[Objective] Riemerella anatipestifer is a Gram-negative bacterium infecting ducks, causing serious economic losses to the duck industry. After infection,R.anatipestifer regulates gene expression to adapt to the 42 ℃ body temperature of ducks. To identify the adaptation mechanism ofR.anatipestifer CH-1 in ducks, we sequenced and compared the transcriptomes ofR.anatipestifer CH-1 at 37 ℃ and 42 ℃. [Methods] R.anatipestifer CH-1 was cultured to the exponential growth phase at 37 ℃ and then subjected to heat stress at 37 ℃ and 42 ℃, respectively, for 1 h. The cells were then collected for the extraction of total RNA. The raw transcriptome data of the bacteria cultured at 37 ℃ and 42 ℃ were obtained by transcriptome sequencing, and differentially expressed genes (DEGs) were screened. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were carried out for the DEGs. The genednaK involved in the response to heat stress was selected for preliminary functional identification. [Results] A total of 234 DEGs were screened out, including 169 genes with up-regulated expression and 65 genes with down-regulated expression. The GO enrichment analysis showed that the DEGs were mainly enriched in the nucleotide metabolic process, glycosyl compound metabolic process, and core RNA polymerase binding transcription factor activity. The KEGG enrichment analysis indicated that the DEGs were mainly involved in oxidative phosphorylation, ribosomes, and bacterial secretion systems. The deletion ofdnaK impaired the growth ofR.anatipestifer CH-1 at 42 ℃, compared with that at 37 ℃. [Conclusion] Compared with that at 37 ℃, the growth ofR.anatipestifer CH-1 was not affected at 42 ℃. The strain up-regulated or down-regulated the expression of heat shock response proteins and other factors to cope with heat stress.

[Objective] Selenium (Se) is an essential trace element playing a critical role in maintaining the physiological metabolism of humans. Among its various forms, selenium nanoparticles (SeNPs) possess higher bioavailability and lower toxicity. The study aims to screen a probiotic strain that can efficiently synthesize SeNPs from selenite. [Methods] Lacticaseibacillus paracasei SCFF20 capable of converting sodium selenite to SeNPs was screened out from 14 strains of probiotics. The SeNPs produced byL.paracasei SCFF20 were purified, freeze-dried, and systematically characterized by scanning electron microscopy coupled with energy-dispersive X-ray (SEM-EDX), dynamic light scattering (DLS), X-ray diffractometer (XRD), Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). [Results] SEM-EDX results revealed that Se was the primary constituent of SeNPs. The synthesized SeNPs were spherical and polydisperse, with an average particle size of 500.62 nm. XRD and Raman spectroscopy confirmed that the SeNPs were amorphous. Additionally, FTIR demonstrated the presence of proteins, exopolysaccharides, and lipids coating the surface of the SeNPs. Moreover, the reduction rate of SeNPs was determined to be 91.42% by inductively coupled plasma-optical emission spectroscopy (ICP-OES). [Conclusion] The findings of this study highlight the potential ofL.paracasei SCFF20 as a probiotic strain capable of producing SeNPs. The strain can be used as a cell factory for the safe production of biogenic SeNPs as nutritional supplements and functional food.

Bacterial infection has become one of the major problems threatening public health, and the abuse of antibiotics has accelerated the development of bacterial resistance. Antimicrobial peptides have attracted extensive attention due to their broad-spectrum antibacterial activity, rapid bactericidal effect, low toxicity, and low risk of drug resistance. However, the natural structures of antimicrobial peptides indicate some limitations, such as easy degradation, instability, low permeability, and high costs, in their application. How to improve antimicrobial peptides is still a problem to be solved. From the sources and structural characteristics of antimicrobial peptides, we analyzed the spatial structures related to the antibacterial activity and corresponding antibacterial mechanisms. In addition, we summarize the existing improvement strategies of antimicrobial peptides to lay a foundation for seeking new improvement schemes. This review provides new ideas and directions for the modification and clinical application of antimicrobial peptides in the future.

[Objective] High-temperature stacking fermentation is a key process in the production of Jiang-flavor Baijiu.Kroppenstedtia is a predominant bacterial genus in the stacked fermented grains, and investigating its growth and metabolic characteristics contributes to comprehensively understanding the crucial role of stacking fermentation. [Methods] The tryptic soy broth was used to screenKroppenstedtia strains from the stacked fermented grains, and the taxonomic status of each strain was determined by morphological observation and 16S rRNA gene sequencing. Furthermore, the growth characteristics and volatile compound metabolism of each strain were investigated by solid-state fermentation experiments on sorghum at different temperatures (45 ℃ and 50 ℃) in combination with pure culture. [Results] Three strains ofKroppenstedtia were isolated from the fermented grains of Jiang-flavor Baijiu and identified asK.eburnea. The liquid culture of strainK.eburnea 1613 significantly enhanced the production of pyrazines, which was 2.66 folds of that in the control group. The volatile compounds of sorghum in solid-state fermentation predominantly consisted of alcohols and acids, the total content of which increased over the fermentation time. The fermentation at 50 ℃ promoted the accumulation of alcohols, acids, and pyrazines, and that at 45 ℃ facilitated the accumulation of esters. The primary metabolites produced by the three strains during solid-state fermentation with sorghum as the substrate were phenethyl alcohol and isovaleric acid. The fermentation withK.eburnea 1615 at 50 ℃ for 15 days produced the highest levels of phenethyl alcohol and isovaleric acid, which reached (31.17±0.14) µg/g and (16.75±0.76) µg/g, respectively. The fermentation withK.eburnea 6E22 andK.eburnea 1613 at 50 ℃ yielded the highest levels of 2, 5-dimethyl pyrazine [(1.67±0.14) µg/g] and hexanoic acid [(3.74±0.19) µg/g], respectively, after 15 days. The accumulation of aldehydes and ketones was significant in sorghum fermented at 50 ℃. The partial least squares-discriminant analysis (PLS-DA) revealed significant influences of temperature and time on the volatile compound composition of sorghum fermented by the three strains. [Conclusion] K.eburnea contributes to the production of flavor compounds in fermented grains, particularly the characteristic flavor compounds such as alcohols, acids, and pyrazines of Jiang-flavor Baijiu.

[Objective] To explore the effect and mechanism of the antimicrobial peptide CATH-B1 on extraintestinal pathogenicEscherichia coli (RS218)-induced inflammatory response in microglia. [Methods] We used RS218-infected mouse microglial BV2 cells as the inflammation modelin vitro and set three groups: Mock, RS218 infection, and CATH-B1 pretreatment+RS218 infection. The Cell Counting Kit-8 (CCK-8) was used to determine cell viability. The colony counting assay was used to examine the growth, adhesion, and invasion of bacterial cells. Enzyme linked immunosorbent assay (ELISA) was employed to determine the concentrations of interleukin (IL)-1β, IL-6, IL-12, and tumor necrosis factor (TNF)-α in the supernatant of cell culture. Quantitative real-time PCR (RT-PCR) was performed to determine the mRNA levels of IL-1β and IL-6. Western blotting was employed to determine the protein levels of nuclear factor-kappa B (NF-κB) P65, the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), and their phosphorylated forms. [Results] CATH-B1 inhibited the RS218-induced secretion of IL-1β, IL-6, and IL-12 and mRNA expression of IL-1β and IL-6. However, CATH-B1 did not affect bacterial adhesion or invasion. In addition, CATH-B1 inhibited the expression of phosphorylated P65 and ERK. [Conclusion] CATH-B1 plays a vital role in reducing inflammation by inhibiting the activation of NF-κB and MAPK signaling pathways. The finding provides a basis for elucidating the mechanism of antimicrobial peptides against neuroinflammation.

[Objective] Soft rot is the main disease ofAmorphophallus konjac K. Koch in konjac production areas. Crop failure can be caused by the wide spreading of soft rot, because the serious destructiveness of the disease can not be effectively prevented and controlled at present. The occurrence and explosive spreading of soft rot are closely related to the microflora and pathogenic bacteria in konjac. This study explored the main pathogenic bacteria and dominant microbial species in rotten corms and rhizosphere soil, and analyzed the structural characteristics of microflora in the samples infected by soft rot in two main konjac production areas of Yunnan, aiming to provide theoretical support for the prevention and control of soft rot in konjac production. [Methods] The konjac samples infected by soft rot were collected from Fuyuan and Yongping in Yunnan. The Illumina NovaSeq 6000 platform was used for metagenomic sequencing, and the sequencing data were analyzed. Meanwhile, the pathogenic bacteria and dominant microorganisms in the rotten corms were isolated by multistage purification using selective medium, and observed by an electron microscop. [Results] Large quantities of microorganisms were detected in the rotten corms of diseased konjac from the two main production areas, including 15 721 species that belonging to 2 502 genera of 107 phyla.Pectobacteriumcarotovorum was the main pathogen in all of the diseased samples infected by soft rot. The main characteristic feature of microflora in rotten corms was that the abundances of pathogen andStenotrophomonas maltophilia were higher than all the other microorganisms. The composition of microflora present great differences between diseased tissue samples and soil samples, in the same production area, whereas, the microflora composition in the diseased tissue samples or soil samples had slight differences between the two production areas. [Conclusion] The microflora composition showed low correlations between the diseased corm and rhizosphere soil, in the two production areas. The regional difference of microflora in soil was larger than that in the diseased tissue. The results showed that the dominant pathogens and saprophytes played a key role in forming the microflora structure by breaking through the regional impact, and resulted in the microbial ecosystems in the konjac corms infected by soft rot were highly similar in two main production areas.

Arbuscular mycorrhizal fungi play important roles in ecosystems and plant growth. The physiological and ecological roles of arbuscular mycorrhizal fungi, especially in promoting plant growth, have attracted much attention. The colonization characteristics of arbuscular mycorrhizal fungi, as vital indicators of the symbiotic relationship between arbuscular mycorrhizal fungi and plants, are key parameters to evaluate the ecological adaptability of plants. However, systematic and comprehensive studies remain to be carried out regarding the colonization characteristics of arbuscular mycorrhizal fungi in the plants growing in China. [Objective] This study explored the colonization parameters of arbuscular mycorrhizal fungi in the plants growing in China, as well as their distribution in different ecosystems and climate regions, aiming to provide fundamental data to advance the research on mycorrhizae. [Methods] A database of arbuscular mycorrhizal fungi colonizing plants growing in China was established with the data from FungalRoot and 605 Chinese papers. Furthermore, the database was supplemented with 47 700 data sets including mycorrhizal colonization characteristics, ecosystem types, climate regions, plant types, and plant growth years. The analysis in this study was then performed based on this database. [Results] In China, 69.7% and 30.3% plants exhibited the arbuscular mycorrhizal fungal colonization rates ranging from 0 to 55% and from 55% to 100%, respectively. The majority of plants showed the arbuscular mycorrhizal fungal colonization intensity, hyphal abundance, vesicular abundance, and arbuscular abundance below 40.0%. Among the symbiotic forms of arbuscular mycorrhizal fungi, type A was the most prevalent, existing in 56.3% plants. Arbuscular mycorrhizal fungi demonstrated similar colonization rates in farmland, desert, and grassland ecosystems (51.8%, 51.6%, and 51.8%, respectively) and a low colonization rate (40.4%) in forest ecosystems. The plants in desert ecosystems showcased the highest arbuscular mycorrhizal fungal colonization intensity, hyphal abundance, vesicular abundance, and arbuscular abundance, which reached 46.0%, 47.1%, 37.2%, and 31.2%, respectively. In different climate regions, the colonization level followed the trend of warm temperate zone (53.3%) > tropical zone (50.0%) > temperate zone (45.2%) > subtropical zone (42.2%). The colonization level was higher in herbaceous plants than in woody plants, and higher in perennial plants than in annual plants. Among woody plants, shrubs had the highest colonization rate at 46.3%, followed by trees and vines, both at 43.9%. Herbaceous plants had higher colonization intensity (30.2%) and hyphal abundance (32.5%) than woody plants, while woody plants had higher vesicular abundance (19.5%) and arbuscular abundance (23.4%). Among the plants colonized by arbuscular mycorrhizal fungi, angiosperms accounted for the majority (90.2%, belonging to 110 families), while ferns, gymnosperms, and lycophytes were less common. [Conclusion] In China, arbuscular mycorrhizal fungi showed the colonization rate below 55% and colonization indicators below 40.0% in most plants. Different ecosystems, climate regions, plant types, and plant growth years affected the colonization status to different degrees.

[Objective] As an important coenzyme in human body, coenzyme Ⅰ (nicotinamide adenine dinucleotide, NAD⁺) plays an important role in maintaining cell growth, differentiation, and energy metabolism and protecting cells. Reduced nicotinamide mononucleotide (NMNH), an effective NAD⁺ enhancer, can efficiently elevate the levels of NAD⁺ in tissues. NADH pyrophosphatase can transform reduced nicotinamide adenine dinucleotide (NADH) into NMNH to promote the regeneration of NAD⁺. The purpose of this study is to construct a NADH pyrophosphatase expression system inBacillus subtilis and realize the synthesis of NMNH by biotransformation. [Methods] NADH pyrophosphatase was successfully expressed inB.subtilis WB600 by vector screening, and promoter engineering was employed to improve the enzyme activity. Furthermore, the industrial application potential of the recombinant enzyme was further investigated by medium optimization and amplified fermentation in a 5 L fermenter. On this basis, the whole cell catalytic system was used for biotransformation to synthesize NMNH. [Results] The initial activity of NADH pyrophosphatase and the yield of NMNH were 1.70 U/mL and 135 mg/L, respectively. After promoter engineering, the enzyme activity was improved by 41%. In addition, the enzyme activity was increased to 5.02 U/mL after optimization of the culture medium and amplified fermentation in a 5 L fermenter, which was 1.09 times higher than that in a shake flask. On this basis, the whole-cell catalytic system was used for biotransformation, and the yield of NMNH reached 1.20 g/L, which was 7.75 times higher than the initial yield. [Conclusion] We built an efficient expression system of NADH pyrophosphatase inB.subtilis and realized the efficient transformation from NADH to NMNH by whole-cell catalysis, providing a new idea for the biosynthesis of NMNH.

[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) ofCorynebacteriumpseudotuberculosis and evaluate the role of this gene in the pathogenicity ofC.pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flankingcp40 ofC.pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred intoC.pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion ofcp40. The roles ofcp40 in the pathogenicity ofC.pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between thecp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WTinvitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WTinvivo. [Results] We successfully constructed thecp40-deleted strain XH02Δcp40 by using the established dual-plasmid CRISPR/Cas9 editing system. Compared with WT (XH02), XH02Δcp40 showed no obvious difference in the colony morphology or growth curve. However, the J774A.1 cells infected with XH02Δcp40 showed decreased lactate dehydrogenase (LDH) release (P=0.06) and propidium iodide (PI) staining ratio (P < 0.01) compared with those infected with XH02. The mortality of XH02Δcp40-infected mice reduced by 50% and the bacterial loads in the liver and kidney of XH02Δcp40-infected mice significantly reduced compared with those of XH02-infected mice (P < 0.001). [Conclusion] The CRISPR/Cas9 gene editing system established in this study can effectively deletecp40 ofC.pseudotuberculosis. The results confirm thatcp40 is a virulence-related gene, providing a foundation for subsequent research on the infection ofC.pseudotuberculosis based on this gene.

[Objective] The biosafety of silver nanoparticles (AgNPs) has been a subject of concern due to the narrow therapeutic window. Expanding the therapeutic window could facilitate the application of AgNPs in the treatment of multi-drug resistant bacterial infections in humans and animals. This study aimed to enhance the biosafety of AgNPs by modifying their surface with alpha-ketoglutaric acid (AKG), a crucial component of the tricarboxylic acid cycle. [Methods] Silver ion was reduced to AgNPs by rutin at room temperature, and then AgNPs were stabilized with 1 mmol/L polyvinylpyrrolidone (PVP) solution to generate PVP-AgNPs. AKG (10 mmol/L) was added to generate PVP-AgNPs@AKG. The prepared AgNPs were characterized by a full-wavelength spectrophotometer, a particle size analyzer, and a transmission electron microscope. The antibacterial activities of PVP-AgNPs and PVP-AgNPs@AKG were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill curve, and post-antibiotic effect. The cytotoxicity of the prepared AgNPs to human cervical epithelial cells (HCerEpic) was examined by the MTT assay and flow cytometry. Furthermore, the effects of the prepared AgNPs on the energy metabolism, oxidative stress, and expression of genes involved in anaerobic respiration ofEscherichia coli BW25113 were studied. [Results] The MIC and MBC of PVP-AgNPs@AKG against Gram-positive and Gram-negative bacteria were 50% or above 50% lower than those of PVP-AgNPs. PVP-AgNPs@AKG and PVP-AgNPs showed no significant difference in the cytotoxicity to HCerEpic cells. Compared with PVP-AgNPs, PVP-AgNPs@AKG at the MIC showed significantly enhanced inhibitory effect on the α-ketoglutarate dehydrogenase inEscherichia coli, increased accumulation of AKG, lowered ATP level, and elevated reactive oxygen species level. Moreover, PVP-AgNPs@AKG significantly up-regulated the expression ofsoxS and down-regulated the expression of genes involved in anaerobic respiration, such asarcA,fnr, andfdnH. [Conclusion] The findings suggested that PVP-AgNPs@AKG disrupted the energy metabolism by targeting α-ketoglutarate dehydrogenase, rending bacteria more vulnerable to oxidative damage. Modifying with AKG would be a potential method to expand the therapeutic window of AgNPs.