[Objective] To alleviate the soil microecological imbalance caused by continuous cropping and improve the quality and yield ofGynostemmapentaphyllum, we studied the effects ofStreptomycesrochei D74 and the newly developed compound microbial agent T3 on the yield, quality, and rhizosphere bacterial community composition ofG.pentaphyllum, aiming to determine the suitable microbial agent for this medicinal plant in continuous cropping and rotational cropping. [Methods] The five-point sampling method was used to determine the yield ofG.pentaphyllum per unit area in the field. The content of main active constituents including flavonoids, polysaccharides, and saponins was determined by UV-VIS spectrophotometry and high performance liquid chromatography. The 16S rRNA gene high-throughput sequencing was employed to reveal the rhizosphere bacterial community structure ofG.pentaphyllum. [Results] In the Z-zone of rotational cropping, the dry weight, total flavonoids, and total polysaccharides of the T3 group increased by 63.44%, 12.50%, and 32.90%, respectively, compared with those in the control group, and T3 outperformed D74 in improving the yield and quality. In the P-zone of continuous cropping, the dry weight, total flavonoids, and total polysaccharides of the D74 group increased by 77.41%, 112.50%, and 23.10%, respectively, compared with those in the control group, and D74 outperformed T3. The differential microorganisms enriched in the T3 group compared with the control group were beneficial microorganisms such asNovosphingobium andRhodanbacter, and those enriched in the D74 group wereBradyrhizobium andNitrospira. [Conclusion] Both T3 and D74 could optimize the microbial community structure in rhizosphere soil and improve the micro-environment for plant growth by recruiting beneficial microorganisms in the soil, thus increasing the content and yield of the active constituents ofG.pentaphyllum. D74 and T3 demonstrate better performance in the fields of continuous cropping and rotational cropping, respectively.

[Objective] To identify the diguanylate cyclase activity of the intracellular domain and its mutants of PA0847 fromPseudomonas aeruginosa and preliminarily probe into its catalytic mechanism. [Methods] Congo red plate staining was employed to verify the diguanylate cyclase activity of the intracellular domain of PA0847. PCR was employed to construct the PA0847 PAS-GGDEF domain and its single-point mutants, and the corresponding proteins were expressed and purified. Gel filtration chromatography was utilized to analyze the aggregation states of proteins in solution. The diguanylate cyclase activity of the proteins was identified byin-vitro enzymatic reactions. Based on thiazole orange fluorescence staining, the production of cyclic diguanylate monophosphate (c-di-GMP) was determined after the enzymatic reactions, and the amino acid residues closely related to the activity of diguanylate cyclase were screened. The structural models of PA0847 PAS-GGDEF and its complex with guanosine triphosphate (GTP) were obtained by structure prediction combined with molecular docking. [Results] PA0847 PAS-GGDEF primarily exerted its catalytic activity as a dimer, with the PAS domain facilitating the dimer formation and increasing the activity of the diguanylate cyclase. Mutant screening revealed a significant increase in the activity of the non-catalytic site mutant Y700A compared with the wild type at a low protein concentration (< 0.6 mg/mL). Gel filtration chromatography indicated that the heightened activity may be attributed to the enhanced GGDEF (Gly-Gly-Asp-Glu-Phe) dimerization driven by Y700A. Structural modeling revealed that PA0847 PAS-GGDEF had a conserved GTP binding site, in which the amino side chain of K722 played an important role in binding to the phosphoryl group of GTP. The aromatic ring of Y700 engaged in a hydrophobic interaction with the alpha-helix containing K722. Therefore, the mutation Y700A may alter the spatial orientation of the K722-containing helix, which promoted the binding of K722 to the substrate GTP and the dimerization of GGDEF. [Conclusion] The non-catalytic site Y700 of PA0847 fromPseudomonas aeruginosa can indirectly regulate the diguanylate cyclase activity.

[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.

[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] 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.

Nattokinase has a variety of physiological functions and serves the treatment of cardiovascular diseases. Menaquinone-7, one of indispensable fat-soluble vitamins in the human body, can prevent diseases such as osteoporosis and Parkinson's disease. [Objective] To enhance the co-production of nattokinase and menaquinone-7 byBacillus subtilis, reveal the co-production mechanism in the recombinant strain, and provide new metabolic engineering strategies for the production of nattokinase and menaquinone-7. [Methods] We constructedB.subtilis 168-ΔbdhA by knocking out the 2,3-butanediol dehydrogenase genebdhA fromB.subtilis 168. RNA-seq was employed to measure the expression changes of key enzyme-coding genes in the nattokinase and menaquinone-7 synthesis pathways. [Results] Compared withB.subtilis 168, the content of 2,3-butanediol inB.subtilis 168-ΔbdhA was 2.76 g/L, which was reduced by 64.0%. The yields of nattokinase and menaquinone-7 were increased by 30.0% and 60.0%, respectively. The expression levels of genes related to central carbon metabolism, oxidative phosphorylation, and the synthesis of nattokinase and menaquinone-7 changed by RNA-seq analysis. The expression level of nattokinase negative regulator genecodY was down-regulated by 2.19-fold in the mutant. The expression ofsecA,tatAD, andtatC involved in protein secretion showed the down-regulation of 0.37-fold, up-regulation of 2.81-fold, and up-regulation of 0.50-fold, respectively. [Conclusion] The knockout ofbdhA blocked the carbon flux of 2,3-butanediol and promoted glycerol uptake, causing more carbon fluxing to the synthesis pathways of nattokinase and menaquinone-7. The down-regulation of the negative regulatorcodY promoted the transcription of nattokinase. The up- and down-regulation of genes involved in protein scretion promoted extracellular secretion of menaquinone-7.

[Objective] Riemerella anatipestifer (RA) is a Gram-negative pathogen that can cause duck serositis. The type Ⅸ secretion system (T9SS) of this bacterium is involved in sliding and pathogenic processes. The previous study showed that the expression ofB739_0093 inR.anatipestifer CH-1 was significantly up-regulated under the iron-limited condition. The sequence analysis showed that the protein encoded byB739_0093 contained a conserved C-terminal domain of the proteins secreted by T9SS, while its function remained unknown. This study aims to identify whether the protein encoded byB739_0093 is secreted by T9SS and the role of this protein in the pathogenesis of this bacterium. [Methods] qPCR was conducted to determine whether the transcription ofB739_0093 was regulated by iron and the ferric uptake regulator (Fur). The recombinant truncated B739_0093 protein was expressed inEscherichia coli, and the polyclonal antibody was prepared. Western blotting was employed to detect whether the protein was secreted by T9SS. Furthermore, we deletedB739_0093 from RA CH-1 and identified the role ofB739_0093 in the pathogenicity ofR.anatipestifer by virulence and colonization tests in ducklings. [Results] The expression ofB739_0093 was significantly up-regulated in the iron-restricted medium, which was mediated by Fur. Western blotting results showed that the protein encoded byB739_0093 was localized in the secretion of the parent strain RA CH-1, while it was localized in the bacterial lysate and not detected in the secretion of the T9SS-deleted strain. Compared with the parent strain RA CH-1, RA CH-1ΔB739_0093 demonstrated attenuated pathogenicity and reduced colonization ability in various tissues and organs of ducklings. [Conclusion] The protein encoded byB739_0093 is secreted by T9SS and involved in the pathogenicity ofR.anatipestifer, and its expression is regulated by iron and Fur.

[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] 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.

[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.