Abstract:Ion transporters play an important role in maintaining intracellular pH homeostasis and ionic equilibrium. Sodium ion transporters and potassium ion transporters exist widely in halophilic and halotolerant microorganisms, and their function of retaining potassium and excreting sodium is one of the two major strategies for microbial tolerance to salt stress. In recent years, new sodium and potassium ion transporters, such as RDD, UPF0118, DUF, and KimA, have been discovered in halophilic and halotolerant microorganisms. The transporters of other metal ions, such as Fe³⁺ and Mg²⁺, have been proved to play a role in microbial osmoregulation by participating in the synthesis of intracellular compatible solutes. This paper reviews the ion transporters associated with salt stress tolerance in halophilic and halotolerant microorganisms, analyzes their molecular structures and working mechanisms, and prospects for their applications in agriculture. Discovering new ion transporters, revealing the structures and mechanisms of ion transporters associated with salt stress tolerance, and analyzing the synergistic effect of coexisting transporter systems and their regulation mechanisms will deepen the understanding of the regulatory mechanisms of salt stress tolerance of halophilic and halotolerant microorganisms and provide new ideas for the improvement of crops in saline-alkali land.

Abstract:Metals like iron, copper, zinc, and manganese are trace elements essential for the survival and growth of diverse organisms. They influence the protease activity, immune response, physiological processes, and anti-infection mechanism in organisms. During bacterial infection, the host can limit or increase the availability of metal ions in the internal environment to inhibit bacterial proliferation. Meanwhile, bacteria have evolved various transport systems to adapt to the changes in metal ion levels in the host. The metal ion efflux systems exhibit distinctive efflux patterns due to variations in the structural and biochemical properties. We reviewed the available articles and our own research findings about the bacterial efflux systems of iron, copper, zinc, and manganese ions, aiming to provide an overview of the progress in the research on the regulatory mechanisms governing bacterial metal homeostasis. This review of metal ion efflux systems across different bacteria highlights the adaptation that enables bacterial survival in diverse host environments.

Abstract:Antibiotics are secondary metabolites produced by microorganisms during the stationary phase. They are widely used in the clinical treatment of bacterial infections because of their ability to kill bacteria or inhibit bacterial growth. In the long-term evolutionary process, bacteria have adopted several strategies to cope with the threats of antibiotics in the environment. In addition to the well-known antibiotic resistance, bacteria can develop tolerance and persistence to antibiotics, which seriously affects the clinical efficacy of antibiotics. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (herein collectively referred to as ppGpp) are the alarmone signal molecules produced by bacteria in response to unfavorable environmental conditions such as nutritional starvation. ppGpp can regulate transcription globally and enable bacteria to survive in unfavorable conditions. An increasing number of studies have shown that ppGpp is closely related to antibiotic stress response. On this basis, this review summarizes the synthesis, hydrolysis, and mechanism of action of ppGpp in bacteria, with emphasis on the role of ppGpp in antibiotic stress response. This review aims to provide new ideas for the development of novel antibiotics.

Abstract:The non-biodegradable nature of heavy metals (HMs) results in their long-term presence in the environment, leading to severe environmental pollution and posing a threat to human health and ecosystems. Compared with physical and chemical remediation techniques, microbial remediation is praised for the low cost, environmental friendliness, and high efficiency. When facing heavy metal stress or nutrient imbalance, microorganisms are stimulated to produce and secrete extracellular polysaccharides (EPSs). Therefore, the production of EPSs is regarded as one of the important strategies employed by microorganisms to combat HM stress. EPSs not only protect microorganisms in extreme conditions such as low temperature, high temperature, high salinity, or exposure to toxic compounds but also facilitate the communication and transfer of information and substances both inside and outside the cells. EPSs serve as a protective barrier to restrict the entry of HM ions into the cells and as a medium for communication. EPSs contain multiple negatively charged functional groups capable of complexing with HM ions, undergoing ion exchange, and participating in redox reactions, thereby reducing the bioavailability and toxicity of HMs. Microbial EPSs play a significant role in the remediation of HM-contaminated environments. However, there is currently a lack of a systematic review on the synthesis process of microbial EPSs, the mechanisms of the interaction of EPSs with HMs, and the application status of EPSs in the environments with HM stress. This article provides an overview of microbial EPSs and their classification, elaborates on the intracellular and extracellular biosynthesis mechanisms of bacterial EPSs, explores the interactions between microbial EPSs and HMs, and discusses research advances in the use of microbial EPSs for the remediation of HM pollution in water and soil environments. Finally, it looks ahead to the synthesis of EPSs and the role of EPSs in HM remediation, offering support for the further application of microbial EPSs in the remediation of environmental HM pollution.

Abstract:African swine fever (ASF) caused by African swine fever virus (ASFV) is a severe infectious disease affecting both domestic pigs and wild boar. ASFV has a large genome, and the non-structural protein pD1133L is predicted to be one of the six helicases the genome encodes. We used the IP-MASS technology to screen the host proteins interacting with pD1133L and found that vimentin (VIM) is one of the host proteins that interacted with pD1133L. However, it remains unclear how the VIM affects ASFV replication. 【Objective】 To investigate the mutual regulation between ASFV and VIM and disclose the mechanism by which VIM enhances ASFV replication. 【Methods】 We employed the Co-IP assay to examine the interaction between pD1133L and VIM. Furthermore, we examined the effects of VIM on ASFV replication by designing and synthetizing VIM siRNAs and overexpressing VIM. Western blotting and quantitative real-time PCR (qPCR) were employed to determine the impact of ASFV on the protein and mRNA levels of VIM. Western blotting and indirect immunofluorescence assay (IFA) were used to explore the changes in the phosphorylation level and subcellular localization of VIM in macrophages infected with ASFV. The CCK-8 kit was used to determine the optimal concentration of KN-93, a VIM phosphorylation inhibitor, for treatment. The effects of KN-93 on the phosphorylation and subcellular localization of VIM and the replication of ASFV were examined by Western blotting and IFA. 【Results】 The overexpression of VIM promoted the replication of ASFV, while the knockdown of VIM inhibited ASFV replication. In addition, ASFV infection down-regulated both the protein and mRNA levels of VIM in a time-dependent manner. After ASFV infection, VIM was modified by phosphorylation and changed in subcellular localization, thereby promoting ASFV replication. 【Conclusion】 This study confirms the interaction between ASFV and the host protein VIM. After ASFV infection, pD1133L leads to the rearrangement of the subcellular localization of VIM towards paranuclear aggregation, which promotes ASFV replication.

Abstract:【Objective】 A LTA4H-deleted porcine kidney cell line (PK-15) was constructed by CRISPR/Cas9 to study the effect of LTA4H on the replication of foot-and-mouth disease virus (FMDV), with a view to providing a theoretical basis for revealing the functions of LTA4H and the mechanism of regulating virus replication. 【Methods】 Two small guide RNAs (sgRNAs) targeting porcine LTA4H were designed and integrated into the vector pX459-puro-MCS, respectively. The recombinant plasmid was transfected into PK-15 cells, which were then cultured in the medium supplemented with puromycin, and the monoclonal cells were selected by limiting dilution method. Western blotting and sequencing were performed to detect LTA4H knockout, and LTA4H-deleted cells were obtained. Furthermore, Western blotting, RT-qPCR, and virus titer assay were employed to examine FMDV replication and protein expression after LTA4H knockout. 【Results】 Compared with the wild type cells, the LTA4H-deleted PK-15 cells exhibited inhibited FMDV replication. 【Conclusion】 This study successfully constructed a PK-15 cell line with LTA4H gene knockout, demonstrating that LTA4H can promote the replication of FMDV, and the results provided a theoretical basis for the subsequent research on the function of LTA4H.

Abstract:【Objective】 Since the accumulation of polyethylene terephthalate (PET) waste causes a major threat to the health of the natural environment, the degradation of PET has become a global hot issue. Enzymatic degradation of PET has garnered considerable attention because of its eco-friendly properties. However, due to the low catalytic activity, natural PET-degrading enzymes remain to be modified according to specific needs. Directed evolution enables the rapidly enhancement of the catalytic activities of PET-degrading enzymes, in which screening methods are the key for obtaining high-performance mutants. This study develops a novel, efficient, and sensitive screening method and applies it to direct modification of Thermobifida fusca cutinase Tfu-0883 to obtain the mutants with improved PET-degrading activity. 【Methods】 A mutant library constructed by error-prone PCR was coated on phospholipid plates. The mutant with improved PET-degrading activity was screened out based on the size of the hydrolytic circle. The enzymatic properties of the mutant were determined, and the rational modification sites were identified. Finally, a forward mutant was obtained. 【Results】 The single colony with the largest hydrolysis circle, mutant H10 (N2D/D94H/A149E), was selected from the phospholipid plate, with the PET-degrading activity 1.5 times that of the wild type. The mutant H10 exhibited the best performance at 60 °C and pH 8.0. The residues at positions 2 and 149 in the mutant H10 were distantly located from the substrate-binding groove, and any mutation in the residues would result in decreased enzyme stability. The residue at position 94 was situated near the substrate-binding groove, where it underwent a change from negatively charged Asp to positively charged His. This alteration facilitated adsorption onto the negatively charged PET surface and played a crucial role in enhancing the degradation ability of mutant H10. With the wild type as a template, the 94th amino acid residue was mutated to His, Lys, and Arg, which possess positive charges but exhibit reduced steric hindrance. The mutants D94H, D94K, and D94R all exhibited enhanced PET-degrading ability. Notably, among these mutants, D94K demonstrated a 3.6-fold higher rate of PET degradation than the wild type. 【Conclusion】 We developed a method for screening PET-degrading enzymes based on the phospholipase cycle and obtained the mutants with enhanced PET-degrading activity. The 94th residue of the cutinase Tfu-0883 is demonstrated as the first to possess the potential for enhancing the PET-degrading activity.

Abstract:【Objective】 Glucanases serve as one of the main components in feed additives. This study identified and characterized a novel GH9 glucanase gene derived from rumen microbiota in herbivores, aiming to provide a reference for the research and development of feed enzymes. 【Methods】 We obtained the IDSGLUC9-25 gene from the rumen fluid cDNA of Hu sheep and heterologously expressed it in Escherichia coli. The recombinant protein was induced for expression by isopropyl β-D-thiogalactopyranoside, purified, and then subjected to functional characterization. 【Results】 IDSGLUC9-25 encoded a protein consisting of 527 amino acid residues, which included a CelD_N domain and a GH9 family catalytic domain. The recombinant rIDSGLUC9-25 protein exhibited a molecular weight of approximately 62.7 kDa and the highest enzymatic activity at 40 °C and pH 6.0. The enzyme displayed robust catalytic activity within the temperature range of 30–50 °C. After preincubation at pH 4.0–8.0 for 1 h, rIDSGLUC9-25 retained the relative activity over 90%. The substrate spectrum analysis revealed that rIDSGLUC9-25 exhibited specific activities against barley β-glucan, moss lichenan, konjac gum, and xyloglucan, with the activities of (443.55±24.48), (65.56±5.98), (122.37±2.85), and (159.16±7.73) U/mg, respectively. The hydrolysis assay showed that rIDSGLUC9-25 primarily catalyzed the hydrolysis of β-glucan into cellotriose (representing 64.19%±1.19% of total reducing sugars) and cellotetraose (representing 26.24%±0.12% of total reducing sugars). Additionally, the enzyme predominantly generated cellotriose from the hydrolysis of lichenan (representing 78.46%±0.89% of total reducing sugars). 【Conclusion】 This study characterizes IDSGLUC9-25, an endo-β-1,4-glucanase (EC 3.2.1.4) derived from Treponema sp. The enzyme exhibited robust activity in the conversion of polysaccharides into cellotriose and cellotetraose, establishing a foundation for the development of feed enzymes and functional oligosaccharides preparation.

Abstract:【Objective】 To construct a chromosome-plasmid balanced lethal system based on the glutamate racemase (MurI) gene for the expression of exogenous antigens in the attenuated vaccine strain of Pseudomonas plecoglossicida (Pp ΔtssD-1), so as to provide new ideas and methods for the development of multi-component live vaccines. 【Methods】 We constructed a murI-deleted strain from Pp ΔtssD-1 by homologous recombination. First, we replaced the kanamycin resistance gene of the pBBR1MCS-2 plasmid with murI to construct a balanced lethal plasmid. Subsequently, we inserted the green fluorescent protein gene into the multicloning site of the plasmid to examine the expression stability of the exogenous antigen. Finally, we characterized the recombinant strain in terms of the growth curve, plasmid stability, and expression of the exogenous antigen. 【Results】 The murI-deleted strain was unable to grow in the lysogeny broth medium without D-glutamate. The non-resistant complemented strain regained growth capability in the lysogeny broth medium without D-glutamate. However, its growth was slower than that of the starting strain. Exogenously introduced antigens were identified as stable in the absence of antibiotic selection, and distinct green fluorescence signals were observed under a fluorescence microscope. Additionally, the balanced lethal plasmid exhibited high genetic stability within the recombinant strain. 【Conclusion】 A novel chromosome-plasmid balanced lethal system targeting murI was developed in this study. It enabled the expression of exogenous antigens in Pp ΔtssD-1 without the need for antibiotic selection. This system provides a new method for the development of multi-component live vaccines, with no need of antibiotic resistance markers and high plasmid stability.

Abstract:Rhizobia would encounter oxidative stress of reactive oxygen species (ROS) in the process of infecting leguminous plants. Methionine-containing proteins are easy to be oxidized to methionine sulfoxide, leading to changes in protein structure and function. Methionine sulfoxide reductases (Msrs) can reduce methionine sulfoxide to methionine, restoring protein structure and function. We have identified four Msrs in the genome of Mesorhizobium huakuii 7653R that are involved in oxidative stress response, while the mechanism remains unclear. 【Objective】 To identify the substrates of four Msrs and elucidate the roles of the four Msrs in M. huakuii 7653R. 【Methods】 According to the methionine content, we determined the distribution of all the proteins in M. huakuii 7653R. Then, we used the online protein interaction prediction tools to predict the substrates of the four Msrs, and performed gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment for the predicted substrates. Finally, we verified the interaction between them by using the bacterial two-hybrid system. 【Results】 The methionine content followed the normal distribution, with most proteins in the middle and few proteins on both sides. Six antioxidant enzymes and six transcription factors were selected as the candidate substrates of the four Msrs. Finally, the bacterial two hybrid results showed that two antioxidant enzymes and five transcription factors can interact with the four Msrs to different degrees. 【Conclusion】 We provided the proof in illustrating the role of Msrs in the oxidative stress response to M. huakuii 7653R and provided a new idea for the research on the mechanism of rhizobia in response to ROS.

Abstract:【Objective】 To isolate the pathogenic bacteria of Drosophila suzukii and study the mechanism of bacterial pathogenicity towards the host. 【Methods】 The pathogenic bacteria were isolated by the plate streaking method and identified based on the 16S rRNA gene sequences. The acidified ethanol method was employed to measure the ability of the isolate to cause browning. The pathogenicity of the isolate was examined by oral infection. Dihydroethidium (DHE) was used to determine the level of reactive oxygen species (ROS), and brilliant blue staining was used to examine the intestinal permeability. 【Results】 A strain was isolated from the brown culture medium of D. suzukii and identified as Gluconobacter oxydans. The strain reduced the survival rate of D. suzukii to 49.41% on day 8 (P<0.001). Furthermore, it weakened the tolerance of D. suzukii to desiccation and starvation, reducing the survival rate to 58% and 50.9% at the time points of 24 h and 32 h, respectively (P<0.001). Moreover, the flies treated with G. oxydans displayed impaired integrity of the intestine and had higher level of ROS in the guts than the control group (P<0.001). G. oxydans robustly reduced the medium pH (pH 2.0), which compromised the survival rate of D. suzukii and the growth of Lactobacillus plantarum, a probiotic of D. suzukii. 【Conclusion】 G. oxydans was a potent pathogen capable of reducing the survival rate of D. suzukii by lowering the medium pH and inhibiting the growth of probiotics, demonstrating the potential of serving the biocontrol of D. suzukii.

Abstract:Methanotrophs can utilize methane as the only carbon source and energy, and they can survive and participate in material circulation and energy flow in ecosystems. 【Objective】 To unveil the structure and functions of methanotrophs community in the sediments from the Aha Lake Reservoir (referred to as the Reservoir), a typical karst lake reservoir in Guiyang City, Guizhou Province. 【Methods】 We used metagenomics to analyze the sediments collected from the edge and the center of the Reservoir. 【Results】 The dominant aerobic methanotrophs were Methylobacter (0.37%) and Methylomonas (0.12%), and the dominant anaerobic methanotrophs were Candidatus_Methylomirabilis (0.12%), being NC10 denitrifying anaerobic methanotrophs. The gene pmoA encoding particulate methane monooxygenase of aerobic methanotrophs had the relative abundance of 6.16×10⁷ copies/g and the 16S rRNA gene had the relative abundance of 2.84×10⁷ copies/g in denitrifying anaerobic methanotrophs. The diversity of four metabolic functional genes followed a trend of nitrogen metabolism>carbon metabolism>sulfur metabolism>methane metabolism. Kyoto encyclopedia of genes and genomes (KEGG) annotation revealed six functions and 18 complete pathways involving carbon (including methane), nitrogen, and sulfur metabolism. The results of principal coordinate analysis (PCoA) showed huge discrepancies in the distribution and functions of methanotrophs between sediments from the edge and the center of the Reservoir. Moreover, redox potential, conductivity, and sulfate were primary environmental factors affecting methanotroph distribution. 【Conclusion】 Type I aerobic methanotrophs dominated the Reservoir with abundant metabolic pathways. Types I and II methanotrophs exhibited huge discrepancies in terms of their adaptability to O₂. All these fundings are expected to provide theoretical support for lake water environment conservation and microbial utilizationation.

Abstract:【Objective】 Antibiotics as emerging pollutants have aroused wide concern. In view of the shortage of effective tetracycline-degrading strains, this study aims to screen and identify the strains for tetracycline degradation, analyze degradation properties and type, pinpoint the localization of active substances for bio-degradation, and evaluate the physiological toxicity of degradation products. 【Methods】 Tetracycline was used as the sole carbon source to screen out the target strain from tetracycline-contaminated pig sludge. The strain was identified based on colony morphology, physiological and biochemical characteristics, scanning electron microscopy images, and the 16S rRNA gene sequence. Different carbon sources, pH, and removal kinetics were employed to characterize the degradation process of the strain. Different components of the strain were extracted to determine the degradation type of tetracycline by the strain. Furthermore, the intracellular and extracellular fluids of the strain were used to degrade tetracycline, so as to determine the location of the active substance for degradation. Finally, the toxicity of the degradation products was assessed. 【Results】 The strain MEH2305 was screened out and identified as Enterobacter hormaechei, which showed the best degradation performance at pH 7.0 and with tryptone as the carbon source. Strain MEH2305 showed a total tetracycline removal rate of 68% on the 7th day of culture via abiotic degradation and bio-degradation, and the removal rates of oxytetracycline and doxycycline hydrochloride were 53% and 56%, respectively. The tetracycline removal efficiency by the intracellular and extracellular fluids of MEH2305 was 40.77% and 31.18%, respectively. Compared with tetracycline control without MEH2305, the tetracycline degradation products of MEH2305 had reduced physiological toxicity on Gram-negative Escherichia coli K88 and Gram-positive Bacillus subtilis 168. 【Conclusion】 The strain MEH2305 can be used as an effective and safe tetracycline-degrading strain for the treatment of antibiotics in the environment.

Abstract:【Objective】 To search for the strains with improved tolerance, probiotic properties, and safety, we isolated Lactobacillus plantarum HY21 from the intestinal contents of Apostichopus japonicus and evaluated the probiotic potential of the strain. 【Methods】 The shake flask fermentation, environmental factor simulation test, and antibiotic susceptibility test were carried out to study the growth characteristics and acid production of the strain, as well as the antioxidant capacity and aquatic pathogen-inhibiting effects of the fermentation broth. In addition, L. plantarum HY21 was characterized in terms of the tolerance to environmental factors, hydrophobicity, auto-agglutination and co-aggregation, antibiotic susceptibility, and intestinal adhesion ability. 【Results】 L. plantarum HY21 in shake flask culture with the initial pH 8.0 entered the logarithmic growth during the period of 2–10 h. The culture showed a strong acid-producing ability with the minimum pH 3.6 at the time point of 18 h. The fermentation broth of the strain showed an inhibition zone diameter of (13.96±0.30) mm against Vibrio alginolyticus and a 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging rate of 95.45%±1.56%. L. plantarum HY21 had a hydrophobicity rate of 60.42%±2.78%, an auto-agglutination rate of 22.69%±1.36% and a co-aggregation rate of 27.98%±1.45% with V. alginolyticus. Furthermore, the strain showed the adhesion number of (1.66±0.01)×10⁶ CFU/mL and (1.23±0.15)×10⁶ CFU/mL to fish body surface and intestinal mucus protein, respectively. L. plantarum HY21 was susceptible to 13 common antibiotics. The survival rate of L. plantarum HY21 reached 99% and above in simulated gastrointestinal fluid (pH 3.0, pH 6.8) within 3 h, 68.11%±7.98% in 10% fish bile fluid within 3 h, and 97.58%±7.14% in 3% sea salt solutions within 16 h. 【Conclusion】 L. plantarum HY21 is a probiotic strain with strong colonization potential, antimicrobial activity, and antioxidant capacity, demonstrating a high survival rate and high safety in the applied environment. The findings provide a theoretical basis for the development of new probiotic strains as probiotic additives for aquaculture.

Abstract:【Objective】 To explore the mechanism of Neorhizobium petrolearium OS53 combined with alfalfa (Medicago sativa L.) in the remediation of petroleum-contaminated soil. 【Methods】 Illumina and Nanopore were employed to sequence the whole genome of N. petrolearium OS53, and the complete genome map of the strain was constructed. Gene prediction and functional annotation were carried out to analyze the genes involved in nodulation and oil degradation. The abilities of strain OS53 to produce indole acetic acid (IAA), secrete siderophore, and solubilize phosphorus and potassium were tested. The activities of urease, dehydrogenase, polyphenol oxidase, and lipase in soil and the levels of chlorophyll, malondialdehyde, proline, soluble protein, soluble sugar, and superoxide dismutase in alfalfa were measured by kits. 【Results】 The genome of strain OS53 consisted of a circular chromosome of 5.56 Mb and two plasmids of 0.92 Mb and 0.38 Mb, respectively, with the G+C content of 60.2%. The genome encoded a total of 6 968 genes. The strain OS53 and N. petrolearium DSM 26482^T showed the 16S rRNA gene sequence similarity of 99.86%, and formed stable branches on the phylogenetic tree, indicating that strain OS53 and N. petrolearium were the same species. Therefore, OS53 was named as N. petrolearium OS53. The strain OS53 had the ability to produce IAA, and the related genes were identified in the genome. After 120 days of remediation of the soil with the initial oil content of (4 403.30±222.10) mg/kg, OS53 and alfalfa showed the remediation efficiency up to 57.53%, which was 44.26%, 41.69%, and 8.84% higher than that of no inoculation of strain OS53, inoculation of OS53 only, and planting alfalfa only, respectively. In the combined remediation system, alfalfa showed elevated the levels of chlorophyll, soluble protein, and soluble sugar and lowered levels of malondialdehyde, proline, and superoxide dismutase, and the soil showed increased activities of polyphenol oxidase, dehydrogenase, lipase, and urease. 【Conclusion】 The strain OS53 had the ability to produce IAA to promote the growth of alfalfa in the petroleum-contaminated soil, which increased the activity of enzymes involved in oil degradation in the soil. Finally, the combined system improved the remediation efficiency of the soil.

Abstract:[Objective] Nickel (Ni) is one of the heavy metal pollutants to which humans are widely exposed, and nickel exposure activates the cell wall integrity (CWI) signaling pathway, which lowers the level of intracellular histone acetylation. However, whether the CWI pathway is regulated by histone acetylation under nickel stress remains to be fully understood. [Methods] We used the histone-targeted mutant strain H4K5R (mimicking the deacetylated state) to study the regulation of the CWI pathway in Saccharomyces cerevisiae by H4K5 deacetylation under nickel stress, aiming to lay a foundation for unveiling the regulatory role of histone modifications in eukaryotes in response to heavy metal stress. [Results] Compared with the wildtype strain, H4K5R had strong nickel resistance, being able to grow in the presence of 5.0 mmol/L NiCl₂. The results of Western blotting and qRT-PCR showed that the CWI pathway of the wildtype strain BY4741 was activated under 5.0 mmol/L NiCl₂, with the expression of Mnn9 (encoding α-1,6-mannosyl transferase) and Fks1 (encoding glucan synthase) being up-regulated by 3.13 folds and 1.49 folds, respectively. Moreover, the content of mannan and β-glucan were increased, which indicated that the wildtype strain activated the CWI pathway to increase the content of cell wall component. The activation of the CWI pathway in H4K5R was mild under the stress of 5.0 mmol/L NiCl₂. Although the expression of Mnn9 and Fks1 was up-regulated, the changes in mannan content were not significant, and the increase in β-glucan content was less than that of the wildtype strain. [Conclusion] Under 5.0 mmol/L NiCl₂ stress, the deacetylation of H4K5 in the mutant strain regulated the CWI pathway, which affected the changes in cell wall components.

Abstract:【Objective】 Episomal expression vectors typically have higher copy number to achieve strong gene expression than chromosomal expression vectors. Moreover, they are more convenient and flexible for DNA manipulation. However, the episomal plasmids suitable for the application in Rhodosporidium toruloides remain to be determined, and the expression of heterologous genes or CRISPR/Cas9-based genome editing needs to be achieved by integration, which is a key reason for the slow progress in its genetic modification. Thus, this work aims to construct an episomal plasmid of R. toruloides, which facilitates the expression of heterologous genes and promotes the gene editing in a time-saving manner. 【Methods】 First, the possible autonomously replicating sequences (ARSs) in the phenylalanine ammonia-lyase gene (PAL) of R. toruloides were mined. Specifically, PAL and its upstream and downstream sequences were amplified in segments and constructed into a plasmid containing the β-isopropyl malate dehydrogenase gene (LEU2). The recombinant plasmids were then introduced into LEU2-deficient R. toruloides by the electroporation method. An ARS was then identified according to transformation efficiency. Then, the BTS1 gene encoding geranylgeranyl pyrophosphate synthase was selected as the knockout target, and its gRNA was constructed into the episomal plasmid based on the identified ARS. The color change of the transformant was observed to verify whether the episomal plasmid was successfully applied to the CRISPR/Cas9 system of R. toruloides. 【Results】 In this work, an ARS was identified, based on which an episomal plasmid was constructed and applied to CRISPR/Cas9 editing in R. toruloides. Finally, the episomal plasmid-based gene knockout of R. toruloides was successfully achieved. 【Conclusion】 This work enriched the existing tool library and provided a research basis and technical support for the application of R. toruloides in synthetic biology.

Abstract:【Objective】 To generate Listeria monocytogenes strains with lmo0880 deleted and complemented strains, so as to investigate the roles of Lmo0880 in bacterial infection in a host. 【Methods】 The lmo0880-deleted strain was generated by homologous recombination, and the complementary strain was constructed by introducing an integrative plasmid carrying lmo0880 into the lmo0880-deleted strain. The growth, adhesion, invasion, and intracellular proliferation were compared between the lmo0880-deleted strain, complementary strain, and the wild type. 【Results】 The deletion of lmo0880 did not significantly impact bacterial growth or adhesion. However, it led to notable decreases in cell invasion, proliferation, and colonization in the liver and spleen, ultimately diminishing the pathogenicity in mice. 【Conclusion】 The LPXTG-anchored protein Lmo0880 plays a crucial role in bacterial invasion, proliferation, and colonization in a host. These findings provide a solid foundation for deeply understanding the pathogen-host interaction during L. monocytogenes infection.

Abstract:【Objective】 This study explored the proliferation dynamics and persistent spread characteristics of Cnaphalocrocis medinalis granulovirus (CnmeGV) in Cnaphalocrocis medinalis, aiming to enrich the epidemiological knowledge about baculovirus and provide theoretical support for the efficient application of CnmeGV formulations. 【Methods】 Transcriptome sequencing was employed to determine the transcriptional levels of viral genes in the larvae infected with CnmeGV for 96 h and in the emerged adults. A quantitative detection marker based on the specific gene Cmorf123 of CnmeGV was established to measure the proliferation dynamics and persistent spread characteristics of CnmeGV in C. medinalis. 【Results】 All the genes of CnmeGV were transcribed in the infected larvae, with the acetyltransferase gene showing the highest transcription level. However, no transcription of viral gene was detected in the transcriptome of adults. The measurement with the quantitative detection marker of CnmeGV showed that the replication level of viral gene remained stable within 48 h post infection. The copies of viral genes significantly increased 2 and 4 days post infection, and each nanogram of DNA contained 83 copies of viral genes 4 days post infection. After infection, no viral transcription was detected in the adults or eggs, while a few transcripts were detected in the pupae. Viral DNA was detected in more than 86.7% of pupa and pupal slough samples and while 13.3% of adults. Viral DNA was detected in both the eggs and the second-generation larvae after infection, while no viral DNA was detected on the egg after the surface treatment. 【Conclusion】 The proliferation level of CnmeGV in C. medinalis gradually increased and then remained stable within 4 days post infection. CnmeGV particles can be spread for two generations by the egg surface. The eclosion of C. medinalis is the main pathway for clearing CnmeGV.

Abstract:Ferric uptake regulator (Fur) is a key regulatory factor of iron metabolism and virulence in Pseudomonas aeruginosa. Many research groups have failed to construct the fur-deleted mutant of P. aeruginosa, so fur has always been considered to be an essential gene in P. aeruginosa, and the knowledge of its biological function is limited. 【Objective】 This study aims to construct a fur-deleted mutant of P. aeruginosa and analyze its phenotypes. 【Methods】 With P. aeruginosa PAO1 as the parental strain, the fur-deleted mutant was constructed by homologous recombination. After that, we studied the effects of fur on the growth, siderophore biosynthesis, resistance to oxygen stress, flagella formation, biofilm formation, and virulence of P. aeruginosa. In addition, we explored the cause of the growth defect phenotype of the fur-deleted mutant by genetic analysis. 【Results】 The fur-deleted mutant of P. aeruginosa was successfully constructed. The deletion of fur greatly limited the growth of P. aeruginosa and reduced the growth adaptability of P. aeruginosa to the iron-limited environment, while it did not affect the growth adaptability of P. aeruginosa to the iron-rich environment. This growth defect phenotype of Δfur was caused by the slow cell growth and proliferation, rather than by cell death. Interestingly, heterologous fur could completely complement the growth defect phenotype of Δfur, suggesting that the Fur of P. aeruginosa was not functionally unique. Although there was a functional relationship between Fur and the toxin-antitoxin system PacTA, the growth defect phenotype of P. aeruginosa Δfur was not associated with PacT toxin. In addition to affecting the growth phenotype of P. aeruginosa, the deletion of fur also made P. aeruginosa lose the inhibitory effect on siderophore biosynthesis and the ability to form flagella and have increased sensitivity to H₂O₂ and reduced virulence to Galleria mellonella larvae. Moreover, the deletion of fur increased the intracellular cyclic diguanylate (c-di-GMP) level of P. aeruginosa to induce the expression of pelF and pslA, thereby promoting the biofilm formation of P. aeruginosa. 【Conclusion】 fur is a non-essential gene that can be deleted and plays a crucial role in the normal growth, siderophore biosynthesis, resistance to oxygen stress, flagellum formation, biofilm formation, and virulence of P. aeruginosa, which lays a foundation for the development of vaccines and agents against P. aeruginosa.

Abstract:【Objective】 To study the inhibitory effect and mechanism of an antimicrobial peptide (A2M3) derived from alpha-2-macroglobulin identified in the human nasal cavity against Staphylococcus aureus. 【Methods】 The mass spectrometry results of the human nasal liquid were analyzed, on the basis of which bioinformatic tools were used for the screening of potential antimicrobial peptides. The minimum inhibitory concentration (MIC) and time-kill curve of A2M3 against S. aureus were determined by the microdilution method and plate colony counting method. Then, transmission electron microscopy, PI uptake assay, flow cytometry, and determination of nucleic acid protein leakage were employed to study the effects of A2M3 on the membrane integrity and permeability of S. aureus. Finally, the effect of A2M3 on the genomic DNA of S. aureus was investigated by the gel retardation assay and fluorescence spectroscopy. 【Results】 A2M3 showed an MIC of 125.0 μg/mL against S. aureus and killed the bacteria completely within 3 h. A2M3 increased the cell membrane permeability to penetrate into S. aureus cells, leading to leakage of nucleic acids and proteins as well as insertion into DNA base pairs to interfere with the gene function, resulting in the death of the cells. 【Conclusion】 The inhibitory mechanism of A2M3 against S. aureus involves multiple targets. The antimicrobial peptide alters the permeability of the bacterial cell membrane and affects the gene function, thus exerting the inhibitory activity. The findings reveal that antimicrobial peptides could be screened and isolated from human body fluids for potential application.

Abstract:【Objective】 Colony extraction and counting is essential in agriculture, food, and health industries. Currently, most of the available algorithms for automatic counting of colonies use colony culture dishes and has poor applicability to colony count plates. In addition, the current technologies have good performance in conventional segmentation of adherent objects, while their accuracy remains to be improved for the segmentation and counting of adherent colonies due to the unique morphological characteristics of colonies. 【Methods】 To solve such problems, we proposed a colony segmentation and counting algorithm based on target color base and gradient direction matching. Firstly, the color feature of the colony in the image was used as a base to convert the image into a base space to enhance the difference between the colony and the background. Secondly, the gradient magnitude feature of the colony image was used to filter the gradient direction, and then the matching was performed through the gradient direction, thereby segmenting the adherent colonies. Finally, non-maximum suppression was employed to screen and count the colonies. 【Results】 Through experiments, the counting accuracy of the algorithm in this study reaches 98.00%, demonstrating its capability to meet practical requirements. 【Conclusion】 In the context of targeted segmentation and counting of colonies, the algorithm studied in this paper not only exhibits high counting accuracy but also demonstrates good robustness. This algorithm had not only high counting accuracy but also good robustness, producing excellent results in the colony segmentation and counting of colony count plates from different manufacturers. However, it showed decreased counting accuracy in the detection and segmentation of large-area targets. Therefore, this algorithm is suitable for the detection and segmentation of small targets such as colonies.