All the three strains demonstrated self-spinning motion and adherence during the logarithmic growth phase, and strain AP21C1 was unable to grow on agar plates. [Conclusion] This study develops an efficient method for the in vitro culture of zooxanthellae associated with corals and provides a technical and theoretical basis for the research on the germplasm banking and physiological functions of zooxanthellae species associated with corals.

Abstract:[Objective] To prepare the fluorescent stable aneurine hydrochloride (VB₁)-protected copper nanoclusters with VB₁ as a protective ligand and reducing agent for the detection of trace amounts of Fe³⁺. [Methods] We used VB₁ as a protective ligand and reducing agent to synthesize VB₁-protected copper nanoclusters. Then, we characterized the copper nanoclusters by UV visible absorption spectrum, fluorescence spectrum, and particle size, and explored their pH responsiveness, selectivity to Fe³⁺, and linear range in the detection of Fe³⁺. [Results] The prepared VB₁-protected copper nanoclusters had good water solubility, excellent stability, and ultrafine size. As the fluorescent probes to detect Fe³⁺, the copper nanoclusters showed good linearity in the ranges of 0–5 μmol/L and 5–500 μmol/L, with the limit of detection of 0.085 μmol/L. When being used to detect Fe³⁺ in the actual microbial sample of Trichophyton, the copper nanoclusters showed the recovery rate between 95.67% and 107.94%. [Conclusion] We used VB₁ as a protective ligand and reducing agent to prepare fluorescent stable VB₁-protected copper nanoclusters. On the basis of the selective quenching of Fe³⁺ by the copper nanoclusters, we established a simple, rapid, and sensitive method for the detection of Fe³⁺ and successfully applied it to the detection of Fe³⁺ in Trichophyton. This method has a good application prospect in actual microbial samples.

Abstract:[Objective] To establish an electroporation-mediated transformation method for the economical and rapid genetic transformation of Sclerotium rolfsii. [Methods] The fusion protein expression cassette composed of the basta-resistant gene bar and the red fluorescent protein gene DsRed Max, controlled by the promoter of the native gpd gene, was transferred into the wild-type cells of S. rolfsii. The transformants were screened and validated by PCR and fluorescence observation. Further, we tested the transformation efficiencies under the different conditions of field strength, pulse time, and the ratio of foreign DNA fragments to recipient cells, to figure out the optimized electroporation parameters. Finally, we transformed the expression cassettes fusing multiple resistance genes and DsRed under optimized conditions to test their availabilities. [Results] The transformants carrying gene bar, sdhR, and aphI were obtained successfully. [Conclusion] The electroporation-mediated transformation method of S. rolfsii was successfully established. The optimized transformation parameters were field strength of 2 kV/cm, pulse time of 1 ms, DNA/homogenized cell ratio of 3 μg/300 mg, and pulse once.