Abstract:The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has been applied in increasing fields since its discovery.Beyond gene editing,certain Cas proteins such as Cas12 and Cas13 have shown great application potential in rapid detection of pathogenic microorganisms since the discovery of their trans-cleavage activity.The CRISPR/Cas-based pathogenic microorganism detection technology is characterized by high sensitivity,high specificity,and simple operation.The presence/absence of targeted microorganisms can be quickly determined with help of the specific guide RNAs designed by bioinformatic analysis.This review introduces the representative CRISPR/Cas-based pathogenic microorganism detection technologies.Further,we expound the characteristics of guide RNAs required by different CRISPR/Cas complexes and provide guidance on how to design the guide RNAs used in the CRISPR/Cas-based pathogenic microorganism detection systems,given the importance of guide RNA in the CRISPR/Cas-based detection system and the lack of comprehensive summarization in this topic.This review aims to contribute to the development of CRISPR/Cas-based pathogenic microorganism detection system.

Abstract:Lanthipeptide are a group of ribosomally synthesized and post-translationally modified peptides (RiPPs),containing rare structure like thioether cross-links termed lanthionines (Lans) or methyllanthionines (MeLans).Recently,lanthipeptides that originated from the phylum Actinomycetota have been the research hotspot due to their outstanding antimicrobial activities and unusual bioactivities.This review focused on lanthipeptides produced by Actinomycetota,with special attention paid to their unique structure and property.Further discussion involved developing lanthipeptide of Actinomycetota origin to meet practical requirement through biological and chemical modifications of known lanthipeptides,as well as genome mining strategies for the discovery of novel lanthipeptides.Lastly,future application potential of lanthipeptide derived from Actinomycetota were summarized and prospected.

Abstract:High salinity wastewater is one of the most difficult treating industrial wastewater because of its high hardness,poor biodegradability and complex composition.Traditional physical and chemical treatment technologies have many disadvantages,such as high operating cost,low treatment efficiency and serious secondary pollution.Considering halotolerant microorganism and halophilic microorganism can achieve normal physiological metabolism in high salinity environment,exploring economical,efficient and reliable biological treatment technology for high salinity wastewater is expected to become the mainstream of high salinity wastewater treatment.In this review,halotolerant/halophilic microbial strategies,such as salting-in,accumulation of compatible solutes (intracellular small molecules),protein stabilization,and cell surface stabilization,were systematically summarized.However,due to the severe growth conditions of halophilic microorganisms and the scarcity of functional microorganism species,halotolerant microorganisms were more likely to be used in the treatment of high salinity wastewater in the future.Latest research indicated that enhanced regulation technologies (electricity,light and magnetism) could improve the adaptability of microorganisms to high osmotic pressure,and electric regulation technology might be the key research direction of biological treatment for high salinity wastewater in the future.

Abstract:Plant metabolites exuded from roots have been identified as a bridge in plant-microbe interaction,and as a signal player in regulating the structure and diversity of soil microbial community.The altered soil microbial community served as feedback to the plant,resulting in promotion of plant growth,development and resistance.In this article,we reviewed recent advances on plant-microbe interaction mediated by plant metabolites,and sketched the category,function and testing method of the secondary metabolites in the above interaction process.Additionally,we investigated how plants developed themselves in order to adapt to the cultivar evolution and the reproduction by regulating their root metabolites,and illuminated the mechanisms of plants recruiting specific or beneficial microorganisms to the rhizosphere to improve their development and therefore alleviate the biotic and abiotic stresses.Furthermore,the"cry for help"strategy that plants recruit beneficial microbes or traits from the environment using a range of chemical stimuli to enhance their resistance was analyzed.This paper provided ideas and theoretical support for the sustainable agricultural development.

Abstract:Plant pathogenic bacteria coordinate multifaceted virulence determinants through some complex and delicate global regulation networks.In the diverse species,these networks dictate bacterial infection strategies,survival,and the success in infection in the presence of host plant defense systems.In this paper,the major virulence regulation systems in the model pathogens in four genera (Pseudomonas,Pectobacterium,Xanthomonas,and Ralstonia) of plant pathogenic bacteria were reviewed,including the quorum sensing,two-component regulatory systems,transcriptional activators,and post-transcription or post-translation regulation systems.Particularly,the similarities and differences in the virulence regulation mechanisms among some model strains were analyzed and related research was summarized.Moreover,virulence regulation networks for the four pathogen groups were plotted.The analysis suggested the different functions of the virulence regulation networks among different species,subspecies,or variants of the pathogens despite some common regulation subsystems.

Abstract:Viral diseases pose great threats to human and animal health.In light of the limitations of vaccination and antiviral therapies available,developing safe,broad-spectrum,and inexpensive novel antivirals is an urgent task.Probiotics are viable microorganisms with a lot of benefits to the body.The antiviral effects and underlying mechanisms of probiotics are research hot spots at the moment.This review summarizes the multiple antiviral modes of probiotics,such as promoting the tight junction of intestinal cells and producing beneficial substances to maintain the mucosal barrier of the host,competing with viruses for targets or directly inhibiting and killing viruses,stimulating the immune system and regulating innate and adaptive immune response,or secreting antiviral metabolites to exert antiviral effects.The review is expected to provide insights into the development of probiotics-based antivirals.

Abstract:[Objective] The thioredoxin family plays an important role in the response of the foodborne bacterial pathogen Listeria monocytogenes to the oxidative stress during the adaptation to environment.Here,we explored the biological characteristics of the thioredoxin Lmo1609 in the oxidative stress tolerance and infection of L.monocytogenes.[Methods] We constructed the lmo1609-deleted and-complemented strains and then studied the growth,motility,oxidative stress tolerance,and pathogenicity of the strains both in vitro and in vivo.Moreover,we purified the recombinant Lmo1609 protein and then studied the oxidoreductase activity of the protein in vitro.[Results] The deletion of lmo1609 did not affect the bacterial growth but significantly reduced the swimming motility in vitro.Surprisingly,it enhanced the tolerance of this bacterium to H₂O₂ stress while showing no impact on bacterial infection.[Conclusion]This study indicates that the thioredoxin family member Lmo609 exhibiting the oxidoreductase activity contributes to the motility and oxidative stress tolerance but not in vivo infection of L.monocytogenes.

Abstract:[Objective]To isolate abundant uncultured Bacteroidia species from pit mud and analyze its physiological metabolism and ecological functions in the cases of pure culture and co-culture.[Methods] The relative abundance of Bacteroidia species was improved by subculture.Pure Bacteroidia species were isolated with the agar dilution method,and the metabolic characteristics of the dominant Bacteroidia species and the interaction with the dominant caproate-producing bacteria were explored based on fermentation test.[Results] Petrimonas sulfuriphila LBM11005 was screened out.Acetate and propionate were the main metabolites of this strain,and glucose promoted its growth.The metabolites ofLBM11005 interacted with those of Caproicibacterium sp.LBM19010,the dominant caproate-producing species in pit mud,regardless of substrate competition.To be specific,LBM19010 utilized propionate,the metabolite of LBM11005,for chain extension to produce odd-numbered carbon fatty acids of pentanoate and heptanoate.[Conclusion] This study reveals the fundamental physiological and metabolic characteristics of P.sulfuriphila LBM11005,one of the dominant Bacteroidia species in pit mud.This strain interacts with the dominant caproate-producing bacterium to contribute to the synthesis of longer odd-numbered carbon fatty acids.

Abstract:[Objective]Corynebacterium pseudotuberculosis,a facultative intracellular pathogen,infects a variety of animals and human,causing chronic suppurative inflammation. This paper aims to further evaluate the role of phospholipase D gene (pld) in C.pseudotuberculosis infection.[Methods] We employed homologous recombination to construct the traceless pld deletion strains (Δpld) of C.pseudotuberculosis without introducing foreign genes.Then,we compared the colony morphology and growth curves of deletion strains and wild-type strains,observed the influence of pld deletion on lactate dehydrogenase (LDH) release of C.pseudotuberculosis-infected macrophages and the proliferation of the pathogen in macrophages,and determined the death rates of and levels of pro-inflammatory cytokines in mice infected with the wild type and the deletion strain,thereby dissecting the relationship between pld and pathogenicity of the bacterial species.[Results] Traceless deletion of pld had no obvious effects on colony morphology and growth of C.pseudotuberculosis.Compared with ATCC 19410 and XH02,ATCC 19410Δpld and XH02Δpld showed no synergistic hemolysis with Rhodococcus equi ATCC6939.The LDH release of the macrophages infected with ATCC 19410Δpld and XH02Δpld was significantly less than that of the macrophages infected with the wild strains,and pld deletion decreased the intracellular bacteria in macrophages.The death rate,bacterial load in the liver and spleen,and the levels of pro-inflammatory cytokines in ascites and organs of mice infected with ATCC 19410Δpld were lower than those of mice infected with ATCC 19410.[Conclusion]The traceless pld deletion strains of C.pseudotuberculosis were developed.We confirmed that pld played an important role in death of C.pseudotuberculosis-infected macrophages and pathopoiesis of C.pseudotuberculosis in mice.

Abstract:[Objective] To analyze the antibiotic-resistant bacteria and antimicrobial-resistant genes in Penaeus monodon (African).[Methods]We collected P.monodon from Beihai New Area,Binzhou,Shandong,and then tested the number,proportion,and species of antibiotic-resistant bacteria in the intestine.Meanwhile,we analyzed the distribution of genes related to the resistance to four antibiotics by quantitative reverse transcription PCR (qRT-PCR).[Results] The total number of culturable bacteria in the intestine was about 1.45x10⁵-2.13x10⁶ CFU/g,and bacteria resistant to tetracycline,nalidixic acid,florfenicol,and gentamicin were all detected,among nalidixic acid-resistant bacteria (35.00%) and gentamicin-resistant bacteria took up the largest and smallest proportion,respectively.The sensitivity to 10 antibiotics was analyzed and the result showed that the intestinal bacteria were highly susceptible to six antibiotics such as gentamicin and florfenicol,moderately susceptible to tetracycline and kanamycin and resistant to nalidixic acid,penicillin,and amoxicillin.The culturable antibiotic-resistant bacteria mainly belonged to Vibrio,and the species of bacteria resistant to different antibiotics varied.Moreover,species of the same genus may be resistant to multiple antibiotics.The abundance of genes related to the resistance of the four antibiotics was different.Significant correlation between the relative copy number of tetA gene and the proportion of tetracycline-resistant bacteria and between the relative copy number of floR gene and the proportion of florfenicol-resistant bacteria was detected (P<0.01),but no significant correlation was observed between the relative copy number of qnrA gene and the proportion of nalidixic acid-resistant bacteria,and between the relative copy number of aadA gene and the proportion of gentamicin-resistant bacteria (P>0.01).[Conclusion] The intestine of P.monodon (African) harbors antibiotic-resistant bacteria and antimicrobial-resistance genes.

Abstract:[Objective] To study the role of OmpR in biological characteristics and pathogenicity of Vibrio parahaemolyticus.[Methods] The ompR gene deletion mutant (ΔompR) and complementary strain (CΔompR) of Vibrio parahaemolyticus were constructed using homologous recombination.The differences of growth characteristics,motility and biofilm formation ability of each strain were analyzed.Furthermore,the effects of various strains on cell adhesion,cytotoxicity and mouse pathogenicity were compared.[Results]The ompR deletion had no significant effect on the growth characteristics,motility and cytotoxicity of V.parahaemolyticus.However,compared with the wild-type (WT) strain,ΔompR showed markedly reduced biofilm formation ability.Mice infected with ΔompR had a 25% higher survival rate and fewer lesions.The bacterial load of ΔompR in mouse heart,liver and kidney was significantly lower than that of the WT strain,whereas the complementary strain restored the virulence to the WT level.[Conclusion]OmpR is involved in the pathogenic process in mice.It is a potential virulence factor of V.parahaemolyticus.

Abstract:[Objective] To identify the laboratory-isolated strain ZH-356 and evaluate its biocontrol effect on plant pathogenic fungi,so as to provide theoretical guidance for the development of biocontrol agents against plant fungal diseases.[Methods] The antimicrobial spectrum of strain ZH-356 and its species were determined by the plate confrontation assay and 16S rRNA gene sequence analysis,respectively.The biocontrol effect of strain ZH-356 was evaluated by the infection prevention test of Valsa malion isolated apple tree branches and the control test of apple valsa canker.[Results]Strain ZH-356 was identified as Streptomyces and had the highest similarity with Streptomyces rectiviolaceus at 99.71%.The antimicrobial spectrum test showed that strain ZH-356 exerted strong inhibitory effect on a variety of plant pathogenic fungi,such as V.mali,Fusarium graminearum,Bipolaris sorokinianumand Alternaria solani,which could lead to the thickening,cross-twisting,infrequent branching and easy fracture of the mycelia of V.mali.In addition,the antimicrobial active substance produced by strain ZH-356 was highly stable to temperature and pH,which only existed in its cells and was secreted in the presence of plant pathogenic fungi to inhibit their growth.In the prevention test of apple valsa canker,strain ZH-356 controlled more than 94% of V.mali infection,and in the control test of apple valsa canker,ZH-356 agents had 100% control of apple valsa canker.[Conclusion] Strain ZH-356 has a wide antimicrobial spectrum and good antagonistic activity against plant pathogenic fungi.It can be used as a biocontrol strain against plant fungal diseases,laying a foundation for the development of ZH-356-based biocontrol agents and the control of plant fungal diseases such as apple valsa canker.

Abstract:[Objective]Polycyclic aromatic hydrocarbons (PAHs) in soil can be absorbed by vegetable roots and accumulated in edible parts,thus threatening human health through the food chain.Inoculation of endophytic bacteria can reduce the accumulation of PAHs in vegetables,but their effects on the accumulation of PAHs in subcellular components of vegetables are rarely reported.[Methods]In vitro experiment was carried out to study the effects of Diaphorobacter sp.Phe15 inoculation on phenanthrene accumulation in the subcellular components of water spinach as well the response of activities of enzymes related to PAHs metabolism.[Results]Diaphorobacter sp.Phe15 inoculation accelerated the degradation of phenanthrene in subcellular components of water spinach stems and leaves,and the content of phenanthrene in water spinach was significantly reduced,with the degradation rate in subcellular components reaching more than 90%.In addition,Diaphorobacter sp.Phe15 inoculation affected the activities of the enzymes related to PAHs metabolism in subcellular components of water spinach.The activities of peroxidase (POD),polyphenol oxidase (PPO) and catechol 2,3-dioxygenase (C23O) were increased overall at subcellular level,and there was a negative correlation between enzyme activities and phenanthrene accumulation in water spinach.[Conclusion]Diaphorobacter sp.Phe15 inoculation improved activities of enzymes related to PAHs metabolism,and lowered the accumulation of phenanthrene in water spinach.The results provided a certain reference and theoretical basis for the use of functional endophytic bacteria to reduce PAHs pollution in vegetables.

Abstract:[Objective] In this study,we fused a catalase KatA from Bacillus subtilis with a self-assembling amphipathic peptide to improve its adaptability in industrial production.[Methods]S1vw-PT-katA and katA were cloned into pHT254 to yield the constructs pHT254-S1vw-PT-katA and pHT254-katA,respectively,which were separately introduced into B.subtilis WB800N for expression.The recombinant enzymes were then purified and characterized.[Results] The purified enzymes were acquired from the extracellular crude extract of the engineering bacteria through a four-step procedure consisting of ethanol precipitation,DEAE anion exchange chromatography,hydrophobic chromatography,and gel filtration chromatography.The fused enzyme S1vw-PT-KatA and natural enzyme KatA exhibited maximum activity at 30℃ and pH 11.0.However,the relative activity of the S1vw-PT-KatA incubated at pH 12.0 for 30 min was 77.3%,which was 14.9 times that of KatA under the same conditions.The relative activities of S1vw-PT-KatA incubated at 65℃ and 70℃ for 30 min were 19.8% and 17.5%,respectively,which were 1.8 and 1.7 times that of KatA.The relative activity of S1vw-PT-KatA stored at 4℃ for 14 days was 88.6%,while that was only 44.3% for KatA.Meanwhile,the k_cat/K_m value of S1vw-PT-KatA was 2.3 times that of KatA.[Conclusion]Fusing with a self-assembling amphipathic peptide S1vw can improve the pH stability,thermostability,storage stability,and catalytic efficiency of recombinant KatA.This finding provides a potential strategy for the modification,large-scale production,and application of catalase.

Abstract:[Objective] The study aimed to explore the dynamic changes of bacterial community in bulk soil at different stages of tomato bacterial wilt under a continuous monoculture system and clarify the response mechanisms of bulk soil bacteria to disease stages.[Methods] For all the bulk soil samples in the 1st,3rd,5th,and 7th crops,quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the number of Ralstonia solanacearum at different disease stages, and high-throughput sequencing of 16S rRNA V4‒V5 region was used to study the diversity,composition,biomarkers and assembly processes of bacterial community.[Results] The number of R.solanacearum at the peak stage of disease (1.28× 10⁷ copies/g) was higher than that at the final stage of disease (1.77×10⁶ copies/g).In addition,the influence of disease stages on the diversity of bacterial community was enhanced with increasing continuous monoculture time.Alpha and beta diversities of bacterial community between different stages in the 3rd and 5th crops were significantly different.Linear discriminant analysis effect size (LEfSe) analysis showed that samples from the final and peak stages of disease during continuous monoculture comprised distinct biomarkers.Additionally,microorganisms that assisted R.solanacearum in inducing diseases gradually accumulatedwith the increasing continuous monoculture time.Microbacterium and Nitrosospira were observed with positive effect for tomato bacterial wilt at the peak stage of disease,while they were Sphingobium,norank f norank o SBR1031,and norank f Rhodothermaceae at the final stage of disease.The analysis of community assembly process revealed that bacterial community at the two stages of disease were both dominated by stochastic processes,especially the dispersal limitation,but the bacterial community at the peak stage of disease was more homogeneous than that at the final stage of disease.[Conclusion] The bacterial communities at the final and peak stages of tomato bacterial wilt were significantly different in diversity,composition and structure in 3rd and 5th crops,and these changes might be related to different community assembly mechanisms.This study facilitated the understanding of the development of tomato bacterial wilt during continuous monoculture system and provided guidance for the control of bacterial wilt.

Abstract:[Objective]Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture.BmNPV infection blocked the cell cycle at G2/M phase in BmN-SWU1 cells.CyclinB is an important cyclin that regulates the transition from G2 phase to M phase of cell cycle.Therefore,studying the changes of CyclinB after BmNPV infection is of great significance to elucidate the mechanism of viral regulation of cell cycle and meanwhile,exploring the viral proteins interacting with CyclinB during this process can provide molecular targets for the construction of transgenic strains of Bombyx mori.[Methods] Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of BmCyclinB after BmNPV infection.The localization change of BmCyclinB after viral infection was observed by immunofluorescence and verified by cytoplasmic and nuclear protein separation experiment.Co-immunoprecipitation was employed to catch the viral proteins that interacted with BmCyclinB.The proportion of BmCyclinB into the nucleus was observed by knocking down BmNPV IAP1 after BmNPV infection.[Results]The transcription level of BmCyclinB was down-regulated after BmNPV infection and BmCyclinB was mainly located in cytoplasm before BmNPV infection and in nucleus after infection.BmNPV infection of BmN-SWU1 cells promoted the nuclear accumulation of BmCyclinB.A total of 7 viral proteins interacting with BmCyclinB were identified.The interaction between BmNPV IAP1 and BmCyclinB was confirmed by co-immunoprecipitation and cell co-localization.The amount of BmCyclinB entering the nucleus was significantly reduced after BmNPVIAP1 was knocked out during BmNPV infection. [Conclusion]BmNPV IAP1 can promote the accumulation of BmCyclinB in the nucleus by interacting with BmCyclinB.

Abstract:[Objective] Culturable bacteria were isolated from camel and the distribution of bacteria was analyzed.On this basis,the acid-producing and enzyme-producing bacteria were screened out.This study is expected to provide resource and technical support for the development and utilization of camel-derived probiotics.[Methods] Serial dilution-coating method was used to screen bacteria in milk,saliva,and rectal feces of lactating Bactrian camels in Jimunai area of Xinjiang in winter,and the strains were identified by 16S rRNA gene sequencing.Strains producing acid and enzyme were identified with the transparent circle method,and the ability of producing organic acid,amylase,cellulase,and protease was further evaluated.[Results]A total of 63 bacterial strains were cultured,with 35 strains from camel feces (mainly Psychrobacter and Acinetobacter),21 from camel milk (dominated by Pseudomonas and Leuconostoc),and 7 from saliva (mainly Bacillus).A total of 11 acid-producing strains were screened out,with the feces-derived Leuconostoc mesenteroides 2F11M yielding the most lactic acid (up to 3.93 mg/mL),milk-derived L.lactis 2N5M achieving the highest acetic acid yield (12.73 mg/mL),and the feces-derived Enterococcus Mundtii 2F17M topping the bacteria in propionic acid yield (10.36 mg/mL).According to the transparent circle,the enzyme-producing strains were mainly from camel feces and camel milk.Among them,17,10,and 15 strains produced amylase,cellulase,and proteinase,respectively.The milk-derived Bacillus velezensis Nai1 had the highest amylase activity (509.07 μg/min/mL) and feces-derived Acinetobacter lwoffii 2F5N had the highest cellulase activity (156.87 μg/min/mL).In addition,milk-derived B.subtilis 2N2N achieved the highest protease activity (3.59 μmol/min/mL).[Conclusion] A variety of acid-producing and enzyme-producing strains with high activities were screened from lactating Bactrian camels in Xinjiang,which have the potential for the preparation of microecologics.

Abstract:[Objective] To isolate and characterize the bacteriophage from the intestinal contents of Marmota himalayan.[Methods]A lytic phage was isolated from the host Escherichia coli in intestinal contents of M.himalayan with the double-layer agar method.The morphological characteristics were observed by transmission electron microscopy (TEM).Meanwhile,the optimal multiplicity of infection (MOI),one-step growth curve,pH stability,and the host range of the phage were investigated,and the genome was analyzed as well.[Results] The lytic bacteriophage was named vB_EcoM_TH18 and the phage plaque was transparent and round with a clear boundary.According to TEM,the phage had the head of (90±5) nm in diameter and tail of (115±5) nm in length.The optimal MOI was 1.According to the one-step growth curve,vB_EcoM_TH18 had a latent period of 20 min and the lysis period of about 110 min with the average burst size of about 15 PFU/mL.vB_EcoM_TH18 can keep stable titer at pH 4.5-9.5.Additionally,it can lyse various pathotypes and serotypes of E.coli and Shigella sonnei,while Salmonella,Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumonia,and Acinetobacter baumannii can't be lysed.The genome of the vB_EcoM_TH18 was 133 882 bp with the G+C content of 39.95%.It comprised 210 coding sequences (CDS) and 13 tRNAs genes,without any virulence genes or antibiotic-resistant genes.BLASTn alignment showed that genome of vB_EcoM_TH18 shared 95.17% identity to E.coli phage Av-05.According to the polygenetic trees for the whole genomes,major capsid proteins,and terminase large subunits of vB_EcoM_TH18 and otherMyoviridae phages,respectively,vB_EcoM_TH18 was a novel phage in Avunavirus of Myoviridae.[Conclusion] In this study,we isolated and identified a novel broad host-range E.coli phage vB_EcoM_TH18.It can lyse various pathotypes and serotypes E.coli and S.sonnei.

Abstract:[Objective] To address the low efficiency of biological nitrogen removal from high-temperature coal chemical wastewater.[Methods] In this study,a mesophilic ammonia-oxidizing bacterial strain A1 and a mesophilic denitrifying bacterial strain D1 were isolated from the activated sludge of the coal chemical wastewater treatment system of an energy and chemical group Co.Ltd.in Shanghai.[Results] Based on the morphological,physiological,and biochemical characteristics and 16S rRNA gene sequence,strain A1 was preliminarily identified as Aquamicrobium ahrensii and strain D1 as Pseudomonas stutzeri.The single factor experiments showed that the optimal growth temperatures of strains A1 and D1 were as high as 42℃ and 40℃,respectively.Then,we simulated the wastewater treatment at the initial NH₄⁺-N concentration of 100 mg/L and 42℃,and established a co-culture composed of strains A1 and D1(W/W,20%/10%) to explore the effects of the co-culture on shortcut nitrification and denitrification and N₂O release at different pH and C/N ratios.At 42℃,pH 9.0–10.0,and initial C/N ratio of 2:1,the co-culture showed a nitrogen removal rate above 99.0% and the maximum N₂O yield of 51.3%.[Conclusion] This study can provide technological support and strain resources for the biological treatment of high-temperature coal chemical wastewater,and help to reveal the law of N₂O release in the treatment of high-temperature wastewater.

Abstract:[Objective]The mechanosensitive channel of small conductance (MscS) in bacteria releases solutes and water when a hypo-osmotic shock raises the pressure in the cells,thereby enabling the survival of bacteria.Given its wide distribution in various bacteria and no homologues found in mammals,MscS is considered a novel antibiotic target.A hallmark of MscS is that it enters a tension-insensitive inactivated state upon prolonged mechanical stimulation,thereby avoiding the loss of a large amount of cell content and preventing cell death.This study aims to identify the key residues related to the inactivation of MscS,which is expected to serve as a reference for the development of MscS-targeting drugs.[Methods] The cysteine mutants of MscS Cyto-helix (P166−I170) were prepared with molecular cloning method.The thiol compound MTSET⁺ binds to cysteine and thus modify cysteine's side chain group.In this study,osmotic downshock assay was used to examine the viability of Escherichia coli expressing cysteine mutants of MscS Cyto-helix (P166−I170) upon hypotonic stimulation without or with MTSET⁺ treatment and screened for cysteine mutant that significantly affected the channel function.The inactivation of MscS mutants before and after MTSET⁺ treatment was examined by electrophysiological experiments.In addition,the inactivation mechanism of MscS was further explored by eletrophysiology combined with site-directed mutagenesis.[Results] MTSET⁺ led to a great decrease in the survival rate of E.coli expressing G168C-MscS upon hypotonic stimulation.G168C-MscS lost its inactivation property after binding to MTSET⁺ and remained open,resulting in great loss of intracellular contents and bacterial death.The inactivation properties of G168Y-MscS,G168L-MscS,and G168K-MscS mutants were consistent with WT-MscS,while the inactivation rates of the three mutants G168D,G168V,and G168I were significantly reduced,especially G168I-MscS which lost the inactivation properties.Therefore,MscS G168 affected channel inactivation,and the channel inactivation characteristics were related to the size and charge of the residue side chain group at 168 site.[Conclusion]MscS G168 is a key residue that affects the inactivation of MscS.

Abstract:[Objective] Corynebacterium glutamicum is an important amino acid producer.In this study,we explored the mechanisms of interaction between SigE and ZAS family protein CseE,particularly the interaction between CseE mutants and SigE.[Methods] SigE and CseE proteins from C.glutamicum ATCC 13032 were used.The sigE-overexpressing and cseE-overexpressing strains were developed with the genetics method.Through RT-qPCR,we explored the influence of SigE on the transcription of sigE and cseE.At the same time,we tested the binding of CseE proteins to Zn²⁺ and SigE through isothermal titration calorimetry (ITC) and His pull-down experiments,followed by functional domain analysis and multiple sequence alignment of CseE protein to study the effect of key amino acid sites in the domain on the binding ability of SigE.Then,we conducted molecular docking and molecular dynamic simulation of SigE and CseE proteins to analyze the mechanisms of key amino acids affecting their binding.[Results] SigE of ATCC 13032 regulated the transcription of sigE and cseE and its activity was regulated by the CseE protein.CseE,a ZAS family protein,bound to Zn²⁺.CseE_His83A,CseE_Cys87A,and CseE_Cys90A mutantsdid not affect the binding ability to SigE,while the binding ability of CseE_C87A-C90A and CseE_{His83A-C87A-C90A} mutants to SigE decreased slightly.Molecular dynamic simulation showed that the SigE-CseE_C87A-C90A binding energy and SigE-CseE_{His83-C87A-C90A} binding energy were -17.23 kcal/mol and -14.06 kcal/mol,respectively,22.8% and 36.9% lower than the binding energy between SigE and CseE,respectively.[Conclusion]SigE regulates the expression of sigE and cseE by aggregating RNA polymerase,and its protein activity is regulated by CseE.CseE protein belongs to the ZAS family,which binds to Zn²⁺ and inhibits SigE activity by interacting with SigE protein.CseE_C87A-C90A and CseE_{His83A-C87A-C90A} affect the ability to bind to SigE and weaken the control of SigE activity.The three-dimensional structures and the identified key amino acid sites in this study lay a theoretical basis for further exploring the mechanism of SigE and CseE in C.glutamicum in response to environmental stresses.

Abstract:[Objective]In addition to rhizobia,non-rhizobia have been simultaneously isolated from the same root nodules,and the existence of non-rhizobia in root nodules of Fabaceae plants was a universal phenomenon.Their colonization meaning and potential ecological functions have been unknown so far.Moreover,the real relationships between rhizobia and non-rhizobia remain unclear.Therefore,the interactions and mechanism of their co-evolution should be investigated intensively and further studied.This paper was designed to reveal the interactions between rhizobia and non-rhizobia in root nodule microbiome of Sophora davidii and to further explore the shift in the interaction pattern between them under different culture conditions.[Methods] The rhizobia and non-rhizobia isolated from the root nodules of wild Sophora davidii in arid areas of Northern Shaanxi Province was taken as the subjects.The interacting strains were screened based on co-culture,two-compartment culture and pure culture,and were investigated for the tolerance to pH and NaCl and the utilization of various nitrogen sources.The colony diameter,growth curve and polysaccharide production of the rhizobia were used to evaluate the interaction effects,so as to further explore the transformation mechanism underlying the interaction effects of strains under saline-alkali and nutritional stresses.[Results] Under saline-alkali stress,the interaction effects of the non-rhizobia Pseudomonas oryzihabitans BT-147 and Priestia aryabhattai BT-59 on Rhizobium azibense BT-170 changed from inhibition under normal culture conditions to promotion.The colony diameter of R.azibense BT-170 increased by 0.803 mm and 1.034 mm,respectively in test groups as compared with the condition in control group.When Bacillus siamensis BT-9-1was co-cultured with Mesorhizobium metallidurans YC-39 under saline-alkali stress,the interaction pattern changed to promotion from inhibition under normal culture conditions.Compared with control group,the test group had increased colony diameter of M.metallidurans YC-39 by 1.019 mm,and the exopolysaccharide production rose from 1.088 μg/mL to 2.555µg/mL.When Glutamic acid was used as the only nitrogen source,the non-rhizobia Pseudomonas oryzihabitans BT-147 and Priestia aryabhattai BT-59 had promotion effects on R.azibense BT-170,and the difference in colony diameter of R.azibense BT-170 between test group and control group reached 1.348 mm and 2.196 mm,respectively.The exopolysaccharides yield of R.azibense BT-170 increased from 0.559 μg/mL in control group to 0.821 μg/mL and 3.341µg/mL,respectively.[Conclusion] Under saline-alkali and nitrogen source stresses,the interaction effects of the non-rhizobia Pseudomonas oryzihabitans BT-147 and Priestia aryabhattai BT-59 on R.azibense BT-170 changed to promotion from inhibition under normal culture conditions and enhanced the exopolysaccharides production of R. azibense BT-170(P<0.05).The shift in the interaction pattern between rhizobia and non-rhizobia under different culture conditions improved the stress resistance of rhizobia and expanded the range of nitrogen sources available to rhizobia,revealing the role of non-rhizobia in root nodule microbiome and simplifying the complex interactions in root nodule microbiome in vitro.

Abstract:[Objective]To study the regulation of eIF2α-mediated translation initiation complex eIF4F after Newcastle disease virus (NDV) HBUN/LSRC/F3(hereinafter referred to as NDV F3) induces ribosomal stress in cervical cancer HeLa cells.[Methods]Flow cytometry and cell counting Kit-8(CCK-8) were used to detect cell apoptosis,quantitative real-time polymerase chain reaction to examine c-Myc gene expression,flow cytometry to analyze cell cycle,Western blotting to assess the expression of c-Myc,RPS7,Bcl-2,NP,eIF4E,and eIF2α proteins,and Western blotting and immunofluorescence staining to locate NP and eIF4E proteins.[Results]Compared with the negative control group,NDV F3 inhibited the proliferation of HeLa cells and induced apoptosis.We observed G₀/G₁ arrest,inhibition of c-Myc expression in a time-dependent manner,decrease in protein expression of c-Myc and Bcl-2 in 0-48 h,generation of NP protein at 24 h followed by the increasing trend,and the increase in content of RPS7,eIF4E and eIF2α proteins followed by a decrease during 0-48 h.The results of Western blotting and laser confocal microscopy showed that NP protein was mainly in cytoplasm and that NP co-localized with eIF4E.[Conclusion] NDV F3 induces apoptosis of HeLa cells and triggers ribosomal stress.NP interacts with eIF4E to inhibit the formation of eIF2α-mediated translation initiation complex eIF4F,which blocks the connection with host mRNA and promotes the expression of NDV F3 mRNA,ultimately resulting in the inhibition of host protein translation.

Abstract:[Objective]To identify the insecticidal activity of wild-type Bacillus thuringiensis strain Bt S2480-1 and explore the insecticidal gene resources.[Methods]The whole genome of Bt S2480-1 was sequenced by Illumina Hiseq2000.The genome information,genes,and insecticidal genes of the strain were analyzed with bioinformatics methods.The total proteins of Bt S2480-1 were analyzed by LTQ-Obitrap nano-LC-MS/MS and the biological activity of Bt S2480-1 against Culex quinquefasciatus and Spodoptera exigua larvae was determined.[Results]The genome of Bt S2480-1 was 6.2 Mb,with GC content of 35.11%,and one nucleoid and three large plasmids were yielded through assembly,containing 6 297 coding genes with 12 encoding insecticidal proteins.Meanwhile,a total of 1 500 proteins of Bt S2480-1 were identified by LTQ-Orbitrap MS/MS,among which 11 were insecticidal proteins.The total proteins of Bt S2480-1 showed strong activity against C.quinquefasciatus larvae but relatively weak activity against S. exigua larvae,with LC₅₀ of 27.636 μg/mL (95% FL:12.559-61.707 μg/mL) and 496.833 μg/mg (95% FL:320.134-776.964 μg/mg),respectively.[Conclusion] Bt S2480-1 contains 12 genes encoding insecticidal proteins and displays insecticidal activity against C. quinquefasciatus and S.exigua larvae.

Abstract:[Objective]To study the regulatory effect of (S)-equol on human gut microbiota and the metabolic effect of human gut microbiota on (S)-equol.[Methods] The interaction between (S)-equol and human gut microbiota was detected by batch fermentation in vitro,bacterial 16S rRNA gene high-throughput sequencing,gas chromatography,high performance liquid chromatography (HPLC),and mass spectrometry.[Results](S)-equol had no significant effect on the overall structure of human gut microbiota or the concentrations of short chain fatty acids (SCFAs).No significant change in the relative abundance of bacteria was detected between the 0.45 mmol/L (S)-equol group and the control group.However,0.90 mmol/L (S)-equol significantly increased the relative abundance of conditional pathogens such as Enterobacteriaceae and reduced that of potential probiotics Coprococcus.The concentration of (S)-equol in the fermentation broth decreased by 15%-30%,which suggested that (S)-equol may be degraded or modified by gut microbiota.[Conclusion] From the perspective of regulating gut microbiota in vitro,0.45 mmol/L (S)-equol is safe,while 0.90 mmol/L (S)-equol may disturb the balance of gut microbiota.(S)-equol can be further metabolized by human gut microbiota,and the structure and function of the specific metabolites and the biological safety of (S)-equol in vivo remain to be studied.

Abstract:[Objective] A bacterial strain likely affiliated with the Bacillus cereus group was isolated from the ascites of Chinese giant salamander from a giant salamander farm in Shaanxi Province and was investigated for the biological characteristics.[Methods]The diseased giant salamander was aseptically dissected,and the samples taken from intestine,ascites,skin and other parts were homogeneously diluted,separated and purified.A pure strain of suspected B.cereus group bacteria was obtained from the ascites,which was named SHOU-BC01.The strain was identified for morphological and staining characteristics,culture and biochemical characteristics,biofilm formation ability,spore formation,drug susceptibility,and whole genome sequencing.Based on the sequencing results,average nucleotide identity (ANI),digital DNA-DNA hybridization (dDDH),multilocus sequence typing (MLST),whole-genome SNP based clustering and virulence factor analysis were performed.[Results] Strain SHOU-BC01 is a Gram-positive bacillus with rough surface,with protease,lecithinase and fibrinolysin activities.It can ferment L-arabinose,D-ribose,D-xylose and other carbohydrates,and can utilize tryptophan and pyruvate,with strong biofilm formation ability.Its spore formation rate reached 70.60% in 120 h.The strain was resistant to 15 antibiotics including penicillin G,cephalothin and vancomycin,and sensitive to 25 antibiotics including piperacillin,cefazolin and gentamicin.According to biological characteristics combined with ANI,dDDH and whole-genome SNP based clustering analysis,strain SHOU-BC01 was identified as B.paranthracis,and the MLST typing showed that the strain belonged to ST205 sequence type.It contains virulence factors such as sphingomyelinase,CytK and NheC toxin genes,polysaccharide capsular,PlcR-PapR quorum-sensing system and type Ⅶ secretion system.[Conclusion] B.paranthracis was successfully isolated from the ascites of Chinese giant salamander,which enriched the data of B.paranthracis.

Abstract:[Objective]To explore the inhibitory effects of Pseudomonas 2-4 and its product on algae based on ecological risk assessment.[Methods]Algicidal bacterial strain 2-4(Pseudomonas sp.) was isolated,and the suppression of strain 2-4 and its product on algae was examined based on orthogonal test and kinetic stimulation.Acute toxicity test was conducted to evaluate the ecological risks.[Results] Under the optimal conditions,strain 2-4(V/V=15%) removed 92.81% of the Microcystis aeruginosa in 4 days,and the algal inhibition could be characterized by the first-order kinetic model (t_1/2=126 h).Strain 2-4 had a broad-spectrum inhibitory activity and the inhibitory effects of Pseudomonas sp.on Ulothrix sp.,Chlorella pyrenoidosa,and Tetradesmus obliquus were reported for the first time.The product (<500 Da) from strain 2-4 had inhibitory effects on algae,but could be destroyed by high temperature and strong acid and alkali.The results of acute toxicity test showed the bacterial liquid at>1.5%(V/V) was toxic to Daphnia magna and the product at>2%(V/V) was toxic to D.magna and Gobiocypris rarus but nontoxic to Photobacterium phosphoreum.At the safe concentration,strain 2-4(V/V=1.5%) and its product (V/V=10%) decreased chlorophyll a in the lake with algae bloom by 4.83%–42.94% and 30.62%–68.69%,respectively.[Conclusion] We analyzed the algicidal effect of strain 2-4 and its product in the safe concentration and clarified the relationship between the ecological toxicity and the inhibitory effects of strain 2-4,hoping to provide a reference for biocontrol of algae.

Abstract:The pepT gene of Vibrio parahaemolyticus encodes a metal dependent peptidase T (PepT) that catalyzes the N-terminal amino acid of tripeptides with high specificity,and thus it is also called aminopeptidase T.Studies have found that most aminopeptidases are involved in protein metabolism of bacteria and regulating the activity of tripeptides,but little is known about PepT in bacterial virulence and pathogenicity.[Objective] In this study,PepT was chosen to explore the effect of gene deletion on the biological characteristics and pathogenicity of V.parahaemolyticus.[Methods] The pepT gene deletion mutant ΔpepT and the complementary strain CΔpepT were generated and their differences of various strains in motility,biofilm,environmental tolerance and cytotoxicity were compared.[Results] Compared with the wild-type strain,ΔpepT had significantly decreased transcription level of polar flagella and reduced swimming mobility.Furthermore,the biofilm formation ability of ΔpepT was weakened,but there were not statistically significant in the swarming mobility and environmental tolerance.In addition,the pepT gene deletion resulted in remarkably lowered cytotoxicity and virulence of V.parahaemolyticus in mice.[Conclusion] The pepT gene deletion affects the swimming motility and biofilm formation ability as well as the pathogenicity of V.parahaemolyticus.

Abstract:[Objective] To observe the dynamic changes of gut microbiota in a pair of newborn twins with cyanotic congenital heart disease after surgery and antibiotic treatment and explore the variation of drug resistance of Enterobacter cloacae during treatment. [Methods] The fecal samples of the twins were collected at different stages of treatment and analyzed by culturomics and 16S rRNA gene sequencing.The susceptibility of ten Enterobacter cloacae strains isolated from the elder twin at different stages to antibiotics was determined.[Results] Culturomics results suggested that antibiotics decreased the flora richness of fecal samples before surgery and only Enterococcus,Acinetobacter and Enterobacter were isolated.Upon the long-time exposure to the hospital environment,opportunistic pathogens of several genera were isolated,including Enterobacter,Klebsiella,Acinetobacter and Pseudomonas.After 4 months of breast feeding,richness of fecal flora increased and the composition changed.To be specific,probiotics such as Lactobacillus and Bifidobacterium were isolated.16S rRNA gene sequencing results showed that α diversity indexes and the genus with highest relative abundance were different depending on treatment stages under the influence of various factors.The susceptibility tests indicated that E.cloacae strains gradually developed resistance to piperacillin during treatment.[Conclusion]Antibiotic treatment,exposure to hospital environment,and breast feeding all changed the gut microbiota structure of the twins during treatment and recovery.Even if only one antibiotic was used at different stages of treatment,the resistance of E.cloacae to different drugs might change.