Abstract:Lawsonia intracellularis (LI), an intracellular pathogen, is a common cause of lethargy, poor appetite, hemorrhagic diarrhea, or sudden death of pigs, posing a threat to pig industry. LI induces a distinct immune response with unique immune patterns in pig intestine. Diagnosis of this intracellular species is dependent on the characteristics of immune response in animals at different stages of infection. In this review, we described the characteristics of immune response upon LI infection and the detection methods, in an attempt to provide a reference for the development of new diagnosis techniques and prevention and control of related diseases.

Abstract:The gut harbors the microbiota with complex structure and diverse functions, which plays an important role in host immunity, nutrient absorption, and metabolic regulation. The rapid development of sequencing technologies such as 16S rRNA gene sequencing and metagenomic sequencing has generated massive data of gut microbiota, among which many unassembled sequences are considered as the microbial dark matter. Through the combination of a variety of culture methods and high-throughput sequencing, a large number of microorganisms have been isolated from the guts of human, mouse, and pig in recent years, which has significantly enriched the bacterial strain resources and provided a basis for the analysis of microbial dark matter and the research on the functions and application of gut microbiota. Although the culturability of microorganisms is affected by many factors and most microorganisms are still uncultured, the acquisition of microbial resources is indispensable for the study of etiology and the analysis of physiological and genetic characteristics of bacterial strains. The isolation and culture of gut microorganisms are of great significance for the deep research on gut microbiota from association study to functional verification, causality elucidation, and functional strain development. This article summarized the factors that affect the culturability of microorganisms, introduced the culture methods of gut microorganisms, and reviewed the progress and application of gut microorganism culture, aiming to give new insights into this field.

Abstract:Biological denitrification is cost-effective, efficient, and environmentally friendly, which thus has broad prospects in the wastewater treatment. At the moment, most of the available denitrifying microorganisms in wastewater treatment are mesophilic bacteria. However, the denitrification is significantly inhibited in the case of low temperature. The cold-adapted denitrifying bacteria, with tolerance to low temperature and high efficiency in denitrification, have attracted the interest of scholars. Moreover, the nanoparticles (NPs) are widely used in biology, agronomy, medicine and other fields, which, however, are inevitably released to water and soil in the production, storage, and usage of NPs-containing products. Massive accumulation of NPs in the water will hinder the denitrification of microorganisms, posing a challenge to wastewater treatment and arousing the concern of scholars. The denitrification process of cold-adapted bacteria, the toxicity of NPs to the denitrification, and the countermeasures have been studied. On this basis, this article describes the cold-adapt mechanism and denitrification process of the cold-adapted bacteria and discusses the toxicity of NPs to the denitrification process and the countermeasures, which is expected to lay a theoretical basis for the use of microorganisms to treat nitrogen-polluted wastewater containing NPs in a low temperature environment.

Abstract:Candida normally colonizes mucosal surfaces without causing any problems. Sometimes, it causes mucosal infections such as oropharyngeal candidiasis and vulvovaginal candidiasis in individuals with compromised immunity or microecological imbalance. For the treatment of the infections, antifungal agents are indispensable, and the host՚s immunity, especially mucosal epithelial cells as the first line of defense against Candida infection, plays an important role. In this paper, we summarize the research on the Candida-epithelial interactions.

Abstract:The complexity and variability of marine environment make marine corrosion an increasingly serious global problem. Marine corrosion not only causes huge economic losses but also brings serious environmental pollution and personnel safety problems, becoming a key problem that must be solved for marine economic development. According to statistics, 20% of corrosion in the marine environment is caused by microorganisms, which exist on metal surfaces in the form of biofilm, mainly including bacteria, archaea, fungi, and algae. This paper reviewed the research progress in the four types of marine microorganisms, involving the species, community structure influencing factors, and functioning mechanisms. Furthermore, we elucidated the influencing factors and summarized the mechanisms of the microorganisms to promote or inhibit corrosion of metal materials and introduced the prevention and control methods of microbial corrosion in the marine environment. Finally, we discussed the trend of research and prevention on microbial corrosion in marine environment, aiming to provide reference for the study of corrosion mechanism and the implementation of corrosion prevention work.

Abstract:Nutritional limitation is one of the common environmental stresses for microorganisms. In addition to natural environments such as oceans, glaciers, deserts, and deep surface severely deficient in nutrients, more and more artificial environments also present the characteristics of nutritional limitations, such as various micro-polluted water bodies and wastewater biological treatment systems with stricter discharge standards. Substrate concentration greatly affects the growth, metabolism, and community structure of microorganisms including bacteria, and eventually leads to changes in their functions. In order to survive with limited nutrients, microorganisms first need to perceive the reduction of nutrient supply, then regulate metabolic processes globally via genes, proteins, signal molecules, and metabolites, and finally change substrate affinity, growth rate, motility, and morphology to adapt to malnutrition. Intracellular signaling molecules and the responses triggered by them are the key for microorganisms to deal with nutritional stress. We sorted out the essential signal products, receptor proteins/regulation process and response results of microorganisms represented by bacteria when dealing with carbon and nitrogen source limitation, and then analyzed the interaction of carbon and nitrogen limitations in the response process. This review provides a theoretical basis for the cognition of microorganisms in extreme environments and the application of microorganisms under nutrient limitations, especially in the biological treatment of low-concentration pollutants and biological monitoring.

Abstract:Pathogens exhibit strong antibiotic resistance, a major cause of which is biofilm (BF) formation. The eradication of bacteria is extremely difficult once biofilms have formed as biofilms can lead to persistent infections in patients and trigger a variety of chronic diseases, which cause a heavy burden on the global healthcare system. Pillararenes are novel macrocyclic compounds with unique pillar-shaped architectures, which have attracted wide attention owing to their potential applications in the development of functionalized and bioactive materials. In addition, they have broad application prospects in the prevention and control of antibiotic resistance. This article reviewed the activities and mechanisms of pillar[5]arene derivatives against bacterial pathogens, especially the inhibitory effect on biofilms. On this basis, researchers can explore new antibacterial and bactericidal strategies and employ non-traditional methods to solve antibiotic resistance. This review is expected to provide a theoretical basis for the development of new agents to control biofilm or treat bacterial infections.

Abstract:Antibiotic-resistant bacteria and antibiotic resistance genes have become new environmental pollutants causing public health problems in the world. The antibiotic-resistant bacteria, especially multidrug-resistant bacteria, has received increasing attention in clinical medicine, animal breeding, and environmental transmission. However, there are few studies about the antibiotic resistance of bacteria in wild animals such as giant panda (Ailuropoda melanoleuca). Giant panda (Ailuropoda melanoleuca) is recognized as a rare wild animal in the world and its population is vulnerable to a variety of diseases, especially intestinal bacterial diseases. With the increasing use of antimicrobials in disease prevention and control, the harm of antibiotic resistance is becoming more and more apparent. By reviewing the research reports on the antibiotic resistance of giant panda-derived bacteria, we introduced the phenotypes, genotypes, and mechanisms of antibiotic resistance as well as the horizontal transmission mechanisms of the bacteria. This review aims to provide a basis for the prevention and control of antibiotic resistance and the reasonable application of antibiotics in clinical practice, so as to facilitate the ex situ conservation of giant panda.

Abstract:Toxin-antitoxin systems (TAs) are prevalent genetic elements in bacteria, archaea, and prophages, which are usually composed of a growth-inhibiting toxin and its cognate antitoxin. Toxins are stable in bacterial cells, while antitoxins are prone to be degraded by the ATP-dependent proteases. Most toxins are proteins and have enzyme activity, which inhibit bacterial growth by affecting important life activities such as protein translation and DNA replication. Antitoxins are either proteins or noncoding RNAs that neutralize the toxicity of toxins to bacteria in diverse ways. The available studies have demonstrated that TAs function as plasmid-stabilization elements, provide defense against phages, and promote biofilm formation. As the research deepens, increasing novel TAs have been discovered, which improves our understanding of TAs. At present, the classification of TAs has been extended to types Ⅰ-Ⅷ. This paper summarizes the recent discoveries of new TAs and focuses on the type Ⅶ TA in which the enzymatic antitoxin chemically modifies the toxin to neutralize it. Since TAs are closely associated with the pathogenicity of pathogenic microorganisms, in-depth study of these TAs can provide new targets for the treatment of drug-resistant microorganisms.

Abstract:[Objective] To obtain the target compounds from the extract of Alternaria panax based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) combined with their fragmentation patterns. [Methods] A. panaxwas fermented in potato dextrose broth(PDB) for 14 days. The filtrate was extracted with ethyl acetate and concentrated under reduced pressure to obtain the crude extract. The chemical composition and fragmentation pattern of the crude extract of A. panax were analyzed by UPLC-Q-TOF-MS/MS (including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), molecular formula, and fragment peak). Semi-preparative high-performance liquid chromatography (HPLC) was employed to further purify the target compounds. The structures of the compounds were identified by nuclear magnetic resonance (NMR), mass spectrometry (MS), and comparison with literature data. [Results] Nine diketopiperazines (1-9) were identified by UPLC-Q-TOF-MS/MS from the crude extract of A. panax and their fragmentation patterns were analyzed. To verify the correctness of these structures, we separated compounds 1-3 and determined their chemical structures. The obtained structures of compounds 1-3 were consistent with the results of UPLC-Q-TOF-MS/MS. [Conclusion] UPLC-Q-TOF-MS/MS combined with fragmentation patterns can rapidly and efficiently identify the target components in an unknown sample, which can guide the targeted isolation as well.

Abstract:[Objective] To explore the tolerance of three fungal species to manganese ion, the optimal conditions for them to adsorb Mn²⁺ in solution, and the mechanism for the adsorption, and thus to provide technical reference for the control of manganese ion pollution.[Methods] The minimum inhibitory concentration (MIC) of Trichoderma harzianum, T. atroviride and T. asperellum was determined and the optimal adsorption conditions were explored. Based on scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) and Fourier transform infrared spectroscopy (FTIR), fungal cells before and after the adsorption were analyzed. [Results] T. harzianum, T. atroviride, and T. asperellum could tolerate the maximum manganese concentration of 1 600 mg/L, 1 800 mg/L, and 2 000 mg/L, respectively. The optimum adsorption conditions are pH 7, adsorption time of 80 h, and temperature of 28℃, and the highest adsorption rate was up to 23.7%. The functional groups involved in the adsorption of T. harzianum were -OH, and -C-N- and-C=O in the amine group, and those of T. asperellum were -OH and -NH. The functional groups of T. atroviride were -C-H, and phosphate groups P=O, P-OH, and PO₄^3-.[Conclusion] The screened T. harzianum, T. atroviride, and T. asperellum showed strong adsorption of heavy metal manganese, which can serve as a reference the treatment of manganese pollution in soil.

Abstract:[Objective] To investigate the mechanism of Jingpixian Tincture (JPXT) on Trichophyton rubrum. [Methods] Microdilution method was employed to determine the minimal inhibitory concentration (MIC), and fluorescence microscopy to observe the effect of JPXT on spore germination and mycelial growth of T. rubidium. Sorbitol was used to detect the effect of JPXT on fungal cell wall. Flow cytometry was employed to determine the intracellular reactive oxygen species (ROS) level of T. rubidium treated by JPXT. A microplate reader was used to determine the leakage of intracellular nucleic acid. High performance liquid chromatography (HPLC) was adopted to determine ergosterol content in the cell membrane. A microplate reader was used to measure the activities of β-1,3-glucan synthase (β-1,3-GS), chitin synthetase (CS), squalene epoxidase (SQLE), and 14-alpha demethylase (CYP51). The mRNA levels of β-1, 3-GS, CS, SQLE, and CYP51 were measured by real-time quantitative PCR (qRT-PCR). [Results] The MIC of JPXT against T. rubidium was determined as 1/8 of the stock solution concentration. JPXT inhibited the spore germination and mycelial growth of T. rubidium. It increased the leakage of intracellular nucleic acid significantly after treatment for 24 and 48 h, elevated the ROS level, and decreased the ergosterol content. It did not affect the activities or mRNA levels of β-1,3-GS and CS, while it inhibited the activities and down-regulated the mRNA levels of SQLE and CYP51. [Conclusion] JPXT may inhibit the growth of T. rubidium by destroying the cell membrane, increasing the oxidative stress, and affecting the synthesis of intracellular substances.

Abstract:[Objective] Long non-coding RNA (lncRNA) usually functions as competing endogenous RNA (ceRNA) to play crucial regulatory roles. As no study of the function of bee lncRNA is available, the role of lncRNA in the immune response of bee is unclear. This study aims to reveal the regulatory function and mechanism of lncRNA in immune response of Apis ceranacerana larvae to Ascosphaera apis infection. Through deep sequencing and bioinformatics analysis, we have found that lncRNA targets ace-miR-4968 and involves in the response of A. c. cerana larvae to A. apis infection. [Methods] Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the expression of lncRNA13164 in larval guts of A. c. cerana after A. apis inoculation. ILoc-LncRNA was employed to predict subcellular localization of lncRNA13164. RNAhybrid, Miranda, and TargetScan were applied to predict target miRNAs of lncRNA13164 and miRNA-targeted mRNAs. PCR and RT-qPCR were performed to validate expression of lncRNA13164 and ace-miR-4968 as well as their potential binding relationship. The larvae which had been infected with A. apis were fed with dsRNA for RNAi of lncRNA13164 in larval guts, followed by determination of the silencing effect on lncRNA13164 and relative expression of ace-miR-4968 and three immune genes (stk, e3ul, and or1). [Results] The expression of lncRNA13164 was up-regulated in guts of 4-day-old larvae and significantly up-regulated in guts of 5- and 6-day-old larvae in the inoculation group as compared with that in the non-inoculation group. LncRNA13164 targeted 15 miRNAs including ace-miR-4968, which formed a regulatory network. ace-miR-4968 putatively targeted 79 genes which were involved in 17 gene ontology (GO) terms and 85 Kyoto encyclopedia of genes and genomes (KEGG) pathways. Both lncRNA13164 and ace-miR-4968 were expressed in A. c. cerana larval gut. The expression of lncRNA13164 in guts of both 5- and 6-day-old larvae was significantly down-regulated as compared with that in the dsRNA-egfp-fed group, and silencing efficiencies were 66.05% and 56.45%, respectively. After the silencing of lncRNA13164, the expression of ace-miR-4968 was up-regulated (P<0.01) in guts of 5-day-old larvae, while expression of stk, e3ul, and or1 was down-regulated (P<0.05). [Conclusion] lncRNA13164 in A. c. cerana larval guts can be silenced through the feeding of specific dsRNA. lncRNA13164 regulates the expression of serine/threonine-protein kinase Doa isoform X4 gene stk, E3 ubiquitin-protein ligase (MYLIP) gene e3ul, and oxidation resistance protein 1 isoform X6 gnen or1 via ace-miR-4968 and further mediates the immune response of host to A. apis invasion.

Abstract:[Objective] To screen out a surfactant-producing microbial strain Bacillus subtilis SX-20 from the soil contaminated by crude oil for a long time and then extract and analyze the products of the strain. [Methods] We used cetylpyridinium chloride-brominated thymol blue combined with blood agar plate for the screening and obtained a strain capable of producing lipopeptide. The crude fermentation product of the strain was extracted by acid precipitation, methanol extraction, and rotary evaporation, and it showed good inhibitory effect on Propionibacterium acnes. Its composition was analyzed by Fourier transform infrared spectroscopy (FTIR), amino acid analysis, and liquid chromatography-mass spectrometry (LC-MS). [Results] The surfactant-producing strain screened out was identified as Bacillus subtilis and named SX-20. The product of the screened strain was a cyclic lipopeptide containing a C15 fatty acid chain and 7 amino acid residues. [Conclusion] This study provided a theoretical basis and technical route for the screening of biosurfactant-producing strains, which is conducive to the subsequent acquisition of high-yield and low-cost biosurfactants.

Abstract:As one of the essential nutrients of living organisms, phosphorus plays a key role in substance metabolism, signal transduction, and energy storage. [Objective] To explore the role of transcription factors related to phosphate metabolism in the filamentous fungus Podospora anserina and further study the regulatory mechanism of phosphorus uptake in eukaryotic microorganisms.[Methods] Two phosphorus-metabolization-related transcription factors PaPho1 and PaPho2 in P. anserina were knocked out by homologous recombination, and a double mutant ΔPaPho1ΔPaPho2 was constructed by genetic hybridization. The changes in mutant strains were analyzed by phenotypic analysis, inorganic phosphorus content determination, and acid phosphatase activity determination. The expression of phosphorous metabolism-related genes was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). [Results] The double mutant ΔPaPho1ΔPaPho2 could not grow in the medium with inorganic phosphate as the only source of phosphorus. There was no significant difference in the growth of ΔPaPho1ΔPaPho2and the wild-type strain in the medium with organic phosphate. In the medium supplemented with organic and inorganic phosphates, the inorganic phosphate content and acid phosphatase activity of ΔPaPho1ΔPaPho2were decreased by 25.0% and 61.9%, respectively, as compared with the wild-type strain. The expression level of inorganic phosphate transporter genes in ΔPaPho1ΔPaPho2 decreased significantly. [Conclusion] In P. anserine, PaPho1 and PaPho2, as transcription factors regulating phosphate metabolism signaling pathway, play a vital role in the absorption of inorganic phosphate, but they do not participate in the metabolic regulation of organic phosphate. This study provides references for the regulatory mechanism of the filamentous fungus P. anserine in the absorption of inorganic phosphate.

Abstract:[Objective] As a common type of chemical modification, nucleic acid methylation has significant biological functions. However, it also brings technical difficulties to some nucleic acid-related studies. Massive methylations on tRNAs will block reverse transcription and decrease the efficiency of real-time fluorescence quantitative PCR (RT-qPCR) and high-throughput sequencing for the determination of tRNA levels. The AlkB from Escherichia coli is a multi-functional dealkylase. It can remove methylation as well as other modifications on DNA and RNA and thus has the potential to solve the problem mentioned above.[Methods] Here we expressed the E. coli sourced AlkB in E. coli and Pichia pastoris. After purification of the protein, we measured its enzyme properties. Finally, two tRNAs represented by tRNAIle UAU were used to examine the effect of AlkB treatment on the detection performance of real-time PCR for tRNA levels. [Results] AlkB mostly presented as inclusion bodies localized in E. coli, however, it was successfully expressed in and secreted by P. pastoris. After being purified by Nickel column, the AlkB protein showed the purity above 95%. The optimum conditions of this enzyme were 25℃ and pH 6.5, at which it showed the V_max of 0.39 μmol/(L·min), K_m of 3.23μmol/L, and specific activity of 1.08 U/mg. When RNA sample was treated by AlkB, real-time PCR could detect the tRNA level more accurately. [Couclusion] AlkB could beefficiently expressed and purified in P. pastoris.By treating RNA sample with purified AlkB, the real-time PCR method is able to detect tRNA levels more accurately. Additionally, the enzyme properties of AlkB have a significant value for relevant theoretical research and application.

Abstract:[Objective] The effect of plant growth-promoting rhizobacteria (PGPR) on bacterial wilt is unstable, which poses a challenge to the application of beneficial microorganisms. Thus, developing a stable and efficient PGPR flora against bacterial wilt is the key to the biocontrol. [Methods] The 8 anti-Ralstonia solanacearum (Rs) strains of PGPR (112, 114, Ba-S, TLZZ, LX4, LX7, Ps-S, and VC110) that had been screened out by our research group and Rs were studied. Based on the composition of tobacco root exudates, the microbial restriction microsystem, microplate, green fluorescent protein labeling, quantitative PCR, rod model design, and other methods were employed to explore the mechanism of PGPR against Rs in tobacco rhizosphere. In addition, experiment on the disease-resistant and growth-promoting effects was carried out in the field. [Results] LX4, Ba-S, and LX7 can make full use of amino acids and carbohydrates in tobacco root exudates as carbon sources to inhibit the growth of Rs. LX7 and 112 suppressed Rs with all acid carbon sources, and the highest antibacterial rates were 40.12% (LX7+lactic acid) and 35.15% (112+citric acid), respectively. The basal niche breadth (Bsw) of Ps-S, 112, and VC110 was 41.9%, 41.0%, and 38.1% higher than that of Rs (Bsw=2.56), respectively. The basal niche overlap index (NOI) of Ba-S was 4.88%-61.76% significantly higher than that of any other PGPR strains, and this strain obviously competed with Rs for nutrients. The average disease index was 27.01% and the average count of Rs in tobacco rhizosphere was 1.1×10⁴ CFU/g in the treatments with the combinations of four PGPR strains, which were significantly lower than those in the treatments with one PGPR strain and two strains. The tobacco plants treated with 8 strains of PGPR were free from the wilt, with the average Rs count of 3.5×10² CFU/g. The utilization efficiency of different carbon sources by combination 32 (consisting of LX4, Ba-S, LX7 and VC110) was significantly higher than that by Rs and particularly the utilization efficiency of alcohols and carbohydrate by the combination was 1.62 and 1.41 folds that by Rs. The field effect of combination 32 against Rs was significantly stronger than that of other treatments. Especially, the Rs-controlling rate was 27.18%, 60.05%, and 54.80% higher than that of combinations 39 (Ba-S, VC110, 114, and TLZZ), 40 (LX4, LX7, VC110, and 112), and 43 (Ba-S, VC110, 114, and 112), respectively. The yield and output value in the treatment with combination 32 were the highest among all treatments, which were 67.50% and 73.53% higher than those of the control treatment, separately. [Conclusion] Via the nutrient competition or antagonistic characteristics, different PGPR strains can be fully utilized to develop PGPR flora against R. solanacearum. The diverse PGPR floras have significantly stronger ability to resist the invasion of pathogenic bacteria.

Abstract:[Objective] To identify the PilZ domain-containing receptor(s) that sense the second messenger cyclic di-GMP (c-di-GMP) produced by the diguanylate cyclase SadC in Pseudomonas aeruginosa and investigate the functions and regulatory mechanisms of the identified receptor(s). [Methods] We constructed the strains in which sadC gene was deleted or overexpressed and tested their ability to swim by using a plate-based approach. We then added sadC in multicopy in each deletion mutant of the eight PilZ domain-containing receptors and screened for the mutants with alleviated swimming repression compared to the wild-type PA14 overexpressing SadC. For the mutations screened out, single gene knockout and overexpression strategies were used to explore the function of the identified receptor(s). Furthermore, site-directed mutagenesis and genetic complementation were employed to test whether the identified receptor's role in SadC-mediated swimming repression requires its c-di-GMP-binding motif. [Results] The SadC-mediated repression of swimming motility was associated with flagellar malfunction rather than flagellum formation. Two PilZ domain-containing receptors, PilZ and FlgZ, were identified to be involved in SadC-mediated swimming repression. The deletion of gene pilZ or flgZ increased the swimming motility, while overexpression of them significantly impaired swimming. A R10A substitution in the conserved c-di-GMP-binding motif of PilZ, or a R140A substitution in FlgZ, resulted in a variant that was no longer able to repress swimming in ΔpilZ or ΔflgZ overexpressing SadC, indicating that the conserved residue required for c-di-GMP binding is critical for PilZ or FlgZ to repress swimming in response to SadC-derived c-di-GMP. [Conclusion] PilZ and FlgZ are the effector relay proteins that respond to SadC c-di-GMP signaling to mediate swimming repression in P. aeruginosa.

Abstract:[Objective] To investigate the effect of thymol and carvacrol on diarrhea, short-chain fatty acids (SCFA), and microbial composition of Tan lambs, thus to provide plant extracts-derived additives for the treatment of lamb diarrhea, and lay a theoretical basis for the treatment. [Methods] A total of ten diarrhea lambs (diarrhea had lasted 2-3 days) were randomized into diarrhea group (CON) and thymol and carvacrol group (SCAT). The lambs in the SCAT group were supplemented with a mixture of thymol and carvacrol (1:1) at 200 mg/d for 7 days. [Results] Thymol and carvacrol improved the daily weight grain, decreased the diarrhea index, diarrhea rate (P<0.05), and concentration of serum pro-inflammatory factors (P<0.05), and increased the concentration of intestinal butyric acid (P<0.05) of the diarrhea lambs. Thymol and carvonol also changed the gut microbial composition of diarrhea lambs, raised the relative abundance of Bifidobacteriaceae and Akkermansiaceae, and decreased the relative abundance of Enterobacteriaceae.[Conclusion] Thymol and carvonol can improve the relative abundance of gut probiotics and butyric acid concentration and reduce the relative abundance of pathogenic bacteria, therefore, it can effectively inhibit lamb diarrhea, and can be used as a new type of anti-antibody product for research and development.

Abstract:[Objective] The heme of host is an important iron source for pathogenic bacteria. However, excessive heme can cause damage to bacteria. Bacteria can reduce the heme toxicity through regulation, efflux, and chelation. Riemerella anatipestifer is a Gram-negative bacterium that infects ducks and other birds. The available studies have demonstrated that R. anatipestifer encodes a heme transport system to obtain heme from host hemoglobin. However, we are not clear whether this bacterium encodes heme detoxification protein or not. In this study, we analyzed the roles of B739_RS00825encoding nitric oxide synthase in heme detoxification, oxidative stress resistance, and host colonization. [Methods] We constructed the B739_RS00825-deleted strain and studied its roles in heme detoxification, oxidative stress resistance, and host colonization through establishing the growth curve and determining the survival rate under H₂O₂ stress, the lethality to ducklings, and the colonization ability in ducklings. [Results] Compared with that of R. anatipestifer CH-1, the growth of CH-1ΔB739_RS00825 in the medium supplemented with excessive heme was not affected. However, compared with CH-1Δfur, CH-1ΔfurΔB739_RS00825 showed significantly inhibited growth in the medium containing excess heme and weakened resistance to H₂O₂. The transcription of B739_RS00825 was significantly upregulated under oxidative stress and in CH-1Δfur. Compared with CH-1, CH-1ΔB739_RS00825 did not show attenuated lethality or colonization in ducklings. [Conclusion] Gene B739_RS00825 was involved in the heme detoxification and oxidative stress resistance of R. anatipestifer and it was regulated by ferric uptake regulator (Fur). However, the gene was not involved in the lethality or colonization of R. anatipestifer in ducklings.

Abstract:[Objective] Lithogenic fungi play an important role in the biological weathering of carbonatite. It is of great significance for understanding the weathering effect of fungi on rocks to investigate the community structure of fungi in weathering crust of carbonatite weathered for different time in typical karst areas of central Guizhou. [Methods] The abandoned carbonatite headstones in the south of Huaxi District in central Guizhou were selected and the weathering crust of carbonatite weathered for different time was sampled, followed by metagenomic sequencing of the samples. Moreover, statistical methods were used to analyze the structural and functional characteristics of fungal communities. [Results] A total of 1 087 fungal species were identified from 18 weathering crust samples, which belonged to 538 genera, 44 classes, and 9 phyla. The number and composition of fungal communities varied greatly among different samples. During the weathering of carbonatite, Ascomycota dominated the fungi, and the average relative abundance was >95%. The abundance showed significant decreasing trend with the weathering. According to the Shannon index and Simpson index, the diversity of fungi community in the crust decreased first, then increased, and finally reduced with the weathering. A total of 3 379 478 genes related to KEGG pathway level 3 were detected from all samples, which were mainly involved in the metabolism and transportation of materials and energy. The main microbiota related to carbon cycle, nitrogen cycle, and sulfur cycle belonged to Ascomycota, which showed a decreasing trend with the weathering. The results of redundancy analysis (RDA) suggested that ferric oxide (Fe₂O₃), total nitrogen (TN), and total phosphorus (TP) were important environmental factors affecting the community structure succession of fungi on the crust. [Conclusion] The weathering of carbonatite intensifies over time, which allows for the formation of microhabitat on rock surface, thus the material accumulation, and colonization of microorganisms, especially fungi. There were significant differences in the fungal communities of carbonatite weathered for different time. Fungal communities on carbonatite surface also change from r strategy to K strategy with the weathering.

Abstract:[ Objective] To understand the contribution of microbial community assembly in plant rhizosphere to the stability of farmland ecosystem. [ Method s] High-throughput sequencing and bioinformatics tools were employed to explore the relationship between the bacterial community in tobacco rhizosphere and soil properties in the eight major tobacco-planting ecotopes in China. [ Result s] The most abundant bacterial classes were Actinobacteria, Alphaproteobacteria, Gammaproteobacteria, and Thermoleophilia. The composition of bacterial community presented a clustering pattern according to ecotopes, and the similarity of bacterial community among samples had a significantly negative correlation with spatial distance. The co-occurrence network of bacterial interactions indicated a higher proportion of positive links than that of negative links between bacteria. The network of Wuling-Qinba mountains (WQM), Huanghuai plain (HHP), Nanling hills (NLH), and Yimeng hills (YMH) presented high modularity. Micromonospora as the network hub in NLH and HHP contributed to the stability of microbial network. Bryobacter and Arenimonas were identified as module hubs in NLH and their characteristics rather than relative abundance determined their role in stabilizing bacterial co-occurrence network. The results of redundancy analysis showed that pH, available iron (availFe), exchangeable magnesium (exchMg), and available manganese (availMn) remarkably affected the bacterial community assembly in tobacco rhizosphere. [ Conclusion ] The homogenization and habitat specificity of bacterial community assembly in tobacco rhizosphere were affected by soil pH, availFe, exchMg, and availMn. Micromonospora, Bryobacter, and Arenimonas played an important role in the bacterial community of tobacco rhizosphere.

Abstract:[Objective] Chlamydomonas nivalis thrives in polar regions and snowfields of high mountains. C. nivalis is characterized by tolerance to cold and drastic temperature fluctuation. However, the mechanism underlying this tolerance is unknown.[Methods] On the basis of physiological responses of C. nivalis to cyclic temperature fluctuation, we selected 10 sampling times for RNA sequencing. The weighted gene co-expression network analysis (WGCNA) yielded 17 modules, and we picked up 5 modules that were significantly associated with the sample treatment. Then we analyzed the differentially expressed genes under cyclic temperature fluctuation. The selected gene sets were analyzed by functional annotation. [Results] The gene expression on C. nivalis remarkably changed under cyclic temperature fluctuation. Genes coding key enzymes in fructose and mannose metabolism, starch and sucrose metabolism, glutathione metabolism, ascorbate and aldehyde acid metabolism pathways were up-regulated at 4℃. We also found that protein quality control system, photosynthesis system, DNA damage and repair system may play important roles in adaptation to temperature fluctuation. [Conclusion] This study provides important clues to unravel the molecular mechanism of C. nivalis in adaptation to temperature stress and enriches stress-resistant gene resources.

Abstract:[Objective] Cysteine is an important sulfur-containing amino acid. Biosynthesis of cysteine has recently attracted increasing attention owing to its widespread application in cosmetics, medicine, food and other industries. Development of an efficient biosensor is indispensable for the screening of increased cysteine producers among a mutation library. In this study, on the basis of comparative transcriptome analysis, we screened and characterized some promoters from Escherichia coli that show significant response to changes in the cysteine concentration. [Methods] E. coli W3110 was cultured in LB medium with different concentrations of cysteine, and genes which showed significant improvement at the transcription level were screened. Then, the promoter fragment was amplified and fused with the fluorescent reporter gene egfp to construct a promoter library. Furthermore, the fluorescence intensity of recombinant bacteria with different promoters under different cysteine concentration was determined with a multi-functional microplate analyzer. [Results] A total of 27 genes, whose transcription levels increased significantly with the rise of cysteine concentration, were screened out and identified. The promoter P_E2 with specific response to changes in cysteine concentration was singled out. Subsequently, random mutation of AAAT was carried out in the P_E2 promoter -35 spacer region and we found that mutant promoter P_E2-33 had a higher specific response to the cysteine concentration range of 1-7 g/L. [Conclusion] The obtained promoter P_E2-33 in this study is a cysteine-specific response promoter, which lays a foundation for the construction of cysteine-specific biosensor and the high-throughput screening of cysteine-producing bacteria.

Abstract:[Objective] To study the interaction between Escherichia coli O157:H7 effector protein EspF and host ANXA6 protein and its pathogenic mechanism, we constructed a stable Caco-2 cell line with the knockout of anxa6 using CRISPR/Cas9 system.[Methods] We designed and synthesized three small guide RNA (sgRNA) which can specifically recognize anxa6 gene. We constructed Lenticrisprv2-sgRNA recombinant plasmid and transfected it into 293T cells to prepare sgRNA-Cas9 lentivirus. Then we infected Caco-2 cells with lentivirus, and applied puromycin to screen the positive cells. We isolated the monoclonal cells by limiting dilution and sequenced the cells to evaluate the knock-out of gene anxa6 and the off-target effect. Western blotting was employed to detect the expression level of ANXA6, cell counting kit 8 (CCK8) assay to determine cell proliferation, and immunofluorescence to detect the distribution of tight junction protein ZO-1. [Results] The anxa6 gene in Caco-2 cell line was knocked out, and no off-target effect in the 10 predicted sites was found. The knockout of anxa6 had no significant effect on cell proliferation. ZO-1 of Caco-2 and Caco-2^anxa6‒/‒ cells displayed continuous distribution along the cell membrane, with complete structure. After transfection with EspF plasmids, the distribution of tight junction was incomplete with clear gaps and crack-like appearance. [Conclusion] We successfully constructed the Caco-2 cell line with the knock-out of anxa6. The cell line was used to preliminarily explore the role of ANXA6 protein in distribution of tight junction protein. This study provides an effective tool for exploiting the molecular mechanism of O157:H7 in mediating intestinal barrier injury through EspF-ANXA6 interaction.

Abstract:[Objective] To explore the effect nitrogen/phosphorus (N/P) ratio on growth, arsenic (As) metabolism, and microcystins (MCs) release of Microcystis aeruginosa in arsenate (As⁵⁺)-polluted water when d-glucose-6-disodium phosphate (GP) was the only phosphorus source. [Methods] The experiment was carried out on N- and P-starved algal cells in As-containing water with different N/P ratios. The algal cell density (OD₆₈₀), chlorophyll a (Chla), actual photosynthetic yield (Yield), superoxide dismutase (SOD) activity, and the content of As species and MCs were measured. Thereby, the physiological response of M. aeruginosa to As stress and the metabolic pathways of As were analyzed. [Results] The N- and P-starved algal cells can well adapt to the low N/P ratio in the case of high GP level (0.1 mg/L), and high N/P ratio in the instance of low GP level (0.02 mg/L) can significant improve the OD₆₈₀, Chla, and Yield of the algal cells in the early stage of culture. The effect of N/P ratio on SOD activity was more significant at the beginning and end of culture. After 8 days of culture, arsenite (As³⁺) changed into the dominant As species in the medium with N/P ratio of 10:0.1, which made up 78.8% of the total As (TAs) in the water, but As⁵⁺ was still the main As species in the media with other N/P ratios. Meanwhile, As⁵⁺ was the dominant As species in algal cells for different N/P ratios, and in the case of N/P ratio at 1:0.1, the proportion of organic As in TAs in algal cells was the largest. As⁵⁺ metabolism in M. aeruginosa cells was significantly affected by GP level, and the high amount of metabolized As per cell was found in the instance of high GP level (0.1 mg/L). In the case of N/P ratio of 10:0.1, As metabolism was dominated by the reduction of As⁵⁺ and the release of As³⁺, and methylation level of As was elevated in the case of low GP level (0.02 mg/L). The concentration of MCs in the medium was related to GP level and the concentration was the lowest in the case of low N/P ratio and high GP level. [Conclusion] The results are of great significance for comprehensive understanding of harmful algal blooms and scientific management of As in As-polluted water under organic phosphorus conditions.

Abstract:As a plant-derived aromatic compound, isopiperitenol has a high economic value. Little is known about the production of isopiperitenol in Escherichia coli engineering strains. [Objective] To construct the engineering strains of E. coli for the biosynthesis of natural isopiperitenol. [Methods] We modified the limonene-3-hydroxylase (LIM3H) gene PM2 of cytochrome P450 family from Mentha×piperita by truncating the N-terminal hydrophobic region or adding 17α short peptide or 2B1 short peptide of LIM3H to increase its hydrophilicity. The activity of LIM3H was detected by CO differential spectroscopy. The NADPH-cytochrome P450 reductase gene with truncated N-terminal hydrophobic region (trATR) from Arabidopsis thaliana was fused with the modified LIM3H (Modi-LIM3H) gene and the fusion gene was expressed in E. coli. The obtained fusion protein was used to catalyze the production of isopiperitenol from limonene. [Results] LIM3H expression and isopiperitenol production were detected in all the strains with the LIM3H N-terminus modified in three different ways. The LIM3H modified by addition of 17α short peptide showed the highest protein level, and the strain carrying this modified gene had the highest yield (1.94 mg/L) of isopiperitenol.[Conclusion] In this study, exogenous LIM3H and ATR were introduced into E. coli and the fusion protein catalyzed the production of isopiperitenol from limonene, which provided a new method for the production of natural isopiperitenol.

Abstract:The homologous genes of invasion-associated locus B (ialB) are conserved in Rhizobiales including Brucella spp., while little is known about this gene in Brucella. According to the limited reports, ialB of Brucella may be associated with the invasion into host cells and the adaptation to intracellular stress. [Objective] To explore the role of ialB gene in the Brucella adhering to and invading host cells and the intracellular survival of Brucella. [Methods] We constructed the ialB-deleted strain ΔialBof Brucella suis S2 by using homologous recombination method and the complemented strain CΔialB by transforming the expression plasmid containing the open reading frame of ialB into ΔialB. Subsequently, we compared the growth phenotype and stress resistance of different strains and assessed the effect of ialB deletion on the expression of genes associated with the polar cell elongation of Brucella. Additionally, we determined the effect of ialB deletion on the invasion and proliferation of Brucella in RAW264.7 murine macrophages. [Results] ΔialB and CΔialB were constructed successfully. Compared with B. suis S2, ΔialB showed suppressed growth and decreased cell viability. The deletion of ialB decreased the resistance of Brucella to acid stress, hypertonic stress, hypotonic stress, and polymyxin-B stress and increased the resistance to oxidative stress. Furthermore, the deletion changed the cell morphology of Brucella, which was manifested as shortened cell length and increased cell diameter. The mRNA levels of the genes associated with the polar cell elongation of Brucella were down-regulated in ΔialB. Compared with B. suis S2, deletion of ialB did not change the adhesion or invasion of Brucella and decreased the proliferation in RAW264.7 cells. [Conclusion] ialB is associated with the cell viability and polar growth of Brucella, playing a role in the stress resistance and proliferation of Brucella in host cells.

Abstract:[Objective] The thioredoxin family plays a key role in the oxidative stress response of the foodborne bacterial pathogen Listeria monocytogenes (LM) during environmental adaptation. Here, we studied the biological role of the thioredoxin Lmo1903 in oxidative stress tolerance.[Methods] The phylogenetic relationship and key active sites of Lmo1903 were analyzed by bioinformatics tools. The recombinant Lmo1903 protein was expressed and purified. The oxidoreductase activity of the recombination protein was determined with insulin as the substrate and the cellular localization was predicted after preparation of mouse polyclonal antibody with the recombinant protein. The oligonucleotide-directed site-specific deletion of cysteine from Lmo1903 protein was carried out to analyze the key sites of enzyme activity. The lmo1903-deleted strain Δlmo1903 was constructed by homologous recombination, and the complementation strain CΔlmo1903was constructed with the integrated complement plasmid pIMK2. The growth, motility, and oxidative stress tolerance of the strains were examined in vitro. [Results] Lmo1903 had a classical CX₁X₂C motif and was close related to the thioredoxin family member TrxA from Bacillus subtilis. It was mainly located in the cytoplasm of bacteria and possessed strong reductase activity. Cysteine was the key site for the enzymatic activity of Lmo1903. Deletion of lmo1903 did not affect bacterial growth, while it significantly weakened the tolerance to oxidative stress in Cu²⁺ stress environment. Furthermore, the deletion of this gene affected the transcriptional levels of the genes involved in bacterial flagellar formation and reduced the swimming motility. [Conclusion] The thioredoxin family member Lmo1903 exhibiting the reductase activity contributes to the oxidative stress tolerance and motility of L. monocytogenes.

Abstract:[Objective] To investigate the protective effects of Bacillus licheniformis HJ0135 on the liver index, inflammatory cytokines, antioxidant function, and related gene expression of the weaned piglets exposed to lipopolysaccharide (LPS). [Methods] A total of 24 healthy 28-day-old weaned piglets (Duroc×Landrace×Yorkshire) were randomly assigned into 4 groups:control (CON) group, 500 mg/kg B. licheniformis HJ0135 (BL) group, LPS stress (LPS) group, and LPS+500 mg/kg B. licheniformis HJ0135 (BL-LPS) group, with 6 piglets in each group. On day 28 of the experiment, the piglets in the LPS and BL-LPS groups were injected intraperitoneally with 1.5 mL of 1 mg/mL LPS, and those in the CON and BL groups with equal amounts of saline. All the piglets were sacrificed after 1.5 h. [Results] Compared with the CON group, BL treatment decreased the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), increased the total antioxidant capacity (T-AOC), the activities of catalase (CAT) and total superoxide dismutase (T-SOD), reduced the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the liver, and up-regulated the expression of CAT, superoxide dismutase 1 (SOD1), SOD2, glutathione peroxidase 1 (GSH-Px1), nuclear factor erythroid 2-related factor (Nrf2), NADPH:quinone oxidoreductase 1 (NQO1), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-𝜅B (NF-𝜅B) in the liver. Compared with the LPS group, BL-LPS treatment decreased liver index and serum AST and ALT levels, increased the levels of CAT, GSH-Px, and T-AOC, and reduced the content of malondialdehyde (MDA), interleukin-1β (IL-1β), IL-6, and TNF-α. In addition, BL-LPS treatment up-regulated the mRNA levels of CAT, SOD2, GSH-Px1, Nrf2, TLR4, MyD88, and NF-𝜅B in the liver. [Conclusion] Dietary B. licheniformis HJ0135 can activate the expression of Nrf2 and TLR4-related genes, reduce liver damage and inflammatory cytokine secretion induced by lipopolysaccharide, and improve the antioxidant capacity in weaned piglets.