Abstract:Algicidal bacteria can kill algae or inhibit algae activity in aquatic environments.This paper focused on the application of algicidal bacteria.To be specific,we described the taxa,combination strategies,application methods,and application scenarios of algicidal bacteria in 78 genera and 5 phyla,and the target algae in 56 genera and 7 phyla.Among the bacteria,Bacillus spp.,Streptomyces spp.,and Pseudomonas spp.are most widely reported,demonstrating broad algicidal spectrum (inhibiting 23,15,and 19 genera of target algae,respectively).The mechanisms are diverse and most (83.1%) of the algicidal bacteria kill algae by secreting algicidal substances.The application methods,such as direct spilling,extracting functional substances,fixing on carriers,inoculating in a reactor,are selected based on scenarios.However,most experiments were carried out in laboratories,and the effect in the field is unclear.In the future,for the purpose of algal bloom control,studies can be carried out on the functions,application technology,and ecological safety evaluation of algicidal bacteria.

Abstract:Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 6(NLRP6) is a member of the NOD-like receptor (NLR) family.It participates in inflammasome assembly to mediate the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18.In addition,it plays an important role in host innate immune response to pathogen,maintaining intestinal epithelial integrity,and regulating metabolic diseases and neuroinflammation.In this paper,we reviewed the activation and regulation mechanism of NLRP6 inflammasome,as well as the role of NLRP6 in infectious and non-infectious diseases.

Abstract:Nurturing numerous microorganisms,oral cavity is the window into the physiological functions of the body.Oral microbiota is a mirror of the interaction between the host and environmental factors,which influences human health and disease progress.As the early colonizers of the oral cavity,the symbiotic Streptococcus and Veillonella contribute to the development of biofilm in human oral cavity.Mounting evidence suggests the key role of the symbiont in the development of many oral disorders,such as dental caries and periodontal disease.In certain state,they can also break through or invade the digestive barrier to colonize in remote organs,thereby affecting human health.They have been used as potential biomarkers for the occurrence,development,and prognosis of many systemic diseases.Thus,this paper reviewed the pathogenic mechanism of the Streptococcus-Veillonella symbiont,analyzed the characteristics of them in the microecosystem related to common systemic diseases,and proposed the representative diagnostic combinations,which would be expected to lay a scientific basis for the diagnosis and prevention of systemic diseases.

Abstract:Hand-foot and mouth disease (HFMD),as a disease of global concern,occurs frequently in Asia and its harm cannot be underestimated.The multiple transmission routes,strong infectivity,induction of complications,diverse pathogenic strains and easy variation bring challenges to the prevention and treatment of HFMD.Enterovirus 71(EV71) is one of the main pathogens causing HFMD.Since there is no specific drug against EV71,it is of great significance to find appropriate therapeutic drugs.We summarize the research progress of drugs and vaccines against EV71,aiming to give insights into the prevention and control of HFMD.

Abstract:The environmental and health problems caused by the massive use of synthetic pigments in recent years increase the demand for safe,non-toxic natural pigments.Natural pigments are mainly derived from plants and microorganisms.However,the large-scale application of plant-derived pigments is limited by the long growth cycle of plants.Compared with plants,microorganisms are easy to be cultivated on a large scale in a short time and thus microorganism-derived pigments have a broad application prospect.We systematically summarized the production mechanisms,reviewed the research progress in the antibacterial,anti-oxidation and anti-cancer activities,and put forward the opportunities and challenges in the development of microorganism-derived pigments.

Abstract:The transmission of antibiotic resistance genes (ARGs) is a potential threat to human health.ARGs are present as both intracellular and extracellular fractions of DNA in the environment.Intracellular ARGs (iARGs) and extracellular ARGs (eARGs) have different distribution and transmission characteristics in different environments.In this paper,we first categorized chromosomal,plasmid-borne,phage-associated ARGs into iARGs and eARGs with regarding to their occurrence states,and then summarized the methods for the isolation and detection of iARGs and eARGs from soil,water,and the atmosphere.Afterwards,we described the abundance distribution of iARGs and eARGs in livestock and aquaculture farmlands,wastewater treatment plants,rivers,oceans,and the atmosphere and compared their transmission methods and ability.This paper is expected to provide a theoretical reference for the control and assessment of the health risks of ARGs in the future.

Abstract:Streptomycetes produce a variety of secondary metabolites,which are highly valuable in clinical practice,agriculture,animal husbandry,biotechnology,etc.The research on the regulatory networks of secondary metabolites in Streptomycetes will improve their yields and facilitate the discovery of novel secondary metabolites.The biosynthesis of secondary metabolites in Streptomycetes can be regulated in a global or pathway-specific manner.The global regulators can target multiple pathway-specific genes and biosynthesis genes,playing a universal and complicated role.Therefore,studying the global regulation will improve the understanding of the complicated regulation networks in Streptomycetes.DasR,a global regulator,plays a key role in the nutrient sensing,morphological changing,and secondary metabolism.In this review,we summarized the structure,sequence,and function of DasR,and reviewed the research findings on role of DasR in the growth,secondary metabolism,nutrient sensing,and primary metabolism of Streptomycetes.

Abstract:Botulinum neurotoxins (BoNTs) secreted by Clostridium are the most toxic biotoxins known.BoNTs are classified into seven serotypes (BoNT/A-G),of which BoNT/A is the most frequent cause of botulism.Due to the strong toxicity and easy preparation,BoNTs are classified as the class A bioterrorism agents.At present,botulism is mainly treated by early administration of antibotulinum serum.However,the antibotulinum serum has limited effect and cannot reverse the paralysis caused by botulism when the toxin enters nerve cells.Therefore,it is necessary to develop more effective drugs for treating botulism.Because small-molecule inhibitors can overcome the limitation that antitoxin cannot neutralize intracellular toxins,they have become a popular direction in the development of novel drugs for treating botulism in the past decades.As the structure of BoNTs and the mechanism of botulism have been gradually elucidated,more and more relevant targets are being used to develop effective inhibitors.This paper reviewed small-molecule inhibitors of BoNTs in recent years according to their mechanisms.

Abstract:Polysaccharides are the main components of macroalgae,phytoplankton,and microorganisms.Polysaccharide degradation products are the main source of marine organic matter.Carbohydrate-active enzymes (CAZymes),the typical functional enzymes for the degradation,modification,and generation of glycosidic bonds of carbohydrate compounds,are the basic functional units in carbohydrate metabolic pathways.Most heterotrophic bacteria are endowed with a complete CAZymes-encoding system,which is the main driving force for carbohydrate metabolism.However,owing to the diversity,complex structures,and functional specificity of CAZymes,there is a lack of a systematic understanding of the members in this family and the molecular mechanisms of action.The development of carbonhydrate chemistry,molecular ecology,microbiology,and bioinformatics allowed an in-depth understanding of the structures of polysaccharides,the degradation pathways,and the action mechanisms of the CAZymes.In this paper,the research advances of CAZymes were summarized.At first,this review introduced the types of polysaccharides in the marine environment and the corresponding methods of qualitative and quantitative analysis.Secondly,we summarized the latest progress of CAZymes and the database status and discussed polysaccharide utilization loci (PULs) and molecular degradation mechanisms of several marine polysaccharides.Finally,we presented a new conceptual model linking polysaccharide structural complexity to bacteria-driven polysaccharide utilization,and introduced the relationship between the polysaccharide degradation by heterotrophic bacteria and the algal bloom dynamics.The purpose of this paper is to enhance the systematic understanding of the molecular functions and ecological significance of CAZymes and deepen the understanding of carbohydrate metabolism in the marine environment.

Abstract:Prokaryotes defend against the invasion of foreign nucleic acids through the adaptive immune system named clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems.During the adaptive immune responses,prokaryotes integrate partial fragments of foreign nucleic acids into their own CRISPR arrays,and then express and process CRISPR RNA and related Cas protein to form a nucleoprotein complex for the recognition and cleavage of corresponding foreign nucleic acids.Although the working mechanisms of CRISPR-Cas systems have been extensively studied,little is known about the transcriptional regulation.In recent years,researchers have investigated the transcriptional regulation mechanisms of type I,II,and III CRISPR-Cas systems,revealing diverse regulation models of CRISPR-Cas systems in different prokaryotic species.This review expounds the transcriptional regulation mechanisms of type I CRISPR-Cas systems,and briefs the research progresses in the transcriptional regulation mechanisms of type II and III CRISPR-Cas systems.Finally,this review puts forward the questions to be addressed in this field.

Abstract:Using wastewater to cultivate microalgae can reduce cultivation cost and pollution.Proteins,polysaccharides,and lipids of microalgae are major factors affecting the utilization of microalgae resources.The research progress on microalgae cultivation with wastewater as the substrate was reviewed.Centering on component accumulation,we expound the selection basis of microalgae species,the factors affecting microalgae growth,the methods to improve microalgae yield,and the synthesis mechanism of components in microalgae.Finally,we put forward the challenges and possible solutions for microalgae cultivation in the future.

Abstract:Gut microbiota plays an essential role in intestinal homeostasis and brain health.Serotonin is an important monoamine neurotransmitter in the brain.More than 90% of serotonin is converted from tryptophan metabolism in colonic enterochromaffin cells and exerts system-wide regulations.Recent studies have demonstrated that the effects of serotonin may be affected by gut microbiota.Specific microbes in the gut have the ability to produce serotonin.The gut microbiota and its metabolites,such as butyric acid,can regulate the level of serotonin by affecting the activity of tryptophan hydoxylase or the metabolism of tryptophan.In turn,serotonin and serotonin reuptake inhibitors can modulate the composition and function of gut microbiota.The gut microbiota-serotonin interaction is of great significance to the host health,and the mechanism underlying the interaction remains to be elucidated.This article reviews the research progress on gut microbiota,serotonin,and their interaction,aiming to provide references for in-depth investigation on how the gut microbiota-serotonin interaction regulates gut and brain health.

Abstract:The bioaerosol harbors diverse microorganisms.The sampling of microbial aerosols and the identification of microorganisms in aerosols are the key to understanding the biological characteristics of microbial aerosols,which is essential for preventing bioaerosol-mediated pathogen transmission.This article briefly reviewed the biological characteristics and potential hazards of microbial aerosols and introduced the general methods for microbial aerosol sampling with respect to their performance,operating mechanisms,advantages,and limitations,in an aim to assist the design of best practices for bioaerosol sampling.Three major (culture-dependent,culture-independent,and single-cell) techniques for the identification of bacteria,fungi,and viruses in bioaerosols were expounded.Finally,the future techniques for microbial aerosol collection and microbial identification were prospected.The cross-disciplinary interaction will facilitate the development of novel methods for bioaerosol sampling and detection.With this review,we hope to provide a theoretical basis for the development of sampling methods and comprehensive characterization of bioaerosols.

Abstract:Alanine racemase belonging to the isomerase family has great research value.It can be used as a target for the screening of new antibacterial drugs and is one of the key enzymes in the enzymatic synthesis of D-amino acids.[Objective] To mine the alanine racemase gene resources from uncultured microorganisms in the gastrointestinal tract of animals,and clarify the functions and properties of alanine racemase genes from different microorganisms.[Methods] On the basis of sequence analysis and gene function annotation,alanine racemase genes were cloned from the metagenome of the fecal microorganisms of Nomascus concolor and heterologously expressed in Escherichia coli BL21(DE3) for the identification of enzyme functions and properties.[Results] Two alanine racemase genes,NCalr 1 and NCalr 6,were obtained,each of which had a length of 1 170 bp and encoded 389 amino acids.The deduced proteins of NCalr 1 and NCalr 6 had the molecular weights of 43.58 kDa and 43.94 kDa,respectively.The recombinant enzymes had the optimal performance at pH 12.0 and 40/37.The relative activities of the recombinant enzymes treated at pH 9.0-12.0 for 1 h were above 90%,indicating good alkali resistance.The recombinant enzymes had good stability at 30-45 relative activity above 85% fter being treated at 30-45 for 1 h.The optimal PLP concentration was 10 μmol/L for the two enzymes,and the incubation with 10 μmol/L PLP increased the relative activity of the enzymes by about 30% and 7%,respectively.However,the mutation from lysine to alanine at the PLP binding site reduced the relative activity of the two enzymes to only 1% and 27% of the initial activity,respectively.The K_m values of the recombinant enzymes were (14.81±1.66) mmol/L and (25.87±1.95) mmol/L,respectively.The recombinant enzymes were inhibited by 10 mmol/L Hg²⁺,Ag⁺,Zn²⁺,and SDS while activated by Fe³⁺,and 10 mmol/L Fe³⁺ increased their activities by 2.6-5.1 times.[Conclusion] The study obtained two new alanine racemases with good application prospects from the fecal microbial metagenomics of N. concolor for the first time.

Abstract:[Objective] Using protein domain engineering to construct and optimize the enzymatic activity of chitinase BtChiA derived from Bacillus thuringiensis BMB 171,and constructing qualitative and quantitative analysis of the product of the enzyme hydrolyzing colloidal chitin.[Methods] According to the sequence of BtChiA,the composition of its domain was analyzed.The expression strain of BtChiA and mutant protein with partial domain deletion in Escherichia coli was constructed and then the protein was expressed and purified.Analyzing the type and content of chitinase hydrolysates by HPLC.[Results] BtChiA (full-length protein) and BtChiA△50(truncated chitin binding domain CBD and fibronectin domain FnIII) were obtained by heterologous expression and purification.The vitality of BtChiA△50 to hydrolyze colloidal chitin is 1.64 times higher than that of BtChiA.The hydrolyzed products of both are chitin monosaccharide GlcNAc and chitobiose (GlcNAc)₂.The content of (GlcNAc)₂ is 0.97 times lower than GlcNAc in the product of BtChiA,the content of (GlcNAc)₂is 1.79 times higher than GlcNAc in the product of BtChiA△50.[Conclusion] Protein domain engineering is a feasible strategy to improve the activity of chitinase BtChiA and optimize the composition of the hydrolysates.

Abstract:[Objective] Bacillus thuringiensis(Bt) D18 is virulent to agricultural pests of Lepidoptera and Coleoptera.This study aims to improve the virulence of D18 by compound mutagenesis.[Methods] After four rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and one round of ARTP-N-methyl-N'-nitro-N-nitrosoguanidine (ARTP-NTG) mutagenesis,highly virulent mutants were screened out by microscopy and virulence bioassay.To reveal the reason for the increase of virulence,we detected the Cry proteins by SDS-PAGE and key cry genes by qRT-PCR.[Results] Two highly virulent mutants,An-L5-1 and An-L5-7 were screened out,compared with the wild strain D18,An-L5-1 and An-L5-7 demonstrated slightly early decline,improved ability of sporulation,80.47% and 88.31% rise of Cry protein expression,and significantly stronger virulence to Plutella xylostella and Mythimna seperata.[Conclusion] The increase in the virulence of An-L5-1 and An-L5-7 was mainly attributed to the significantly up-regulated expression of cry1Aa,cry2Aa,and sigK.

Abstract:[Objective] This paper aims to reveal the bacterial community structure in the Three Gorges of the Yangtze River,the topological properties of co-occurrence network and its hub microorganisms,and the metabolic functions of the bacterial community.[Methods] Water samples were collected from the mainstream and four tributaries (Xiangxi River,Daning River,Zhuyi River,and Meixi River) of the Yangtze River.PacBio 16S rRNA high-throughput sequencing and bioinformatics analysis were performed to elucidate the bacterial community structure and Tax4Fun was employed to predict the metabolic functions of the community.[Results] (1) The dominant phyla in the upper reaches of the Three Gorges of the Yangtze River were Proteobacteria(66.4%),Bacteroidetes(13.2%),and Cyanobacteria(6.2%).In Proteobacteria,Gammaproteobacteria was most abundant and followed by Alphaproteobacteria.The diversity index and richness index implied the rich microbial species in the study area.(2) Co-occurrence network formed a "small world" topology,and Sphingomonadaceae,Chloroflexi,Gemmataceae,Flectobacillus,and Cryomorphaceae were the central microorganisms which played a crucial part in maintaining the community structure and diversity.(3) A total of 6 functions in level 1 and 42 functions in level 2,which mainly involved in metabolism and genetic information processing,were found in Tax4Fun function prediction,suggesting the active metabolism of the bacterial community.[Conclusion] Bacterial community in the upper reaches of the Three Gorges was of high diversity,and the hub microorganisms were mainly rare microorganisms which played an important role in maintaining the community structure.The bacterial community in the upper reaches of the Three Gorges featured abundant metabolic pathways,which was beneficial to the health of the water body.Clarifying the distribution and interaction characteristics of microbial community could lay a scientific basis for the healthy ecosystem development,water resource utilization,and water environment improvement of the Yangtze River.

Abstract:[Objective] To explore the effect of the knockout of aceE gene which encodes pyruvate dehydrogenase on the growth characteristics,tricarboxylic acid cycle,and pyruvate metabolism of Streptococcus suis.[Methods] The growth curves of the wild-type strain and strain ΔaceE were established with the OD₆₀₀ values of the bacterial liquids.The content of acetyl CoA,succinate CoA,fumarate,oxaloacetate,pyruvate,lactate,and ATP in the tricarboxylic acid cycle and the pyruvate metabolism bypass was measured.Quantitative RT-PCR was employed to determine the gene expression levels of citrate synthase,malate dehydrogenase,succinate dehydrogenase,isocitrate dehydrogenase,pyruvate decarboxylase,lactate dehydrogenase,alcohol dehydrogenase,and acetaldehyde dehydrogenase.[Results] Compared with the wild-type strain,strain ΔaceE showed decreasing OD₆₀₀ value during the platform phase.The OD₆₀₀ value of strain ΔaceE in the plateau phase significantly increased after the addition of 1 g/L acetate.Compared with the wild-type strain,strain ΔaceE showed increased content of pyruvate while decreased content of ATP,acetyl CoA,succinate CoA,and fumarate.Moreover,it showed up-regulated expression levels of citrate synthase and malate dehydrogenase,while down-regulated expression levels of succinate dehydrogenase and isocitrate dehydrogenase.For the proteins involved in pyruvate metabolism bypass,pyruvate decarboxylase,lactate dehydrogenase,alcohol dehydrogenase,and acetaldehyde dehydrogenase presented up-regulated gene expression levels in strain ΔaceE.[Conclusion] Strain ΔaceE has reduced activity of tricarboxylic acid cycle.Although part of pyruvate can be decomposed into acetate through the pyruvate dehydrogenease bypass and further converted into acetyl CoA to enter the tricarboxylic acid cycle,the ATP content has not been restored to the wild-type strain level.This study provides a theoretical basis for further research on the relationship between metabolic changes and bacterial phenotypes.

Abstract:[Objective] We cloned the bovine lactoferrin N-lobe (BLF-N) into the genome of Pichia pastoris and enabled the heterologous expression of BLF-N through the optimization of its gene codon and fermentation conditions.Furthermore,we studied the antibacterial activity of the recombinant protein.[Methods] With BLF gene as template,BLF-N gene was optimized according to the codon bias of P.pastoris.On this basis,the recombinant expression vector pPIC9K-UBLF-N was constructed and transformed into P.pastoris GS115 by electroporation to yield the recombinant P. pastoris GS115/pPIC9K-UBLF-N.Through high-copy screening and optimization of fermentation conditions,BLF-N expression was improved and then evaluated by SDS-PAGE.The inhibition of the recombinant BLF-N on Gram-positive and Gram-negative bacteria was explored.[Results] SDS-PAGE results showed the efficient soluble expression of BLF-N with molecular weight of about 37 kDa (consistent with the theoretical molecular weight) in P. pastoris GS115.A transformant resistant to 3 mg/mL genomycin G418 was obtained through high-copy screening.The highest recombinant BLF-N yield (50.5 mg/L) was achieved under the following optimized fermentation conditions:0.2%(V/V) methanol,30℃,and pH 5.0.Recombinant BLF-N demonstrated strong inhibition on Escherichia coli and Staphylococcus aureus and higher antibacterial activity than commercial BLF.[Conclusion] Through high-copy screening and optimization of fermentation conditions,we achieved the efficient expression of BLF-N in P. pastoris and the recombinant BLF-N presented significantly higher antibacterial activity than the commercial BLF.This study lays a foundation for the efficient and green preparation and industrial production of BLF-N and provides theoretical guidance for research on the difference in structure-function relationship between BLF and BLF-N.

Abstract:[Objective] Enhancing immunity and producing exopolysaccharide (EPS),Limosilactobacillus fermentum is widely used in food industry and is of huge economic value.Based on population genetics,this paper aims to analyze the genetic background and functional gene characteristics of L. fermentum F-6,which is expected to lay a genetic basis for the development and utilization of the strain.[Methods] Comparative genomics was used to analyze the whole-genome sequences of 23 L. fermentum strains and 1 model strain ATCC 14931^T from NCBI,Roary to identify core-pan genes,and rapid annotation using subsystem technology (RAST) to annotate the genomes.[Results] A phylogenetic tree was constructed based on the yielded 997 core genes and the analysis showed that the clustering had nothing to do with the source.However,strain F-6 was in the same clade with 3 food-derived strains.Genome annotation results revealed that only strain F-6 contained genes involved in branched chain amino acid synthesis pathway (ilvD,leuA,etc.) and thus can provide essential amino acids for the body.Strain F-6 harbored a large number of genes encoding glycosyltransferases and UDP-glucose 4-epimerase,and contained a complete eps cluster.Compared with other L. fermentum strains,strain F-6 had the unique functional gene gadC which is associated to acid resistance.In addition,potential probiotic genes such as genes related to the synthesis of glutathione,antimicrobial peptide,and adhesion molecules were also identified in the genome.[Conclusion] According to the genomic background and functional genome,F-6 is a potential probiotic strain with acid resistance genes,branched chain amino acid synthesis genes,and a complete eps gene cluster,which will contribute to our understanding of the genetic basis of L. fermentum F-6.

Abstract:[Objective] We cloned and expressed a novel β-xylosidase Xyl4900 from Leifsonia sp.ZF2019 isolated from Artipe eryx larvae and then investigated its enzymatic properties,aiming to provide foundations for developing β-xylosidase suitable for industrial application.[Methods] We used bioinformatics tools to analyze the gene of Xyl4900,expressed it in Escherichia coli,and investigated the enzymatic properties of the expressed protein.[Results] Xyl4900 had high homology with the β-glucosidase of GH3 family while had a domain of β-xylosidase.It was a novel β-xylosidase that could specifically hydrolyze 4-nitrophenyl β-D-xylopyranoside.Xyl4900 had the highest activity at 45℃ and pH 7.0,and it still maintained more than 80% of activity after being incubated at pH 6.0-9.0 for 14 h.This enzyme was barely affected by other metal ions (2.5 mmol/L) except Cu²⁺ and had strong tolerance to low-concentration organic solvents (5%,V/V).In addition,the Xyl4900 in 20%(W/V) NaCl or 100 mmol/L xylose solution showed the activity higher than 50%,demonstrating good salt or xylose tolerance.The K_m,V_max,and xylose inhibition constant K_i of Xyl4900 were 0.80 mmol/L,36.10 U/mg,and 150.12 mmol/L,respectively.In particular,we found that Xyl4900 could well degrade xylobiose and xylotriose.[Conclusion] Xyl4900 showed good pH stability and tolerance to xylose and salt,and thus could be used in hemicellulose degradation and other industrial production practices.

Abstract:The beneficial Clostridium butyricum,which synthesizes short-chain fatty acids such as butyric acid,can be added to feed to improve animal production,feed utilization efficiency,and the antioxidant ability of animals.[Objective] This paper explored effect of C.butyricum as a replacement of chlortetracycline on quality and antioxidant function of broiler leg muscle.[Methods] A total of 360 healthy 1-day-old Arbor Acres broilers were randomly selected and classified into three groups,with 8 replicates each group.Negative control group (CON) was fed with basal diet,antibiotics group (ANT) basal diet supplemented with 75 mg/kg chlortetracycline,and C.butyricum group (CB) basal diet supplemented with 500 mg/kg C. butyricum(viable bacteria,10⁹ CFU/g).The experiment lasted 42 days.[Results] Compared with CON group,CB significantly increased weight,24h redness (a^*),pH_{45 min},pH_{24 h},crude protein content,and ether extract,decreased 24 h brightness (L^*),drip loss,cooking loss,and malondialdehyde (MDA) content,improved the total-antioxidant capacity (T-AOC),and the activity of catalase (CAT),total superoxide dismutase (T-SOD),and glutathione peroxidase (GPx),and up-regulated the expression of nuclear factor erythroid 2-related factor 2(Nrf2),GPx-1,SOD-1,and SOD-2 of broiler leg muscle (P<0.05).ANT raised the weight and pH_{45 min},reduced the drip loss and MDA content,and up-regulated the expression of Nrf2,GPx-1, and SOD-1 of broiler leg muscle compared with CON group (P<0.05).CB group demonstrated lower drip loss and L^* after 24 h and higher ether extract and CAT activity of broiler leg muscle than the ANT group (P<0.05).[Conclusion] C. butyricum can replace chlortetracycline for broiler feed,activate the expression of Nfr2 signaling pathway related genes,and which improves the quality and antioxidation of broiler leg muscle.

Abstract:[Objective] This paper aims to analyze the gut microbiota structure of Furuili 2(Cyprinus carpio) from different culture systems.[Methods] The 16S rRNA high-throughput sequencing was performed to analyze the composition and abundance of gut microbiota in Furuili 2 carps from the rice-fish co-culture system in Hani Terraces (RCIC),monoculture system in conventional pond (YPIC),and the monoculture system in cement pond (HPIC).[Results] The high-throughput sequencing yielded 2 345,238,and 118 operational taxonomic units (OTUs) for the gut microbes in carps from RCIC,YPIC,and HPIC groups,respectively.Sobs and PD indexes in RCIC group were higher than those in YPIC group (P<0.05) and HPIC group (P<0.01),and the diversity indexes in YPIC group were higher than those in HPIC group (P<0.05).The dominant phyla (abundance>10%) were Proteobacteria (39.39%),Fusobacteria(38.55%),and Firmicutes(15.4%) in RCIC group,Proteobacteria(21.87%) and Fusobacteria(58.27%) in YPIC group,and Proteobacteria(46.63%) and Fusobacteria(53.14%) in HPIC group.At the genus level,Cetobacterium and Aeromonas were dominant in all samples.LEfSe analysis (LDA>3) suggested 17 biomarkers,among which 8 were abundant in RCIC group:Romboutsia,Clostridium_sensu_stricto_1,Enterobacter,Paraclostridium,Methylobacterium,norank_f__norank_o__Chloroplast,Edwardsiella,and Epulopiscium.[Conclusion] Different culture modes enabled significantly different microbiota structures in carps.In particular,RCIC group boasted higher richness and diversity of gut flora of carps and had more beneficial bacterial markers.As a result,the intestines of carps were healthier.

Abstract:[Objective] To explore the genetic diversity,phosphorus- and potassium-solubilizing ability,stress resistance,and effect on rice seedling growth of indoleacetic acid (IAA)-producing endophytic bacteria isolated from Aconitum carmichaelii Debeaux and to provide scientific support for the sustainable development of A.carmichaelii industry.[Methods]Culturable endophytic bacteria were isolated from surface sterilized healthy plants of A.carmichaelii,and their IAA-producing capacities were measured by Salkowski spectrophotometry.The genetic diversity and phylogenetic status of these bacteria were studied via 16S rDNA-restriction fragment length polymorphism (16S rDNA-RFLP) and 16S rDNA full-length sequence.The phosphorus- and potassium-solubilizing abilities and stress resistance of them were studied by plate culture method.The effects of IAA-producing endophytic bacteria on the seed germination rate of rice were tested by semi-solid culture method after the seeds were immersed in bacterial liquids for 30 min.The effects of the endophytic bacteria on rice seedling growth were investigated by hydroponic experiment in our laboratory.[Results]Twenty-four endophytic bacterial strains were isolated from healthy A.carmichaelii,with the IAA-producing capacities ranging from 21.39 mg/L to 84.43 mg/L.The 16S rDNA-RFLP analysis indicated that these 24 strains were classified into 12 groups.The phylogenetic tree constructed based on 16S rRNA gene sequences showed that the representative strains belonged to Enterobacter,Klebsiella,Pantoea,Pseudomonas,Microbacterium,Bacillus,Sphingobacterium,and Chryseobacterium,which demonstrated high genetic diversity.Twelve strains showed strong potassium-solubilizing capacities.Most of the strains could grow normally within wide ranges of pH,temperature,and NaCl concentration,while they showed little resistance to different antibiotics.Ten strains significantly improved the germination rate of rice seeds,and 16 strains significantly promoted the growth of rice seedlings.We screened out 3 strains JY1-1S,JY3-6S,and JY10-1L that could not only improve the seed germination rate but also promote the seedling growth of rice.[Conclusion] A.carmichaelii harbored rich and diverse IAA-producing endophytic bacteria.We screened out 3 endophytic bacterial strains that could promote the seed germination and seedling growth of rice.This study laid a foundation for the development of plant growth-promoting biological fertilizer in the future.

Abstract:[Objective] Laccases can oxidize a wide range of compounds and have great application value in diverse industries.Lac15,a microbial laccase with considerable application potential,can be modified by protein engineering to expand the application range.[Methods] By structure-based analysis,we chose some residues supposed to contribute directly or indirectly to electron/proton transfer or substrate interaction for site-directed mutagenesis,and measured the activities of the mutants for various substrates and the enzymatic properties.[Results] Some of the mutants showed significantly enhanced activity towards particular substrates.To be specific,the catalysis efficiency of mutant D216N toward 2,2'-azino-bis (3-ethylbenzthiazoline-6-suphonic acid)(ABTS) was 1.0 fold higher,and that of mutants R178V and K433T toward dopamine was 1.2 and 11.1 folds,respectively,higher than that of Lac15.Moreover,the mutants maintained the advantages of Lac15 in application,such as the neutral optimum pH and salt activation.[Conclusion] By structure-based semi-rational engineering,the specific activities of Lac15 toward some substrates can be improved.A conserved Asp in laccases,corresponding to D216 in Lac15,can be a common engineering target for enhancing the activity of the enzymes with ABTS as substrate or mediator.

Abstract:[Objective] The mycelial pellets of Lactarius deliciosus were used to decolorize and degrade the dye Eriochrome Black T (EBT).We investigated the performance and mechanism of the decolorization by the mycelial pellets under different environmental conditions.[Methods] Single factor experiments were conducted to explore the optimal decolorization performance of the fungi.We measured the fungal enzyme activity by spectrophotometry,and performed wheat seed germination test,E. coli contact toxicity test,and Caenorhabditis elegans toxicity test to evaluate the toxicity of the EBT solutions before and after decolorization.[Results] The results manifested that the mycelial pellets of L. deliciosus were slightly affected by the shaker temperature and rotation speed.At pH 5,28℃,and 120 r/min,the decolorization rate for 400 mg/L EBT solution was 97.14%.In the decolorization process,the mycelial pellets of L. deliciosus secreted three ligninases,namely lignin peroxidase,manganese peroxidase,and laccase,which had the maximum activities of (134.15±9.93),(64.1±2.98),and (12.43±0.34) U/L,respectively.We deduced that the mycelial pellets of L. deliciosus removed EBT by the synergistic effects of biosorption and degradation.The multi-level toxicity evaluation demonstrated that the dye wastewater after decolorization had significantly reduced toxicity.[Conclusion] All these results have an important reference value in exploring the process of dye wastewater treatment with biological method.

Abstract:[Objective] To investigate the relationship between rhizobacterial community structure and bacterial wilt occurrence of tomato plants in a substrate culture system with the integration of water,fertilizers,and biocontrol agents.[Methods] After the field survey,the disease incidence (DI) and disease severity index (DSI) of bacterial wilt were calculated.Furthermore,the high-throughput sequencing technology was adopted to compare the rhizobacterial diversity and composition between infected and healthy tomato plants.[Results] The DI (6.17%) and DSI (5.11) of tomato plants at the reproductive stage were significantly higher than those at the vegetative stage (DI of 2.5% and DSI of 1.25,respectively).The number of Ralstonia solanacearum in the rhizosphere of diseased tomato plants at the early infection stage (grades 1 and 2) was much higher than that at the later infection stage (grades 3 and 4).Both Chao 1 and Shannon indexes of bacteria in the rhizosphere of diseased tomato plants were significantly lower than those in the rhizosphere of health plants.Compared with that in the rhizosphere of healthy plants,the relative abundance of Proteobacteria increased while that of Acidobacteriota and Cyanobacteria decreased (P<0.01);the relative abundance of Rhodanobacter and Ralstonia significantly increased,which was positively correlated with the number of R. solanacearum as demonstrated by redundancy analysis;the relative abundance of Bradyrhizobium,Granulicella,Haliangium,and Devosia significantly decreased (P<0.05),which was negatively correlated with the number of R. solanacearum.[Conclusion] Compared with those in the rhizosphere of healthy plants,the bacterial diversity and beneficial bacteria in the rhizosphere of tomato plants infected with bacterial wilt significantly reduced.Therefore,the addition of beneficial bacteria to change rhizobacterial community structure would be an efficient method to control tomato bacterial wilt.

Abstract:[Objective] Endophytes play an important role in the promotion of plant growth and the remediation of heavy metal-contaminated soil.In this study,we isolated the endophytic bacteria from cadmium-contaminated rice seeds and analyzed their cadmium tolerance,plant growth-promoting traits,and effects on rice seed germination and seedling growth under cadmium stress.[Methods] First,we used ethanol and sodium hypochlorite to sterilize the surface of rice seeds and then isolated the endophytic bacteria by standard dilution plating method.Second,we amplified and sequenced the 16S rRNA genes of the strains and identified their taxonomic positions.Third,we analyzed the cadmium tolerance,cadmium removal rate,and plant growth-promoting traits of the endophytic bacteria via microdilution method,inductively coupled plasma mass spectrometry (ICP-MS),and spectrophotometry,respectively.Finally,we infected the rice seeds with bacterial inoculants to observe their effects on seed germination,seedling growth,and cadmium accumulation.[Results] We isolated a total of 133 endophyte strains belonging to 24 species of 12 genera from two rice cultivars.Eighty-three strains were isolated from low-Cd-accumulating caltivar 728B,including Pseudomonas(34.94%),Bacillus(28.92%),and Paenibacillus(10.84%).Fifty strains were isolated from high-Cd-accumulating caltivar BB,including Pantoea(40%),Curtobacterium(22%),and Microbacterium(12%).The minimum inhibitory concentrations of cadmium against 24 representative strains were 80-2 560 μmol/L.Bacillus sp.HNR-4 showed the highest cadmium tolerance.The cadmium removal rate of HNR-4 reached 77.57% after the strain was cultured in the liquid medium containing 5 mmol/L Cd²⁺ for 24 h.Most of the 24 strains were capable of producing plant growth-promoting substances,such as IAA (0.78-40.12 μg/mL),siderophore (1.46-752.74 mg/L),soluble phosphate (0-3.10 mg/L),and ACC deaminase (0-9.22 U/mg).Under cadmium stress,HNR-4 inoculant significantly increased the germination rate and seedling shoot length,while reduced the transfer coefficient of cadmium from root to shoot.[Conclusion] Although the composition of seed endophytic bacteria varied in different rice cultivars,these bacteria all showed different levels of cadmium tolerance and plant growth-promoting activities.Isolation and identification of Cd-resistant endophytic bacteria may help investigate the interaction between endophytic bacteria and host plants exposed to heavy metal contamination,and explore the potential of microbial remediation in farmland.

Abstract:[Objective] To study the effects of flippase DRS2 on the expression and secretion of lignocellulases in Trichoderma reesei.[Methods] The homologous gene drs2 in T. reesei was firstly identified through sequence alignment by BLAST and then deleted via homologous recombination strategy from T. reesei.The growth,protein secretion,and activities of cellulase and hemicellulase were compared between the drs2-deleted strain ∆drs2 and the control strain Cpyr4.Furthermore,the subcellular localization of DRS2 was predicted.[Results] Compared with Cpyr4,∆drs2 showed significantly decreased growth rate in the medium supplemented with glucose,lactose or microcrystalline cellulose (avicel).The total protein secretion and the activities of cellulase and hemicellulase of ∆drs2 were significantly higher than those of Cpyr4.The transcriptional levels of the key cellulase genes were similar between ∆drs2 and Cpyr4. Subcellular localization prediction revealed that DRS2 was located in spitzenkörper of the hypha tip.[Conclusion] The deletion of drs2 gene can significantly improve the production of lignocellulases in T. reesei cultured with cellulose as the sole carbon source. DRS2 may play a role in the secretion rather than the transcription of cellulases.