This article reviews the remarkable achievements of microbiology in both fundamental research and applied technologies. It provides a brief summary of the 32 papers included in the special issue commemorating the 50th anniversary of Microbiology China, highlighting its broad applications across environmental, industrial, medical, and agricultural fields, as well as its future development prospects.

Anaerobic microorganisms, particularly methanogens, are widely distributed in the anoxic environments of earth. They have diverse metabolic potentials, and play indispensable roles in earth materials cycle and maintaining environmental and human healthy. Anaerobes are not only the main species in human intestinal microbiome, but also are the players converting organic waster to bioenergy, methane. I have been engaged in anaerobes especially in methanogenic archaea for more than 30 years, and would like to share my experience and lessons in the research on anaerobes with the young microbiologists at the time of the 50^th anniversary of Microbiology China.

China’s policy of reform and opening up has created opportunities for the vast number of scientific and technological personnel to learn the advanced theories and technologies from other countries. The author and his team are witnesses and beneficiaries of the policy. This article first introduces the author’s experience of going to Utrecht University in the Netherlands to study advanced theories and techniques of virology in his early career, then introduces the process of leading the team to exchange and cooperate with Netherlands scientists for building a high-level scientific research platform. Through cooperative research with Dutch scientists, the author’s team established the world’s first reverse genetic manipulation platform of porcine epidemic diarrhea virus (PEDV). Leveraging this platform, extensive and in-depth research has been carried out, yielding fruitful results. Finally, this article reviews the recent research progress in PEDV and makes an outlook on the future of the research field.

The complex microbiome that resides in the human body plays a crucial physiological role, closely linked to both the maintenance of human health and the onset of diseases. In recent years, microorganisms have been detected in sites previously considered sterile, such as the female upper genital tract. The involvement of these microorganisms in normal physiological processes remains unclear. However, the specific microbiota in the female upper genital tract is strongly correlated with the development of various female diseases. This article reviews the research progress in the microbiota of the female upper genital tract and its associations with normal physiology and genital tract diseases.

A retron, a bacterial genetic element mainly composed of a non-coding RNA and a reverse transcriptase, is involved in the anti-phage defense system. Since retrons can produce multi-copy single-stranded DNAs, combining retrons with proteins of homologous recombination or CRISPR/Cas has achieved efficient and accurate gene editing in bacteria and yeast, without relying on the direct introduction of exogenous DNAs. The optimized retron system can be applied in multiple fields such as mutant library construction, molecular recorder, and continuous evolution of proteins. This review introduces the structures and molecular mechanisms of retrons, summarizes the application of retron-based gene editing in bacteria and fungi, and analyzes the advantages and limitations of this technology. Finally, the future development of gene editing based on retrons is prospected.

Acid mine drainage contains microorganisms with unique characteristics that enable them to survive in highly acidic environments. This paper details the key microbial groups in AMD and evaluates their application potential in bioleaching. It then introduces the principle, reviews the current research status, and offers insights into the future development of bioleaching. Bioleaching has been applied in industrial applications. However, enhancing the efficiency and adaptability of microbial communities for bioleaching remains a focus and challenge in this field at present.

As an abundant and renewable bioresource, lignocellulose after efficient depolymerization can provide raw materials for green bio-manufacturing. Trichoderma reesei is one of the main industrial producers of cellulases. Its outstanding capability of producing cellulases plays an important role in lignocellulose saccharification. In the past few decades, researchers have gained a preliminary understanding of the mechanisms underlying the induced cellulase gene expression in T.reesei, and revealed a complex regulatory network controlling the biosynthesis of various glycohydrolytic enzymes. Here we systematically review the recent progress in the regulation of cellulase gene expression in T. reeseiregarding signal transduction, transcriptional regulation, and chromatin remodeling. Furthermore, we review the latest progress in genetic engineering for improving cellulase production based on the understanding of the above regulatory network.

Candida glycerinogenesis a strain with independent intellectual property right and more than 50 years of research history. Our research group has established the aerobic fermentation technology with this strain for glycerol production and achieved the world’s highest yield. As the research on this yeast is deepening in recent years, researchers have discovered that C.glycerinogenes has strong resistance and high efficiency of sugar metabolism, demonstrating the potential of serving as an industrial chassis cell for synthetic biology. This paper reviews the discovery process and characteristics of C.glycerinogenes, the industrialization of fermentation technology for glycerol production, and the research progress in the molecular operating system, signaling responses, and metabolic engineering of this yeast, and makes an outlook on the future direction of development, aiming to provide reference for establishing an innovative synthetic biology system with independent property right.

Microbiomes have great application potential in industry, agriculture, environment, health, etc. In practice, it is still difficult to construct efficient and stable microbiomes and utilize the engineered microbiomes in various application fields. To summarize and analyze the construction strategies of microbiomes, thus facilitating the engineering of microbiomes for application in different scenarios. We first classified the application scenarios of microbiomes into closed, semi-closed, and open systems according to the application requirements and then reviewed the existing microbiome construction strategies in the three systems, especially focusing on their characteristics and limitations. Finally, according to the characteristics of different systems and the empirical feedback from practical applications, we proposed the microbiome construction strategies. In the closed system, the microbiome is mainly synthesized by the bottom-up method, which has high requirements for microbial individual information and microbiome efficiency. In semi-closed and open systems, the microbiome is constructed by top-down and bottom-up methods, which require high stability and safety of the microbiome. In diverse application scenarios, the strategies and focuses of constructing microbiomes are different, and appropriate strategies could be chosen to construct efficient microbiomes according to the actual situations.

Squalene-hopene cyclase (SHC) is an important member of the triterpene cyclase family. It mainly catalyzes the cyclogenesis of squalene, an important linear precursor in cells, to generate pentacyclic triterpenoids (hopanoids). These compounds mainly exist in prokaryotes and are relatively conserved and widely studied in bacteria. They are important components of bacterial cell membranes and their structures are similar to sterols in eukaryotic cell membranes. Hopanoids participate in physiological processes such as membrane fluidity and permeability, stress responses, and drug resistance. This paper reviews the research advances in SHC and its catalytic products hopanoids in terms of their functions and structures.

Gramineous plants, known for their extensive distribution and substantial economic importance, have garnered significant interests. Epichloë endophytes maintain mutualistic symbiosis with cool-season gramineous plants, demonstrating strict host specificity. This review aims to facilitate the foundational research on Epichloë by summarizing the research progress in their host specificity. Initially, this review delineates the fundamental attributes of gramineous plant endophytes, emphasizing the host specificity of Epichloë through seed transmission and genetic evolution. Endophytic fungi, mycorrhizal fungi, and pathogenic fungi of plants are compared. The pivotal role of noxA in modulating “symbiosis versus pathogenicity” in host plants is expounded, and a potential link in host specificity across these three categories of plant-associated fungi is suggested. It is posited that the host specificity mechanisms of Epichloë might offer insights into the host specificity of both plant pathogenic fungi and mycorrhizal fungi. Finally, this review summarizes the recent advancements in omics-era, expecting that these emerging technologies will advance the discovery of novel genes and deepen the understanding of fungal host specificity. It emphasizes the importance of developing effective artificial inoculation techniques for Epichloë as a means to investigate host specificity. These research efforts are expected to help create new endophyte-host combinations in the future.

Alkenes, unsaturated hydrocarbons with carbon-carbon double bonds, are emitted in large quantities through both natural and anthropogenic processes. These compounds exhibit diverse functions and characteristics due to variations in their structures. Ethylene, for instance, is a crucial regulator of plant growth, while propylene serves as the primary raw material for the industrial production of polypropylene and acrylonitrile. However, some alkenes pose environmental and health risks. 1,3-butadiene, used in the manufacturing of synthetic rubber and plastics, is a known carcinogen. Isoprene, the most abundant non-methane biogenic volatile organic compound, significantly impacts global climate change. Microorganisms play a critical role in the environmental fate of alkenes by mediating their degradation and transformation. Understanding these microbial processes is essential for elucidating the flow of alkenes in the environment and their impacts on geochemical cycles. Furthermore, this knowledge holds great promise for the bioremediation of alkene-contaminated sites. This paper comprehensively reviews the aerobic and anaerobic microbial degradation and transformation mechanisms of five prevalent short-chain alkenes: ethylene, propylene, butene, 1,3-butadiene, and isoprene. Alkene-degrading strains are widely distributed across multiple phyla. Despite the structural differences among alkenes, their microbial degradation pathways share common features. For example, under oxic conditions, short-chain alkenes are typically oxidized by alkene monooxygenases, the products of which are then conjugated with coenzyme M or glutathione. After a series of enzymatic transformations, they ultimately enter the central metabolic pathways of microorganisms. Under anoxic conditions, short-chain alkenes can be transformed by acetogens, methanogens, and other microorganisms via hydrogenation reactions. By elucidating the mechanisms of microbial degradation and transformation of common short-chain alkenes, this study emphasizes the crucial role of microorganisms in bioremediation efforts at alkene-contaminated sites. Moreover, it contributes to a deeper understanding of microbial influences on geochemical cycles and global climate change, ultimately promoting sustainable development and efficient resource utilization.

Cyanobacteria, among the oldest life forms on Earth, are discovered in nearly all habitats ranging from lakes to deserts. As an integral component of the solar spectrum, ultraviolet (UV) is known to exert profound effects on the growth and evolution of cyanobacteria through its radiative impact. This paper reviews the mechanisms by which UV radiation affects cyanobacterial growth, the regulatory role of UV in the growth of cyanobacteria, and the adaptive strategies of cyanobacteria to UV. Additionally, this paper explores the relationship between UV radiation and cyanobacterial evolution and discusses the scientific value of the interactions between cyanobacteria and UV radiation in the research on biological evolution. Furthermore, this paper expounds the environmental factors affecting cyanobacterial resistance to UV and envisions the future research directions concerning the adaptation and evolution of cyanobacteria exposed to UV radiation.

The generation and spread of antimicrobial resistance (AMR) in the environment pose a serious threat to human health. The minimum inhibitory concentration (MIC) is a key indicator for assessing the risk of AMR in environmental settings. Based on a literature review, this study found that among the commonly employed MIC testing methods, broth microdilution was the most prevalent, followed by agar dilution and E-test, and the MIC values observed showed no differences among different testing methods. Furthermore, we collected and analyzed the MIC data of different strains and antibiotics from the EUCAST database. According to the data, the available AMR studies mainly focused on Gram-negative bacteria (G⁻), which had a larger amount of MIC data than Gram-positive bacteria (G⁺). Notably, we observed that G⁺ bacteria exhibited stronger resistance to antibiotics than G⁻. Acinetobacter baumanniiand Enterococcus faecium demonstrated the strongest resistance among G⁻ and G⁺, respectively. Additionally, we found that the research on AMR primarily focused on β-lactams, with limited attention to sulfonamides and peptides. Bacteria displayed the strongest resistance to ampicillin-sulbactam, streptomycin, and fusidic acid among the antibiotics tested. This study reviews the current status of MIC testing methods and data. It emphasizes that existing data are insufficient and recommends expanding the scope of MIC research while promoting the sharing of AMR information.

[Background]The bioleaching performance is closely related to the microbial community structure and its dynamics during the bioleaching process. Understanding the changes in microbial community structure and function during the bioleaching process is crucial for elucidating the role of microorganisms in this process and building efficient microbial combinations.[Objective] To obtain the microbial community with stable and high pyrite-leaching efficiency by domestication, and analyze the structure and function of the microbial community. [Methods]The microbial community was subcultured in the 9K medium with pyrite as the energy source. The iron and sulfur oxidation capacity of the microbial community was measured before and after domestication. The structure of the domesticated microbial community was analyzed by metagenomic sequencing combined with 16S rRNA gene clone library. Differentiation culture experiments were carried out to study the functions of dominant microorganisms in the domesticated microbial community during pyrite bioleaching. The functional annotation of metagenomic sequencing results was carried out to analyze the functional changes of the microbial community before and after domestication.[Results]The microbial community with a stable structure, improved bioleaching ability, and adaptability to the pyrite leaching environment was obtained after domestication. The metagenomic sequencing results showed that the relative abundance of the genus Acidithiobacillusand Sulfobacillus were both increased. Detection results from the 16S rRNA gene clone library showed that the domesticated microbial community mainly consisted of Acidithiobacillus thiooxidans and Sulfobacillus thermotolerans, with the iron and sulfur oxidation capacity increasing by 15.15% and 70.86%, respectively. The leaching effects were compared between the bacterial community and the dominant species alone. In terms of total iron leached from pyrite, the 16-day leaching rate of the domesticated community 5Biol reached 81.18%, while those of A.thiooxidans and S.thermotolerans were 51.86% and 37.65%, respectively. The better leaching ability of 5Biol than that of single dominant species indicated that the existence of synergistic effect between dominant species in the community, which endowed 5Biol with the leaching effect that a single species cannot achieve. The differentiation culture experiments showcased that A.thiooxidans primarily contributed to sulfur oxidation, while S.thermotolerans was mainly responsible for iron oxidation. S.thermotolerans was dependent on A.thiooxidans for iron oxidation. This indicated that different strains within the community may collaborate synergistically, leveraging their respective strengths to enhance the overall leaching efficiency. This is consistent with the increases in the relative abundance of Acidithiobacillus and Sulfobacillus and the enhancement of leaching ability of 5Biol after domestication. After domestication, sulfocyanin-like proteins were the key proteins involved in iron oxidation, while sulfur oxidation included the sox system, sor system, and non-sox subsystems. [Conclusion] Long-term domestication by passages with a specific ore can yield a stable microbial community with enhanced bioleaching performance. The simplified stable microbial community makes it easy to elucidate the functional roles of different microbial species. The findings provide insights into the construction of efficient microbial communities for bioleaching.

[Background] Pseudomonas putidaF1 with simple nutritional requirements, rapid growth, and diverse metabolic pathways is regarded as one of the most promising chassis cells in synthetic biology. However, few studies have reported the application of P. putida F1 as a biodegradable chassis cell by streamlining the genome. [Objective] To construct a chassis cell with high biosafety. [Methods] We employed scarless genome editing based on homologous recombination principle to streamline the genome of P. putida F1 and integrated the conditional suicide system into the genome. [Results] Two lysogenic phage genomic islands PP1 (50.7 kb) and PP2 (58.4 kb), which account for 1.83% of the genome size, were successfully knocked out, and a knockout strain P. putida F1ΔPP12 was constructed. Furthermore, the conditional suicide system controlled by the arabinose promoter was integrated into the site of upp in the genome of P. putida F1ΔPP12, and thus a chassis cell P.putida F1ΔPP12-P_ara-nuc with high biosafety was successfully constructed. [Conclusion] This study increases the application value of P.putida F1 and provides a theoretical basis for the application of this strain or genetically engineered bacteria from this strain in environment remediation in situ.

[Background] The black soil area in northeast China is an important grain production base in China. However, the excessive use of chemical fertilizer has caused serious environmental issues, including nitrogen loss and environmental pollution. Nitrification, one of the key steps in nitrogen cycling, is mainly driven by microorganisms. [Objective] To distinguish the relative contributions of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and comammox to soil nitrification by using nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and acetylene in the black soil fields with low and high nitrogen content. [Methods] The soil samples applied with no nitrogen fertilizer (low-nitrogen soil) and nitrogen fertilizer (high-nitrogen soil) were collected from the black soil area in the long-term fertilization experiment. The effects of nitrification inhibitors on soil available nitrogen content, the abundance of ammonia-oxidizing bacteria, and their relationship were investigated by quantitative PCR of soil microcosms. [Results] Ammonia-oxidizing archaea and comammox Clade B predominated in the low-nitrogen soil, while ammonia-oxidizing bacteria and comammox Clade A predominated in the high-nitrogen soil. Both nitrification inhibitors, DMPP and acetylene, significantly inhibited ammonia oxidation, with more pronounced effects in high-nitrogen soil. Acetylene had a greater inhibitory effect than DMPP on ammonia oxidation. In addition, acetylene significantly reduced the abundance of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and comammox Clade B in low-nitrogen soil, while DMPP significantly inhibited ammonia-oxidizing bacteria and comammox Clade A. The correlation analysis showed that the amoA abundance of ammonia-oxidizing bacteria and archaea was positively correlated with NO₃⁻-N content and negatively correlated with NH₄⁺-N content in low-nitrogen soil. However, the amoA abundance of ammonia-oxidizing microorganisms had no significant correlation with NH₄⁺-N or NO₃⁻-N content in high-nitrogen soil. [Conclusion] The long-term application of nitrogen altered the responses of ammonia-oxidizing microorganisms to environmental changes in black soil.

[Background] T7 phage-derived T7 RNA polymerase (T7 RNAP) is a commonly employed component of the reverse genetic operating system of viruses, due to its high specificity and efficient initiation of transcription. Several cell lines stably expressing T7 RNAP have been constructed for reverse genetics studies of RNA viruses. The grass carp, the freshwater fish with the highest production in China, is susceptible to grass carp reovirus (a double-stranded RNA virus). However, no grass carp cell line stably expressing T7 RNAP has been reported to date.[Objective] To construct the stable cell lines expressing T7 RNAP by using the Tol2 transposon system from grass carp fin (GCF) and grass carp ovary (GCO) cells and measure their viability, thus providing cellular tools for establishing the reverse genetic operating system for grass carp reovirus. [Methods] The T7 RNAP gene was amplified from the genomic DNA of Escherichia coli BL21(DE3) via PCR and inserted into the Tol2 transposon plasmid via homologous recombination. The recombinant Tol2 transposon plasmids carrying T7 RNAP with or without an N-terminal or C-terminal nuclear localization signal (NLS) were constructed. These recombinant plasmids, along with a helper plasmid expressing the Tol2 transposase, were co-transfected into the two grass carp cell lines, followed by selection with hygromycin B. Western blotting (WB) was employed to determine the T7 RNAP expression in the selected cells. An enhanced green fluorescent protein (EGFP) reporter plasmid driven by a T7 promoter was used to validate the functionality of T7 RNAP, and the effects of NLS on the expression and activity of T7 RNAP were studied. [Results] Three recombinant Tol2 transposon plasmids carrying the T7 RNAP gene were successfully constructed. Six stable cell lines expressing T7 RNAP were obtained after selection. WB results confirmed significant expression of T7 RNAP in all the selected cell lines. Transfection with the T7 promoter-driven EGFP plasmid demonstrated high levels of EGFP expression, confirming the transcriptional activity of T7 RNAP in these cell lines. Notably, the GCO cells expressing T7 RNAP with an NLS, especially with the C-terminal NLS, showed higher expression and transcriptional activity than those without NLS. However, no significant difference was observed in GCF cells. [Conclusion] We successfully established stable T7 RNAP-expressing GCF and GCO cell lines by using the Tol2 transposon system. These cell lines demonstrated efficient transcription and expression of genes under the control of the T7 promoter, which could be further enhanced by addition of an NLS. NLS could enhance nuclear accumulation and transcriptional efficiency of T7 RNAP by facilitating its transport into the nucleus, with the effects depending on the cell lines. The results layed a foundation for the application of T7 RNAP in the research on grass carp reovirus.

[Background] The peripheral light-harvesting complex (LH2) performs the function of light energy capture and transfer in photosynthesis and is essential for the phototrophic growth of anoxygenic phototrophic bacteria (APB). The α and β subunits of LH2 are usually encoded by multiple copies of pucBA genes, which reach 5–7 copies inRhodopseudomonas. As a result, the heterogeneous synthesis of LH2 by multiple genes makes LH2 difficult to be separated from each other, which greatly limits the full understanding about the contributions of different pucBAgenes to the structures and physiological roles of LH2. [Objective] To develop an expression system and evaluate the light energy transfer activity in vivo andin vitro for the reconstituted LH2 of R. palustris. [Methods] Using Rhodopseudomonas palustris CGA009 (ΔpucBA) with all five pucBA genes deleted as the host bacterium and the co-expression products of pucBAd and mrfp (red fluorescent protein gene) as the markers, we conducted genetic modification of the expression vector and promoter screening. The spectroscopic method was employed to determine the biomass of recombinant bacteria, the spectral characteristics of gene expression products, and the spectral characteristics, photosynthetic pigments and light energy transfer activity of purified LH2. [Results] The T7 promoter (P_T7) combined with T7 RNA polymerase gene promoted the co-expression of pucBAd and mrfpin ΔpucBA. We replaced P_T7 with P_badR, P_pckA, and P_ars, respectively, and the activities of the promoters followed a descending order of P_pckA, P_ars, P_badR, and P_T7. This strategy increased the mrfp gene expression, the synthesis product of pucBAd (d-LH2), and the phototrophic growth rate in the host bacteria. The expression system ΔpucBA(pucBAd) harboring P_pckA promoter had the highest expression activity of reconstituted LH2. Then, we used pucBAa from strain CGA009 to replace pucBAd in ΔpucBA(pucBAd) and successfully obtained the recombinant strain ΔpucBA(pucBAa). Compared with the pucBAaconstruct, the recombinant strain with pucBAd showed significantly accelerated growth under low light and slightly accelerated growth under high light. The purified d-LH2 and a-LH2 showed typical characteristic spectra of B800-only and B800-850, respectively. The fluorescence quantum efficiency of d-LH2 at ~863 nm was higher than that of a-LH2. However, the light energy transfer efficiency from carotenoids (Car) to bacteriochlorophylls (BChl) of d-LH2 measured by the classical method was lower than that of a-LH2. The inconsistency between the two results was due to the fact that the classical method did not consider the photon absorption difference at ~863 nm by different spectral types of LH2. [Conclusion] The reconstituted LH2 expression system ofR.palustris was successfully constructed. The light energy transfer activities of two different spectral types of LH2 in strains with multiple copies of pucBA were evaluated on the levels of gene-pure LH2 and photosynthetic growth rate of the recombinant bacteria. The results showed that the energy transfer activity of abnormal spectral LH2 (d-LH2) was higher than that of typical spectral LH2 (a-LH2). The present work laid a foundation for comprehensively understanding the molecular regulatory mechanisms of the formation and assembly of LH2 in the bacteria with multiple copies of pucBA.

[Background] The histone acetyltransferase general control nonderepressible-5 (GCN5) is a key epigenetic modifier that plays a role in regulating biological processes in eukaryotes. At present, little is known about the function and mechanism of GCN5 in Botrytis cinerea.[Objective] To analyze the function of BcGCN5 and lay a foundation for elucidating the function and regulatory mechanism of histone acetylation in the growth, development, and pathogenicity of B.cinerea.[Methods] The mutant ΔBcGCN5of B. cinerea was successfully constructed by gene knockout. The phenotype and pathogenicity of ΔBcGCN5 were analyzed with the wild-type strain B05.10 as the control.[Results] ΔBcGCN5 showed slow growth, reduced production of sclerotia and conidia, and diminished acid production, which ultimately led to weakened pathogenicity towards tomato fruits and tobacco leaves. [Conclusion] BcGCN5 positively regulated the growth, development, and pathogenicity of B.cinerea.

[Background] Transglutaminase EC 2.3.2.13 (TGase) catalyzes cross-linking between the γ-carboxamido group of glutamine residues and the ε-amino group of lysine residues. This process leads to the formation of an isopeptide bond, which modulates the conformation and functions of proteins. TGases play a crucial role in food, pharmaceutical, textile, and leather processing industries. [Objective] To mine a high-performance TGase from natural Streptomyces mobaraensis and enhance the titer of this enzyme by recombinant expression in the industrial chassis. [Methods] The TGase production potential of S.mobaraensis (CGMCC 4.266) was evaluated by shake-flask fermentation. The TGase (TGe) was purified by Capto S cation exchange chromatography. The enzymatic properties including optimal pH, pH stability, optimal temperature, thermal stability, and cross-linking ability with casein were evaluated. We knocked out tg from industrial S. mobaraensisand obtained Δtg, in which tge was introduced and expressed. [Results] TGe showcased the optimal pH 5.0, with high activity within the range of pH 4.0–10.0. This enzyme achieved the highest activity at approximately 50 ℃, which was comparable to that of commercially available TGases. TGe exhibited good stability within 4–40 ℃, and its activity surpassed those of commercial TGases at 40–65 ℃. In addition, TGe demonstrated higher cross-linking ability with casein at 50 ℃ than commercial TGases. The recombinant expression of tge in Δtg increased the TGe titer (6.3 U/mL) by 162.5% compared with the wild-type strain, without compromising the catalytic activity of TGe. [Conclusion] High-performance TGases can be mined from natural S. mobaraensis. The heterologous expression of TGases in mature industrial strains gives a novel insight into enhancing the TGase titer of S.mobaraensis.

[Background] The reductive dehalogenation process mediated by organohalide- respiring bacteria is crucial for halogen cycling and the remediation of organohalide- contaminated sites. Reductive dehalogenases (RDases), as terminal electron acceptor reductases in organohalide respiration, play a critical role in dehalogenation. [Objective] To systematically understand the sequence and structural characteristics of RDase proteins and lay a foundation for elucidating the mechanisms and biological functions of RDases and organohalide-respiring bacteria. [Methods] Multiple bioinformatics tools were used to predict the basic physicochemical properties, transmembrane structures, signal peptides, phosphorylation sites, phylogenetic relationship, homology matrix of multiple sequence alignments, conserved motifs, conserved domains, secondary structures, tertiary structures, and disordered regions of 44 RDase proteins. [Results] The physicochemical properties varied among different RDase proteins, but they were conserved to some extent. The phosphorylation sites, distribution of conserved domains, distribution of conserved motifs, and secondary structures were highly conserved among different RDases proteins. The RDases with similar or identical substrate categories tended to have greater similarity in their protein sequences and tertiary structures. The phylogenetic relationships were relatively close among most RDase proteins. RDase proteins were primarily secretory proteins, non-membrane proteins, and intrinsically disordered proteins. [Conclusion] Different RDase proteins are conserved to some extent and thus have similar biological functions. The findings provide a valuable reference for further studies on RDases, particularly offering scientific evidence and theoretical support for the application of organohalide-respiring bacteria based on RDases in site remediation.

[Background] A large amount of crop straw as agricultural waste is produced in northeast China every year. Low temperature leads to slow degradation of straw under natural conditions.[Objective] To screen the bacterial strain capable of degrading lignin efficiently at low temperatures and explore the pathways of lignin degradation. [Methods] The strain was identified by morphological observation and 16S rRNA gene sequencing. The lignin degradation conditions were optimized at low temperatures, and transcriptome sequencing was employed to predict the pathways of lignin degradation.[Results] A strain Sporosarcina sp. N2 capable of efficiently degrading lignin at 15 ℃ was screened out. The degradation rate of lignin was 20.8% after 8 days of low temperature culture and increased to 31.7% after degradation condition optimization. The differentially expressed genes in different time periods were identified by transcriptome sequencing. The functional genes related to cold resistance and lignin degradation presented down-regulated or up-regulated expression in different degradation periods. According to the gene expression pattern, we hypothesized that the strain degraded lignin via the benzoic acid pathway and protocatechuic acid pathway. [Conclusion] This study enriched the microbial resources for degrading lignin at low temperatures and provided data support for enhancing microbial degradation of straw at low temperatures.

[Background] Endophytic bacterial communities in the roots of peach plants have the potential to promote plant growth and enhance stress resistance. Studying the structures and functions of these microbial communities can reveal the role of endophytes in peach root growth and development. The results can serve the subsequent development and utilization of microbial resources. [Objective] To study the structures, diversity, and functions of endophytic bacterial communities in the roots of different peach varieties, laying a foundation for further research on the relationship between peach plants and endophytes. [Methods] We employed 16S rRNA gene amplicon sequencing to study the community structure and diversity of endophytic bacteria in the roots of seven different peach varieties in the National Peach Base in Pinggu District, Beijing. Furthermore, we performed linear discriminant analysis effect size (LEfSe) and used PICRUSt2 for function prediction. [Results] Significant differences existed in the structures of endophytic bacterial communities in the roots among red-skin, white-skin, and yellow-fleshed peach varieties. The dominant phyla were Actinobacteriota, Proteobacteria, and Firmicutes, with Actinobacteriota being predominant in most samples. The Shannon and Simpson indices of the bacterial communities were significantly different among the varieties, indicating the varied and complex diversity of root endophytes among different peach varieties. LEfSe identified the root endophyte biomarkers associated with peach varieties, such as Bifidobacterium, which had higher relative abundance in red-skin and white-skin peach varieties. The endophytic bacteria showcased varied biological functions among different red-skin peach varieties, mainly participating in carbohydrate and amino acid metabolism, which highlighted the potential impact of endophytic bacteria on the physiological functions of host plants. [Conclusion] This study revealed the structures, diversity, and functions of endophytic bacterial communities in the roots of different peach varieties, enriching our knowledge and laying a foundation for the development and utilization of endophytic bacterial resources in peach plants.

[Background] Hygrophorus, belonging to Basidiomycota, Agaricomycetes, Agaricales, Hygrophoraceae, is widely distributed all around the world. [Objective] To investigate the species of Hygrophorus and enrich the knowledge about the species diversity of this genus in Shanxi Province. [Methods] We collected fungal specimens, observed and characterized their morphology. After DNA extraction, the internal transcribed spacer (ITS) was sequenced. Phylogenetic trees were constructed based on the maximum likelihood method and bayesian inference. [Results] Eleven species of Hygrophorus were identified based on the morphological and molecular evidence, including ten known species and a new species. This article described the macroscopic and microscopic morphological characteristics of the new species Hygrophorus brunneodiscoides in detail. The basidiocarp photos of ten known species and a key to the species of Hygrophorus in Shanxi Province were provided. [Conclusion] We identified a new species, H.brunneodiscoides, in China, confirmed 11 species of Hygrophorus in Shanxi Province, and briefed their geographical distribution and associations with hosts according to the field observation.

[Background] Listeria monocytogenes is a major food-borne pathogen, causing the mortality rate of 20%–30% in susceptible populations. [Objective] To screen the lactic acid bacteria with antagonistic effect on L.monocytogenes and explore the physicochemical properties and killing effect of the bacteriocin produced by the isolate on L.monocytogenes. [Methods] The bacteriocin-producing lactic acid bacteria with antagonistic effect on L.monocytogenes were screened by the Oxford cup method. The strain screened out was identified based on morphological characteristics, 16S rRNA gene sequencing, and average nucleotide identity (ANI). The physicochemical properties of the bacteriocin produced by the strain were analyzed and the antagonistic effect of the bacteriocin crude extract on L.monocytogenes was characterized by laser confocal microscopy and scanning electron microscopy. [Results] A strain A9-1A with strong antagonistic effect on L.monocytogenes was obtained, with the inhibition zone diameter of 37.56 mm and antagonistic effects on Listeria and Enterococcus faecalis. It was identified as Enterococcus mundtii. The bacteriocin produced by this strain could tolerate strong acids and alkali (pH 2.0–11.0) and high temperatures (37–121 ℃). It was not sensitive to amylase and lipase but sensitive to proteases. The minimum inhibitory concentration of the bacteriocin crude extract was 16 μg/mL. The results of confocal laser scanning microscopy and scanning electron microscopy showed that the bacteriocin crude extract damaged the cell membrane and led to the formation of holes, causing the leakage of cell contents and thereby killing L.monocytogenes. [Conclusion] A bacteriocin-producing strain E. mundtii A9-1A with strong antagonistic effect on L.monocytogenes was screened out. Its bacteriocin could tolerate strong acids and alkali and high temperatures, demonstrating the application potential in the prevention and control of L.monocytogenes contamination in food production.

[Background] Lack of nitrogen sources always affects the fermentation performance of fruit wine, resulting in poor sensory quality of fruit wine. However, few researchers have reported the effects of supplementing nitrogen sources on the fermentation performance and flavor of passion fruit wine. [Objective] To explore the effects of dibasic ammonium phosphate (DAP) as an adjunct nitrogen source on the fermentation performance and volatile components of passion fruit wine, providing a theoretical basis and data support for the quality improvement of the fruit wine. [Methods] We employed HPLC, GC-MS, gas chromatography-triple quadrupole mass spectrometry, and electronic nose to determine the content of organic acids, amino acids, and volatile compounds, thereby comprehensively analyzing the effects of DAP on the flavor characteristics of passion fruit wine. [Results] Compared with the control (DAP-free), the addition of 400 mg/L DAP to passion fruit juice shortened the fermentation time of the wine by 5 days, and the total sugar content and ethanol content reached (3.35±0.57) g/L and (10.73±0.06)% vol, respectively, at the end of fermentation. In addition, the involvement of DAP increased the content of total phenols ((68.62±2.81) mg/L) and total flavonoids ((23.41±0.65) mg/L) in passion fruit wine. The passion fruit wine produced with or without DAP contained 20 free amino acids. The content of total amino acids was (669.40±56.27) mg/L in the passion fruit wine produced with DAP and (1 017.58±80.53) mg/L in the passion fruit wine produced without DAP, which showed an obvious difference. Specifically, glutamic acid, aspartic acid, and proline were consumed the most after the addition of DAP, which indicated their contributions to the fermentation performance of passion fruit wine. Moreover, 59 volatile compounds were detected in the passion fruit wine by GC-MS. More volatile compounds (about 20) were formed in the DAP-supplemented passion fruit wine than the DAP-free wine, and 19 volatile compounds were observed to be the main aroma substances (odor activity value, OAV>1), including isoamyl alcohol, ethyl butyrate, ethyl hexanoate, isoamyl acetate, and trans-β-ionone. Interestingly, the levels of higher alcohols and acetic acid decreased while the content of terpenes, aldehydes, and ketones increased in passion fruit wine due to the presence of DAP. [Conclusion] The addition of DAP (400 mg/L) not only improved the fermentation performance but also increased the types and content of flavor components, finally improving the comprehensive quality of passion fruit wine.

[Background] β-glucosidase (EC 3.2.1.21) has important application values in food and medicine industries. Bgl2A:V224D, a mutant of a marine microorganism-derived β-glucosidase, is potent in the conversion of soybean isoflavones in the deep processing of soybean yellow slurry water. However, the low stability and low ethanol tolerance limit the application of Bgl2A:V224D. [Objective] To enhance the application potential of Bgl2A:V224D, we adopted a computation-assisted rational design to improve the stability and organic solvent tolerance of this enzyme in the application environment. [Methods] We employed three complementary computational methods to design stable mutants of Bgl2A:V224D by seeking overlapping mutations. Then, sequence coevolution and conservation analyses were performed to determine candidate mutants. Finally, the target mutant enzymes were selected by experimental assays. After the stable mutant enzymes were identified, their stability was further enhanced by combinational mutation. Finally, the application performance of the mutant enzymes for the conversion of soybean isoflavones in the deep processing of soybean yellow slurry water was evaluated. [Results] Several single-point and multi-point mutants with improved stability and maintained or increased enzyme activity were obtained, among which S360K/E408Y had the highest stability, with the half-life at 35 ℃ and pH 6.5 about 6.7 times that of the starting enzyme. Moreover, S360K/E408Y was more tolerant to ethanol than the starting enzyme. The former had the relative activity of about 35% after 20 min of incubation with 20% ethanol at 35 ℃, while the latter had the relative activity of only about 5% after 10 min of incubation under the same conditions. S360K/E408Y was then applied to hydrolyze soybean isoflavones, and its efficiency was significantly increased compared with that of the starting enzyme Bgl2A:V224D. Specifically, the amount of substrate that could be hydrolyzed by the former enzyme was two times that of the latter enzyme under the same conditions. The amount of S360K/E408Y required for converting soybean isoflavones in the deep processing of soybean yellow slurry water was about 1/3 less than that of the starting enzyme. [Conclusion] The stability-enhancing engineering strategy for improving the enzyme tolerance to organic solvents is applicable to β-glucosidases, and the application potential of β-glucosidases in various fields can be enhanced by this strategy.

[Background]Mycoplasma bovis is one of the major pathogens causing bovine mastitis, arthritis, and respiratory diseases, seriously endangering the healthy development of the cattle industry. [Objective] To establish a convenient, rapid, sensitive, and specific method based on loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) for the detection of M.bovis. [Methods] The conserved region was selected by comparison of the P48 specific sequence of M.bovis and used to design primers by Primer Explorer V5 online. The designed primers were compared with the LAMP primers designed with oppD/F,oppD, and urvC as target genes by the fluorescent dye method. The optimal internal primers (FIP/BIP) designed based on P48 were labeled with biotin and 6-carboxyfluorescein, respectively. The reaction temperature, time, and primer concentration ratio were optimized by the single factor method. LAMP was combined with LFD for the detection of M.bovis. Finally, the sensitivity, specificity, repeatability, and clinical application effect of the established method were evaluated. [Results] The LAMP primers designed based on P48 had stronger fluorescence signal, lower Ct value, and higher amplification efficiency, outperforming the reported LAMP primers. The reaction conditions were optimized as 60 ℃, primer concentration ratio (F3/B3:FIP/BIP) of 1:4, and 40 min. The lower limit of detection of the established method was 17.28 fg/μL, which was 1 000 times lower than that of the PCR method, the standard method in the industry. There was no cross-reaction with 9 pathogens causing bovine respiratory diseases, such as Pasteurella multocida and bovine herpes virus (BHV). The results of inter-batch and intra-batch tests were consistent. The detection rate of 39 clinical nasal swabs by the established method was 28.21%, which was higher than that (23.07%) of the PCR method. [Conclusion] A sensitive, specific, and easy-to-use LAMP-LFD method for detecting M.bovis was successfully established, which provided technical support for the prevention and control of M.bovis.

In the context of digital transformation in education, blended teaching which utilizes information tools and digital resources holds significant importance in enhancing teaching quality and efficiency. To address issues such as complex contents, monotonous teaching methods, inadequate student engagement, and unsatisfactory teaching outcomes in Medical Microbiology, we developed a novel online-offline blended teaching mode by integrating Rain Classroom (an online interactive teaching platform) with micro-lectures (short-video teaching resources) under the BOPPPS (bridge-in, objective, pre-assessment, participatory learning, post-assessment, and summary) teaching mode. We then practiced this mode in the teaching of Medical Microbiology and evaluated its pedagogical effectiveness by a questionnaire survey and grade comparison. The results indicated that the BOPPPS teaching mode based on Rain Classroom and micro-lectures significantly enhanced students’ interest in learning and classroom participation, while also improving their academic performance. This study demonstrates the efficacy of this teaching mode in enhancing teaching quality and learning outcomes and gives insights into the teaching reform in medical education.

Microbiology is a basic course of biological sciences, biotechnology, bioengineering, and other agriculture-related majors in agricultural colleges and universities, with the main teaching content of microorganisms and their life activities. Facing the major needs of the country and focusing on the frontiers of agricultural microbiology, the National Key Laboratory of Agricultural Microbiology (NKLAM) of Huazhong Agricultural University has been exploring and practicing the cultivation of innovative talents in microbiology with the support of high-level scientific research. The teachers, also the researchers of the NKLAM, transformed the discipline and research advantages into teaching resources for the standardized, systematic, and cutting-edge research training of students. The NKLAM has used the advantages of first-class disciplines to cultivate students’ interdisciplinary thinking and international cooperation programs to expand students’ global vision. Meanwhile, the NKLAM has strengthened students’ practical skills through production-education-research-learning cooperation and promoted students’ all-round development by carrying forward the cultural spirits of scientific research. In short, by taking various measures, the NKLAM has achieved remarkable effect in talent cultivation, fostering a large number of innovative talents in microbiology-related fields for the bio-industry and rural revitalization.

Cornell University, as one of the internationally renowned first-class universities, has undergone a long and complex evolution in the formation of its curriculum system. Microbiology, one of the branches of biology, offers an integrated curriculum for undergraduates and postgraduates. The curriculum for the undergraduates majoring in microbiology is complete, independent, and practical and it encompasses basic courses, core courses, and elective courses, which promotes the advanced training from low order to high order, from foundation to core, and from exploration to innovation. The teaching management is rigorous and standardized, boosting the advantages of cultivating innovative talents. The undergraduate microbiology curriculum at Cornell University can provide reference for the cultivation of top innovative talents in basic disciplines and the reform of microbiology curriculum and teaching practices in Chinese universities.

The tricarboxylic acid (TCA) cycle is a crucial yet challenging topic in the teaching of Biochemistry due to its complex reactions and dense knowledge points. Conventional teaching method typically follows the sequence of the reactions in the TCA cycle, which often resulting in suboptimal learning outcomes. This paper introduces an alternative teaching method with introduction of decarboxylation clues to simplify the reaction process and product terminology in the TCA cycle, making it easier for students to understand the pathways and mechanisms involved. Firstly, the dissection of decarboxylation demonstrates that pyruvate undergoes decarboxylation to form acetyl-CoA, which reacts with oxaloacetate to produce the 2-carboxymethyl group in citrate. This 2-carboxymethyl group does not participate in decarboxylation during the first round of the TCA cycle. Subsequently, with R representing carboxymethyl for analyzing the TCA cycle, it is found that starting from the condensation of acetyl-CoA and oxaloacetate, the chemical formulas of some intermediates in the TCA cycle can be correspondingly written as follows: citrate (R-2-malate), cis-aconitate (R-fumarate), isocitrate (R-3-malate), α-ketoglutarate (R-pyruvate), succinyl-CoA (R-acetyl-CoA), and succinate (R-acetate). The two decarboxylation steps in the TCA cycle occur in isocitrate and α-ketoglutarate, respectively. To evaluate the effectiveness of this new teaching method, we recruited students to participate in a survey assessing their learning outcomes before and after teaching with this method. Statistical analysis of the feedback results showed that the majority of students taught with this new method found each step of the TCA cycle easier to understand and remember, with significant improvement in learning outcomes. The teaching method with decarboxylation clues enhances learning outcomes by stimulating students’ thinking and analysis and promoting their learning initiative and innovative spirit, thereby leading to better learning results.