Abstract:Since the formation of a committee under the guidance of R. E. Buchanan in 1936, microbiologists have developed and refined a nomenclatural code for bacteriology, the International Code of Nomenclature of Prokaryotes (ICNP). This code has greatly facilitated recent advances in taxonomy and related fields of microbiology. Technical developments in omics have led to a surge in genome-based discoveries of novel uncultured species, many of which play important roles in environmental and medical microbiology. Nevertheless, the ICNP only recognizes cultures as nomenclatural types, thereby preventing the names of the uncultured microorganisms from being validly published. To compensate for the ineffectiveness of the ICNP in creating permanent and stable names of uncultured taxa, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode) was published in 2022. The SeqCode was formed with the intention of complementing and someday merging with the ICNP. However, as the two nomenclatural codes currently work independently, the consequences of the concurrence of both codes on the scientific community are still not clear. Here, we introduce the histories and principles of the ICNP and SeqCode, summarize their advantages and limitations, and call for scholars to both respect and utilize the two nomenclatural codes of prokaryotes, aiming to facilitate the establishment of a more reasonable and beneficial naming system.

Abstract:Botulinum neurotoxins (BoNTs), a group of the most toxic proteins, can cause muscle paralysis and even lead to death in severe cases. BoNTs can be classified into 7 serotypes (BoNT/A-BoNT/G) and further classified into more than 40 subtypes according to the differences in amino acid sequences. BoNTs consist of three basic domains: the C-terminal receptor-binding domain of the heavy chain, the N-terminal translocation domain, and the light-chain catalytic domain. On the surface of motor neurons, the receptor-binding domain binds first to polysialoganglioside and subsequently to synaptic vesicle protein 2 or synaptotagmin to form a two-receptor complex. The functioning of each serotype relies on the binding of the receptor-binding domain to the corresponding receptor. BoNTs have always been a research hotspot in terms of the structure, function, and effect on the host. The role of the receptor-binding domain in promoting the specific binding of BoNTs to motor neurons has become a new research direction. This review summarizes the structural changes of the receptor-binding domains and the differences in binding sites during the binding of different serotypes of BoNTs to receptors. By analyzing the sequences and structural characteristics of the receptor-binding domains of different serotypes and subtypes, we can fully understand the sequence differences and functions of the receptor-binding domain and give insights into the treatment of BoNTs.

Abstract:Bacterial cellulose, a natural biopolymer with higher purity and better mechanical properties than plant cellulose, is expected to be widely used as a new green polymer material. A variety of bacteria have now been proven to have the ability to produce cellulose, in which bacterial cellulose synthase plays a crucial role. Therefore, understanding the catalysis mechanism of bacterial cellulose synthase is a key to the mass production and broad utilization of bacterial cellulose. This paper reviews the basic properties of bacterial cellulose synthase, including the screening of strains, the enhancement of yield, and the cellular localization of the synthase, aiming to promote the research on the catalysis mechanism of cellulose synthase. Further, based on the mechanism of cellulose synthase, this paper detail the influencing factors of in vitro synthesis and review the research progress in the roles of each subunit of this synthesis method. We explore the catalysis mechanism of bacterial cellulose synthase, point out the problems in the current research, and envision the future research directions in this field, with a view to providing a theoretical basis for the large-scale application of bacterial cellulose by deciphering the synthesis mechanism.

Abstract:Urothelial carcinoma of bladder (UCB) is a common malignant tumor of the urinary system and a common pathological type of bladder cancer (BC). With high morbidity, high mortality, and easy recurrence, UCB poses a serious threat to human health. The occurrence of UCB may be associated with smoking and exposure to toxic chemicals. With the deepening of research, researchers have discovered unique microbiota related to the occurrence and development of UCB in the bladder. This review focuses on the involvement of microbiota in the occurrence and development of UCB through urinary tract infection (UTI), affecting epithelial-mesenchymal transition (EMT), and up-regulating the expression of programmed cell death 1 ligand 1 (PD-L1). At the same time, this paper introduces the microbiota characteristics of healthy people and UCB patients as well as the prevention and treatment of UCB. By reviewing the relationship between microbiota and UCB, this paper provides new ideas for further clarifying the promoting effect of microbiota on UCB and developing drugs for treating UCB.

Abstract:Food allergy (FA) with growing incidence has emerged as one of the public health problems around the world as it seriously affects the life quality of children. Recent studies have discovered that there are significant differences in the composition of gut microbiota between the children with FA and healthy children. In-depth studies have reported that gut microbiota can help to maintain immune balance by regulating immune cells such as dendritic cells, helper T cells, regulatory T cells, mastocytes, and granulocytes. In addition, gut microbiota can enhance the intestinal barrier function to inhibit FA in a variety of ways. On the basis of research results from animal experiments, probiotics and prebiotics have been used in the treatment of FA in children, whereas the effect is not ideal. As FA occurs in a growing number of children in the world, this article reviews some mechanisms of gut microbiota in influencing FA and summarizes the application of probiotics and prebiotics in the treatment and prevention of FA in children in recent years. Furthermore, this article proposes new ideas for deciphering the mechanism of gut microbiota in regulating FA and applying probiotics and probiotic metabolites in the treatment and prevention of FA in children, which is of great significance for promoting the research on the treatment of FA in children.

Abstract: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.

Abstract:[Objective] To clarify the promotion effects of different organic compounds on the formation of magnetosomes in Acidithiobacillus ferrooxidans BYM, so as to provide a new idea for safely and effectively improving the magnetosome yield. [Methods]Single-factor experiments were conducted to measure the effects of ten organic compounds on the ferrous oxidation of A. ferrooxidans BYM, and the organic compounds promoting the synthesis of magnetosomes were further screened by a 4 L fermentation system. The classical kinetic models (Logistic, Luedeking-Piret, and substrate consumption kinetic equations) were employed to build the kinetic models for the growth, magnetosome synthesis, and ferrous consumption of A. ferrooxidans BYM by batch fermentation experiments. [Results]The maximum magnetosome yield (2.00×10⁻³ g/L) was achieved with the addition of 10 mmol/L gluconic acid, in the presence of which the bacterial cells were oval and had a smooth surface. With the addition of gluconic acid, the fermentation kinetics of A. ferrooxidans BYM was in accordance with Logistic, Luedeking-Piret, and substrate consumption kinetic equations.[Conclusion] The addition of 10 mmol/L gluconic acid increases the magnetosome yield of A. ferrooxidans BYM by eight times. Gluconic acid changes the cell morphology and surface of A. ferrooxidans BYM. The kinetics models of cell growth, product formation, and substrate consumption can illustrate the batch fermentation of A. ferrooxidans BYM in the presence of gluconic acid.

Abstract:[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 of Escherichia 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 in Escherichia coli, increased accumulation of AKG, lowered ATP level, and elevated reactive oxygen species level. Moreover, PVP-AgNPs@AKG significantly up-regulated the expression of soxS and down-regulated the expression of genes involved in anaerobic respiration, such as arcA, fnr, and fdnH. [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.

Abstract:[Objective] Environmental fungi emerge as the principal agents causing microbial deterioration of museum collections. The varying materials and origins of collections and the different storeroom environments in a museum contribute to high diversity of environmental fungi. Additionally, the air conditioning system within a museum may instigate fungal dispersion among storerooms. Recognizing the compositional differences and seasonal variations of fungal communities in the air across varied storerooms underpins the early warning and prevention against microbial hazards in museum settings. [Methods] Sampling was carried out every two months by the impacting method in the iron and silk storerooms of a museum for one year. Fungal species and community composition were examined by ITS rDNA sequencing. [Results] The amplicon sequencing yielded a broad spectrum of fungal data, encompassing five phyla, 20 classes, and 184 families, over half of which displayed notable seasonal changes. Distinct and comparatively consistent fundamental fungal populations existed in both iron and silk storerooms, exhibiting minimal seasonal variations. However, during summer when it was humid, substantial proliferation of the fungi capable of degrading proteinaceous and cellulosic materials was observed in both storerooms, which presented a latent hazard to the collections. Furthermore, it was noted that certain fungi produced acidic by-products during growth, which could compromise the integrity of the collections. [Conclusion] This investigation provides groundwork for the mitigation and management of environmentally derived fungal threats in museum contexts, underscoring importance for the preventive conservation of museum collections.

Abstract:[Objective] A genetically engineered virus strain rGS15-△2 with deletion of the residues at the positions 519-565 and 628-747 of the non-structural protein 2 (NSP2) had been rescued based on the PRRSV/GSWW/2015 infectious clone. This study aims to construct and rescue an engineered virus strain with the deletion of three sites in NSP2 based on rGS15-△2. [Methods] Based on the infectious clones of rGS15-△2, two recombinant plasmids with the deletion of three sites were constructed by fusion PCR. Specifically, the dominant epitope at the amino acid site 323-364 or 372-433 of NSP2 was further deleted on the basis of rGS15-△2. The recombinant plasmids were linearized and mixed with liposome, which were transfected into Marc-145 cells for virus rescue. The growth characteristics of the engineered virus strains were analyzed by electron microscopy, immunofluorescence assay (IFA), virus titer determination, and growth curve establishment. [Results] The engineered virus strains rGS15-△3-1 and rGS15-△3-2 were rescued successfully. Virions with the diameter from 50 nm to 80 nm can be observed under an electron microscope. The results of IFA confirmed the expression of PRRSV N protein by the rescued virus strains and the parent strain GS15. Furthermore, the rescued viruses were cultured with Marc-145 cells for 40 passages, and the deletion regions were confirmed to be stable by RT-PCR and sequencing. The titers of rGS15-△3-1 and rGS15-△3-2 were 2.00×10^6.0 TCID₅₀/mL and 2.25×10^5.8 TCID50/mL, respectively, which had differences from that of the parent strain (P<0.05). The growth curves showed that the rescued viruses had lower replication levels than the parent strain, and they reached the peak titers 24 h later than the parent strain. [Conclusion] We characterized the growth of the viruses with the deletion of multiple sites in NSP2 of PRRSV. The findings laid a foundation for the development of novel PRRSV-labeled vaccines and provided a new strategy for the prevention and control of porcine reproductive and respiratory syndrome.

Abstract:[Objective] To alleviate the soil microecological imbalance caused by continuous cropping and improve the quality and yield of Gynostemma pentaphyllum, we studied the effects of Streptomyces rochei D74 and the newly developed compound microbial agent T3 on the yield, quality, and rhizosphere bacterial community composition of G. 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 of G. 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 of G. 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 as Novosphingobium and Rhodanbacter, and those enriched in the D74 group were Bradyrhizobium and Nitrospira. [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 of G. pentaphyllum. D74 and T3 demonstrate better performance in the fields of continuous cropping and rotational cropping, respectively.

Abstract:[Objective] To identify the diguanylate cyclase activity of the intracellular domain and its mutants of PA0847 from Pseudomonas 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 by in-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 from Pseudomonas aeruginosa can indirectly regulate the diguanylate cyclase activity.

Abstract:[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 by L. 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 of L. 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.

Abstract:[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 of R. anatipestifer CH-1 in ducks, we sequenced and compared the transcriptomes of R. 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 gene dnaK 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 of dnaK impaired the growth of R. anatipestifer CH-1 at 42 ℃, compared with that at 37 ℃. [Conclusion] Compared with that at 37 ℃, the growth of R. 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.

Abstract:[Objective] To explore the effect and mechanism of the antimicrobial peptide CATH-B1 on extraintestinal pathogenic Escherichia coli (RS218)-induced inflammatory response in microglia. [Methods] We used RS218-infected mouse microglial BV2 cells as the inflammation model in 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.

Abstract: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 by Bacillus 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 constructed B. subtilis 168-ΔbdhA by knocking out the 2,3-butanediol dehydrogenase gene bdhA from B. 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 with B. subtilis 168, the content of 2,3-butanediol in B. 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 gene codY was down-regulated by 2.19-fold in the mutant. The expression of secA, tatAD, and tatC 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 of bdhA 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 regulator codY promoted the transcription of nattokinase. The up- and down-regulation of genes involved in protein scretion promoted extracellular secretion of menaquinone-7.

Abstract:[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 of B739_0093 in R. anatipestifer CH-1 was significantly up-regulated under the iron-limited condition. The sequence analysis showed that the protein encoded by B739_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 by B739_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 of B739_0093 was regulated by iron and the ferric uptake regulator (Fur). The recombinant truncated B739_0093 protein was expressed in Escherichia coli, and the polyclonal antibody was prepared. Western blotting was employed to detect whether the protein was secreted by T9SS. Furthermore, we deleted B739_0093 from RA CH-1 and identified the role of B739_0093 in the pathogenicity of R. anatipestifer by virulence and colonization tests in ducklings. [Results] The expression of B739_0093 was significantly up-regulated in the iron-restricted medium, which was mediated by Fur. Western blotting results showed that the protein encoded by B739_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 by B739_0093 is secreted by T9SS and involved in the pathogenicity of R. anatipestifer, and its expression is regulated by iron and Fur

Abstract:[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 in Bacillus subtilis and realize the synthesis of NMNH by biotransformation. [Methods] NADH pyrophosphatase was successfully expressed in B. 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 in B. subtilis and realized the efficient transformation from NADH to NMNH by whole-cell catalysis, providing a new idea for the biosynthesis of NMNH.

Abstract:[Objective] Soft rot is the main disease of Amorphophallus 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. Pectobacterium carotovorum 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 and Stenotrophomonas 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.

Abstract:[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) of Corynebacterium pseudotuberculosis and evaluate the role of this gene in the pathogenicity of C. pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flanking cp40 of C. pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred into C. pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion of cp40. The roles of cp40 in the pathogenicity of C. pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between the cp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WT in vitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WT in vivo. [Results] We successfully constructed the cp40-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 delete cp40 of C. pseudotuberculosis. The results confirm that cp40 is a virulence-related gene, providing a foundation for subsequent research on the infection of C. pseudotuberculosis based on this gene.

Abstract:Phenolic acids autotoxic substances secreted by plant roots, such as p-hydroxybenzoic acid (PHBA), are the main factors causing continuous cropping obstacles in plants. [Objective] In order to obtain PHBA degrading bacteria and improve its degradation efficiency. [Methods] a PHBA-degrading strain was isolated by screening medium and inorganic salt medium, and it was identified as Microbacterium aurantiacum. The initial content of PHBA, culture temperature, pH and nitrogen source were optimized by single factor experiment and response surface methodology. [Results] The highest degradation rate was obtained when the PHBA content was 0.4 g/L, the temperature was 30 ℃, the pH was 8.0, and the nitrogen source was ammonium sulfate. The optimal degradation conditions were temperature 30.2 ℃, pH 8.3, and PHBA concentration 0.18 g/L, and the degradation rate reached 100%. Combined with pot experiments and high performance liquid chromatography, the results showed that the strain could effectively mitigate the PHBA stress effect in cucumber rhizosphere. [Conclusion] The strain of Microbacterium aurantiacum screened in this study has a high ability to degrade PHBA, and has the potential value in continuous cropping obstacles.

Abstract:The proposal of ban on antibiotics in feed is a turning point for the animal husbandry, marking that the feed industry and animal husbandry have entered a new stage of transformation and upgrading. [Objective] To investigate the effects of adding Bacillus licheniformis HDTN to feed on the growth performance and intestinal flora of ‘817’ broilers. [Methods] B. licheniformis HDTN powder (7.0×10¹⁰ CFU/g) was added to the feed for ‘817’ broilers. The effects of high-, medium-, and low-dose (1 000, 500, and 250 g/t) bacterial addition on the growth performance, serum biochemical indexes, intestinal morphology, and intestinal flora structure of the ‘817’ broilers during 1-56 days were studied, with the group without bacterial addition as the control. [Results] Compared with the control group, the addition of low-dose B. licheniformis HDTN increased the average body weight by 105.47 g (P<0.01) and reduced the feed-to-weight ratio by 0.25 (P<0.05) during days 1-35. The antioxidant indexes in the broiler serum were positively correlated with the dose of HDTN, which reduced oxidative stress in broilers. The content of malondialdehyde was negatively correlated with the dose of HDTN, which suggested that HDTN reduced cellular damage in broilers. The ratio of villus height to crypt depth in the duodenum of the low-dose group was higher than that of the control group (P<0.01). In addition, compared with the control group, medium-dose (P<0.01) and low-dose (P<0.05) groups showed increased relative abundance of Firmicutes, and the medium-dose group showed increased relative abundance of Bacteroides (P<0.05). [Conclusion] Addition of low-dose B. licheniformis HDTN for ‘817’ broilers can improve the growth performance, reduce the feed-to-weight ratio, improve the intestinal morphology, and accelerate the assembly of intestinal flora during days 1-35.

Abstract:[Objective] To construct a recombinant food-and-mouth disease virus (FMDV) strain carrying the genes encoding three topotypes of immunodominant structural proteins of serotype O FMDV by reverse genetic manipulation and evaluate the potential of the recombinant strain serving as a vaccine candidate for porcine food-and-mouth disease (FMD) type O. [Methods] Based on the gene of the recombinant FMDV with the replacement of the VP1 structural protein of O/NXYCh/CHA/2018 epidemic strain, the recombinant full-length plasmid featuring substitution of G-H loop genes of the structural protein VP1 of O/TUR/5/2009 vaccine strain was constructed by gene synthesis. The recombinant virus was rescued after transfection of the linearized recombinant plasmid into BSR/T7 cells expressing T7 RNA polymerase, and then identified by RT-PCR, sequencing, and indirect immunofluorescence. The plaque assay and one-step growth curve building were employed to characterize the recombinant virus. Pigs were vaccinated with the vaccines prepared from the recombinant virus and the parental virus, and then virus neutralization tests were carried out to examine the cross-reactive responses against the epidemic serotype O FMDV isolates of three topotypes. [Results] The recombinant FMDV strain carrying the structural protein genes of three topotypes was successful rescued. The recombinant strain showed similar biological properties to the parental virus. Pigs vaccinated with the vaccines prepared from the recombinant virus and the parental virus produced protective neutralizing antibodies with the mean titer of >1.65log₁₀ against the viruses of the Middle East-South Asia (ME-SA) and South-East Asia (SEA) topotypes. The pigs did not produce protective neutralizing antibodies against the Cathay topotype (<1.65log₁₀). The substitution of O/TUR/5/2009 G-H loop gene improved the cross-reactivity against the viruses of ME-SA and SEA topotypes compared with the parental virus (p<0.05). [Conclusion] This study has guiding significance for the design of FMD vaccines in the future.

Abstract:[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 screen Kroppenstedtia 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 of Kroppenstedtia were isolated from the fermented grains of Jiang-flavor Baijiu and identified as K. eburnea. The liquid culture of strain K. 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 with K. 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 with K. eburnea 6E22 and K. 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.

Abstract:[Objective] To reveal the composition and functions of endophytic bacterial communities in maize under different patterns of tillage combined with straw returning in the Tumochuan Plain, identify the endophytic bacterial resources that promote maize straw degradation under different patterns, and lay a foundation for the selective isolation, cultivation, and functional verification. [Methods] We employed Illumina MiSeq high-throughput sequencing to compare the diversity and community structure of endophytes during the mature stage of maize under different patterns of tillage combined with straw returning in the continuous positioning experiment in the irrigation area of Tumochuan Plain, Inner Mongolia Autonomous Region. [Results] No tillage and deep tillage demonstrated significant effects on the endophytic bacterial diversity of maize. Tillage methods exerted stronger effects on the composition and structure of endophytic bacterial community than straw returning. The structures of endophytic bacterial communities in maize can be classified into two categories: no tillage combined with straw returning and the other seven patterns. The dominant endophytic bacterial genera shared by the nine patterns of tillage combined with straw returning were Pseudomonas, unclassified_f__Enterobacteriaceae, Pantoea, Raoultella, and Rahnella1. Straw returning increased the abundance of Raoultella and unclassified_f__ Enterobacteriaceae. [Conclusion] Different tillage practices alter the diversity, composition, and structure of endophytic bacterial community in maize. Straw returning can increase the relative abundance of Raoultella and Lactococcus, which have positive effects on the degradation of maize straw.

Abstract:l-asparaginase, a key enzyme in amino acid metabolism, is widely used in the food and pharmaceutical industries. The gut microbiota and its product l-asparaginase are closely associated with host health and diseases. [Objective] This study aims to acquire a novel l-asparaginase gene from gut microbiota and explore its enzymatic characteristics and potential applications. [Methods] An l-asparaginase gene was cloned from the metagenome of the fecal microbiota of Nomascus concolor and heterologously expressed in Escherichia coli BL21(DE3). The enzymatic properties of the expressed protein were determined. Furthermore, the potential applications of this protein were explored, including processing potato chips and treating cancer cells. [Results] The cloned l-asparaginase gene, NCasn5, was 996 bp. It encoded the recombinant enzyme NCasn5 with a molecular weight of 37.296 kDa, optimal activity at pH 8.0 and 60 °C, K_m of (3.33±0.21) mmol/L, V_max of (836.30±13.91) µmol/(min·mg), and a serum half-life of about 69 h in vitro at 37 °C. NCasn5 reduced the acrylamide content in potato chips by 69.35% and inhibited the growth of human liver cancer cells (QGY-7703) and human melanoma cells (A-375). [Conclusion] We obtained a novel l-asparaginase demonstrating good thermal stability and a prolonged serum half-life. This enzyme lacks the glutaminase activity and reduces acrylamide levels in potato chips. Moreover, it can induce apoptosis in the cancer cell lines QGY-7703 and A-375. These findings suggest the potential applicability of l-asparaginase in both food processing and pharmaceutical industries.

Abstract:[Objective] To develop a stable and controllable method for isolating and cultivating the eukaryotic microorganism Entodinium caudatum from the rumen of ruminants, which would lay a foundation for the germplasm banking and provide sufficient experimental materials for researching the physiological function of rumen ciliates. [Methods] First, a rumen cannula was used to collect rumen fluid from cows in Wuhan, and E. caudatum was gradually enriched and isolated from the rumen fluid by micro-strainer filtration through different sizes of nylon mesh. Subsequently, the enriched E. caudatum was cultured in anaerobic culture bottles loaded with the modified SP medium. After purification, a single culture of the strain was established. The species was identified by morphological observation and the phylogenetic analysis based on the 18S rRNA gene. Finally, the generation time of the E. caudatum was calculated based on the half-transfer cultivation method. [Results] A single culture of E. caudatum was isolated from the rumen fluid of Holstein cows. The modified SP medium used in this study was composed of SP salt solution, supernatant of the rumen fluid without protozoa, cysteine hydrochloride, antibiotics, starch, and grass powder. In the modified SP medium, the obtained culture grew and proliferated steadily, reaching the highest density of 37 000 cells/mL on day 16 from the initial inoculation density of 320 cells/mL. Morphological features and molecular data indicated that the culture obtained in this study was E. caudatum, named E. caudatum strain WH. The generation time of E. caudatum strain WH was 19.0 h. [Conclusion] This study has successfully developed an in vitro cultivation method for E. caudatum strain WH, a prevalent eukaryotic microorganism from the rumen of ruminants. This work lays a technical and theoretical basis for the germplasm banking and in-depth research on the physiological functions of rumen ciliates.

Abstract: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.