Abstract:Porcine deltacoronavirus (PDCoV), an emerging virus globally prevalent, causes watery diarrhea, vomiting, and dehydration of newborn piglets, posing severe economic losses in the swine industry. Moreover, with the characteristic of cross-species transmission, PDCoV can infect human and farmed animals, posing a serious threat to human health. Pyroptosis is a form of programmed cell death mediated by members of the Gasdermin family, which plays a role in defending and eliminating viruses. Coronaviruses, however, have developed precise strategies to antagonize pyroptosis and evade immune responses. This review expounds the biological characteristics of PDCoV, the process of pyroptosis, and the antagonism of Gasdermin D-mediated pyroptosis by PDCoV, aiming to enrich the knowledge about the antagonism of immune responses by PDCoV and lay a theoretical foundation for the prevention and control of this virus.

Abstract:Chitin is a polysaccharide that is polymerized by N-acetylglucosamine through β-1,4 glycosidic linkages and ubiquitous in the global terrestrial and aquatic ecosystems. Chitin is one of the most abundant organic macromolecular polymers on earth. Chitinases are a class of enzymes that catalyze the degradation of chitin. Chitinases are not only the focus of basic research but also have shown broad application potential in a variety of fields such as agriculture, medicine, and environmental science. This paper systematically reviewed the research progress in fungal chitinases in terms of the classification, distribution characteristics in different fungal taxa, biological functions in yeasts and filamentous fungi, enzymatic characteristics, and applications in agricultural pest and disease control, disease treatment, and production of chitooligosaccharides. Furthermore, we discussed the future research directions of fungal chitinases. This paper provides new perspectives for the study of fungal chitinases.

Abstract:Lignin, the most abundant aromatic biopolymer resource in the nature, is difficult to be degraded by common microorganisms due to its complexity and highly aggregated aromatic structure. Lignin-degrading microorganisms from extreme environments are considered as suitable candidates for lignin bioprocessing. This review summarizes several types of extremophiles capable of degrading lignin and the extremozymes produced by them and elucidates the properties, catalytic mechanisms, and metabolic pathways of the extremozymes. Furthermore, this article discusses the prospects for the identification of novel extremophiles and extremozymes by multi-omics and makes an outlook on the development and utilization methods of extremophiles, with a view to providing a reference for the subsequent screening and development of more efficient lignin-degrading strains.

Abstract:Probiotics refer to live microorganisms that are beneficial to the health of the host when ingested in sufficient quantities, and their beneficial effects have been widely recognized. However, the probiotic function of probiotics is strain-specific. As more and more emerging technologies are introduced into the research on probiotics, the screening of probiotics with specific functions according to individual needs has become a research hotspot. Conventional probiotic screening methods have limitations and cannot meet the current demand for precision medicine. Therefore, a bottom-up precision screening strategy for probiotics based on phenotyping, genotyping, and target is of great practical significance. This article discusses the theoretical basis, methods, and technologies of accurate screening of probiotics from phenotype, genotyping, and target, and the application and safety evaluation of probiotics, aiming to provide reference for the accurate screening of probiotics.

Abstract:Giant pandas, despite having the digestive systems of carnivores, have evolved to thrive on a bamboo-based vegetarian diet, which results in a unique gut microbiota. With the advancement of science and technology, our understanding of the gut microbiota of giant pandas has evolved from simple strain isolation to exploring the intricate relationship between the microbiota and metabolic functions of the hosts as well as predicting the functions of the microbiota. In this review, we summarize the characteristics of the bacterial and fungal communities in giant pandas, examining the effects of the factors such as age, diet, habitat, and health conditions on gut microbiota. Furthermore, we discuss how these factors influence the metabolism of the gut microbiota. This review is expected to provide a theoretical basis for the future research on the intricate structure and functions of gut microbiota in giant pandas. The knowledge can pave the way for innovating the measures to enhance and stabilize the gut microecological environment, ultimately contributing to the conservation of giant pandas.

Abstract:With the increasing emphasis on health, probiotics have garnered increasing attention due to their safe and beneficial features for intestinal health and have become a hot spot in the fields of food and medicine. In terms of oral drug delivery, it is difficult to select a safe, convenient, and stable carrier. Probiotics can be used as carriers to deliver drugs through the complex in vivo environment without damage and have excellent safety and stability. This review describes the structures, advantages, and disadvantages of five common probiotic-based oral drug delivery approaches: display on spore surface, yeast microcapsules, recombinant probiotic expression, bacterium-like particles, and bacterial ghost, aiming to provide options for the application of oral delivery carriers.

Abstract:Airborne microorganisms (AMs), including bacteria, fungi, and viruses, are ubiquitous. They are indispensable components of the atmospheric ecosystem, play an essential role in maintaining the stability and functions of the atmospheric ecosystem. AMs include not only beneficial microorganisms but also pathogenic microorganisms, which pose a threat to human health. Therefore, comprehensively revealing the distribution characteristics and succession patterns of AMs is of great significance for improving air quality, safeguarding human health, and ensuring national biosafety. This review systematically elucidates the sources, distribution characteristics, influencing factors, and categories of pathogenic microorganisms as well as the health risks associated with AMs. It contributes to the in-depth understanding of AMs pollution and its health risks, providing a scientific basis for preventing and controlling the pollution and protecting human health and eco-environment.

Abstract:Nitrogen is an essential element for living organisms on Earth, and it is constantly recycled in the biosphere in different forms. A biohybrid constructed with photocatalytic materials is a new system produced in recent years, which combines photocatalytic materials with electroactive microorganisms, integrating the excellent light trapping performance of photocatalysts and the bioefficient catalytic capability. Therefore, it is of great significance to study how the system plays a role in nitrogen cycling and the related mechanisms. This paper introduces the microbial nitrogen cycling and the nitrogen cycling driven by biohybrids and details several types, advantages and disadvantages, and related mechanisms of electron transfer driven by biohybrids constructed with photocatalytic materials. Finally, this paper makes an outlook on the development prospects in this field from the natures and combinations of photocatalytic materials and microorganisms.

Abstract:Escalating resistance of pathogens, especially Gram-negative bacteria, to antibiotics has become a public health problem arousing worldwide concern because of the abuse of antibiotics. The “Trojan Horse” strategy emerges as a promising approach to the development of new antibacterial agents. This strategy improves the antibacterial activity or broadens the antibacterial spectrum of antibiotics by using the siderophore-mediated bacterial iron transport system. In 2019, cefiderocol as the first siderophore-antibiotic conjugate was approved for marketing, which has garnered wide attention of scientists in this field. Currently, researchers mainly focus on siderophores or utilizing antibiotics with different mechanisms and ignoring linkers in the design of agents based on the “Trojan Horse” strategy. This review will summarize the impact of different linkers of conjugates on antibacterial activity, which could provide reference for the development of new antibacterial drugs and combating bacterial resistance.

Abstract:[Objective] To obtain a stable microbial consortium with a high yield of caproate and achieve high-value carbon recovery from Baijiu-making wastewater (Huangshui). [Methods] We used the plate screening approach to obtain a simplified caproate-producing microbial consortium, evaluated the preferred carbon source of the consortium, and optimized the substrate concentration, pH, and feeding strategy. The metagenomics based on nanopore sequencing was employed to determine the composition and stability characteristics of the simplified caproate-producing microbial consortium. [Results] SimpCom1, a simplified caproate-producing microbial consortium, demonstrated significantly higher conversion rate of lactate than glucose to caproate. When SimpCom1 was used to ferment unsterilized Huangshui, we controlled the working concentration of Huangshui between 30% and 50% and initial pH 5.50 to achieve stable growth and metabolism of the consortium for caproate production. The fermentation was carried out in a fed-batch manner with 50% Huangshui, initial pH 5.50, and pH 6.50-7.00 after 48 h. Within four fed-batch fermentation cycles, the average caproate titer, productivity, proportion of caproate in total acids, and conversion rate of lactate to caproate reached 16.83 g/L, 3.05 g/(L·d), 67.27%, and 0.42 g/g, respectively. The metagenomic analysis showed that Caproicibacterium lactatifermentans, Ligilactobacillus acidipiscis, Clostridium tyrobutyricum, and ‘Butyriproducens baijiuensis BJN0003’ were the core species of SimpCom1. C. lactatifermentans and ‘B. baijiuensis BJN0003’ remained stable growth and metabolism in the unsterilized Huangshui, with the relative abundance of 45.3% and 6.7%, respectively, at the end of fermentation with 50%-diluted Huangshui. [Conclusion] We successfully established an efficient and low-cost approach for producing caproate by fermentation of unsterilized Baijiu-making wastewater with a simplified microbial consortium containing C. lactatifermentans and ‘B. baijiuensis BJN0003’.

Abstract:[Objective] To investigate the effects of hydrogen sulfide (H₂S) on the structures of phyllosphere and rhizosphere microbial communities of soybean plants under drought stress. [Methods] High-throughput sequencing of the 16S rRNA gene was combined with bioinformatics analyses (α and β diversity, species composition, co-occurrence networks analysis, etc.) to study the phyllosphere and rhizosphere microbial communities of soybean plants before and after NaHS treatment. [Results] For the soybean plants under normal moisture conditions, the addition of NaHS decreased the diversity and increased the endemic species of phyllosphere microbial community. The addition of NaHS increased the diversity of rhizosphere microbial community of soybean plants under normal moisture conditions but not under severe drought. In addition, exogenous addition of NaHS altered the bacterial co-occurrence network, and the microbial communities in both phyllosphere and rhizosphere were so aggregated that neither rhizobium inoculation nor NaHS addition had significant influences on them. The addition of NaHS mildly affected the relative abundance of operational taxonomic unit (OTU) in the phyllosphere and decreased the relative abundance of OTU in the rhizosphere, which was particularly pronounced under severe drought. Rhizobium inoculation and NaHS addition enriched different microbial taxa in both phyllosphere and rhizosphere. [Conclusion] Under drought stress, H₂S had an insignificant modulating effect on the microbial community structure in the phyllosphere but a pronounced effect on the microbial community structure in the rhizosphere of soybean plants. H₂S reduced the relative abundance of total OTU in the rhizosphere and altered the bacterial co-occurrence network, thus influencing soybean adaptation to drought stress.

Abstract:[Objective] To study the species and community structure of bacteria in winged and wingless Megoura crassicauda (Mordvilko). [Methods] Illumina MiSeq high-throughput sequencing was carried out for the V3-V4 variable region of bacterial 16S rRNA gene. [Results] A total of 4 746 449 sequences were obtained and annotated to 586 operational taxonomic units (OTUs) belonging to 477 species, 336 genera, 192 families, 106 orders, 46 classes of 25 phyla. Firmicutes, Actinobacteria, Proteobacteria, and Cyanobacteria were the dominant phyla in all samples. The dominant genera included Staphylococcus, Corynebacterium, and Buchnera. At the phylum level, the relative abundance of Proteobacteria, Cyanobacteria, and Deinococcus-Thermus in the winged morph was significantly higher than that in the wingless morph. At the genus level, the relative abundance of Buchnera, Exiguobacterium, Pseudomonas, Glutamicibacter, and Pseudonocardia in the winged morph was significantly higher than that in the wingless morph. [Conclusion] This study clarified the diversity and community structure of the bacteria in winged and wingless M. crassicauda and provided a reference for exploring the role of bacteria in the wing differentiation of aphids and developing new methods for pest control.

Abstract:[Objective] To evaluate the safety and immunogenicity of outer membrane vesicles (OMVs) of Fusobacterium nucleatum prepared under the pH conditions simulating normal intestinal and tumor microenvironments, and to lay a foundation for the later development of OMV-based vaccines against F. nucleatum. [Methods] Ultra-high-speed centrifugation and density gradient centrifugation were employed to isolate the OMVs of F. nucleatum under different pH conditions. Three models of human colorectal cancer cells (HCT116), human colonic epithelial cells (HCoEpiC), and mouse macrophage Raw264.7 were infected by F. nucleatum. The cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell counting kit-8 (CCK-8) assay, and the cell apoptosis were evaluated by flow cytometry and in situ fluorescence of adherent cells. The safety in vivo was detected by blood routine and whole blood index of mice. The enzyme-linked immunosorbent assay (ELISA) was employed to examine the cell immunogenicity, and the adhesion inhibition assay was conducted to assess cell immunoprotection. [Results] Acidic outer membrane vesicles (aOMVs) and neutral outer membrane vesicles (nOMVs) had no significant different effects on the proliferation or apoptosis of HCoEpiC, HCT116, and Raw264.7 cells. Both nOMVs and aOMVs at a concentration of 25 μg/mL did not lead to significant hemolysis of red blood cells, and the routine blood and whole blood test results of the mice treated with aOMVs were within the normal ranges. The results demonstrated that both aOMVs and nOMVs exhibited good immunogenicity and reduced the adhesion of F. nucleatum to colon cancer cells DLD-1. The aOMVs outperformed nOMVs regarding immunogenicity. [Conclusion] Both aOMVs and nOMVs had good safety and immunogenicity, and aOMVs were superior to nOMVs.

Abstract:[Objective] To investigate the changes of microbial communities on loess slopes with different noise conditions under the influences of temperature and time. [Methods] Based on the changes in soil phosphate content, the noise was determined at 70, 90, and 110 dB. The microbial communities presented variations along the temperature gradients of -5, 15, and 35 °C and the noise duration of 2, 4, and 6 h. Subsequently, metagenomic sequencing was carried out for the soil microbial communities. [Results] At the phylum level, Actinobacteria, Mucoromycota, Thermoproteota, and Myxococcota showed differences in the relative abundance among groups (P<0.05). At the genus level, Arthrobacter, Rhizophagus, Pseudarthrobacter, Actinomadura, Kocuria, Rubrobacter, and Corynebacterium demonstrated different relative abundance among groups (P<0.05). At the species level, there were significant differences in the relative abundance of Rhizophagus irregularis, Actinomadura sp. WMMB 499, Rubrobacter tropicus, Arthrobacter sp. PGP41, Arthrobacter sp. 24S4 2, and Arthrobacter crystallopoietes among groups (P<0.05). [Conclusion] Different noise environments have significant effects on the relative abundance of soil microorganisms at the phylum, genus, and species levels.

Abstract:[Objective] This study aims to validate the functions of two ferritin-encoding genes: bacterioferritin (Bfr)-encoding gene (atu2771) and DNA-binding protein from starved cells (Dps)-encoding gene (atu2477), in Agrobacterium fabrum, to determine the open reading frame (ORF) of the Bfr-encoding gene, to investigate the effects of terminal fusion, heme group, and key residues on the function and self-assembly of A. fabrum Bfr, and to explore the potential applications of the two ferritin nano-cages. [Methods] We re-introduced the two ferritin-encoding genes into the ferritin-deficient mutants of A. fabrum respectively via plasmids to verify if the re-introduction could complement the ferritins of the ferritin-deficient mutants and thus validate the functions of the two genes. Native PAGE was employed to separate the ferritins in the crude extract of A. fabrum and potassium ferrocyanide (an iron-specific staining reagent) was used to stain the ferritins. Various peptides or protein were fused to the termini of two ferritins to test if the terminal fusion would affect the functions and self-assembly of the two ferritins. Site-directed mutation was then employed to test the effects of the key residue and heme group on the function and self-assembly of Bfr. [Results] Iron-specific staining on the ferritins separated by native PAGE showed that the Bfr-encoding gene expressed Bfr in all the tested A. fabrum strains, whereas the Dps-encoding gene expressed Dps in none of the tested A. fabrum strains. Complementary experiment with two different Bfr-encoding ORFs (encoding 161 residues and 169 residues) showed that Bfr in the wild type was encoded by the ORF encoding 161 residues. The results demonstrated that terminal fusions with different peptides or protein influenced but did not abolish the function and self-assembly of Bfr. The substitution of Met₆₀, which was predicted to chelate the iron of heme, indicated that heme affected the function and self-assembly of Bfr but was not indispensable. [Conclusion] A. fabrum utilizes Bfr to store iron. The ORF of the Bfr-encoding gene utilizes uug (a rare start code) as its start code and encodes a Bfr composed of 161 residues. The Dps-encoding gene of A. fabrum expressed in none of the tested conditions. The structures of both Bfr and Dps of A. fabrum are stable enough to withstand the terminal fusion with various peptides or protein, suggesting that both Bfr and Dps nano-cages demonstrate great promise for biotechnological applications.

Abstract:[Objective] To develop probiotics for gynecological inflammation, we isolated and screened out Lactobacillus with antimicrobial and probiotic properties from the vagina of healthy women. [Methods] The plate streaking method was employed to isolate the Lactobacillus strains from vaginal samples, and the isolates were then identified based on morphological and 16S rRNA gene sequencing evidence. The growth and adhesion of the five strains were characterized. The Oxford cup method with Escherichia coli and Staphylococcus aureus as indicator strains was employed to assess the antimicrobial activities of the strains. The microtiter plate method was used to measure the inhibitory effects of the strains on Candida albicans. The antimicrobial components were explored by organic acid elimination and hydrogen peroxide elimination methods. [Results] Five strains of Lactobacillus were isolated, including three strains (Q2.1, BHC04, and Q8.5) of Lactobacillus crispatus and two strains (Q6.3 and BHG05) of Lactobacillus gasseri. All the five strains of Lactobacillus had strong growth and high acid production. Strains Q2.1, BHC04, and BHG05 had a short delay period and reached a plateau growth stage after 20 h, and strains BHG05, Q6.3, and Q8.5 had high acid production, with the culture medium finally reaching pH 3.80-4.03. The adhesion capacity (hydrophobicity, self-agglutination rate, and co-agglutination rate with pathogens) of L. crispatus Q2.1, BHC04, and Q8.5 was significantly higher than that of the positive control strain, L. delbrueckii DM8909. The inhibitory effects of the five strains on E. coli, S. aureus, and C. albicans were stronger than those of the positive control (nisin). Strains BHC04, BHG05, and Q8.5 showcased stronger inhibitory effects on E. coli than DM8909. The inhibitory effect of BHG05 on C. albicans was significantly stronger than that of DM8909, with the inhibition rate reaching up to (73.14±0.14)%. The inhibitory effects of BHC04 and Q8.5 on C. albicans were not significantly different from that of DM8909, with the inhibition rates reaching up to (72.80±0.30)% and (72.93±0.10)%, respectively. According to the results above, we selected BHC04 and BHG05 as high quality strains with antimicrobial potential. The five strains produced organic acids and hydrogen peroxide to exert antimicrobial effects. [Conclusion] Two strains of Lactobacillus with antimicrobial effects and excellent probiotic properties were screened out. They can be used as candidate strains of antimicrobial probiotics for the prevention and treatment of gynecological inflammation caused by E. coli, S. aureus, and C. albicans.

Abstract:[Objective] To study the effects of endoplasmic reticulum stress on the regulation of the lifespan and autophagy of Saccharomyces cerevisiae by the protein O-mannosyltransferase 1 (PMT1). [Methods] The double deletion strain (pho8Δ60 pmt1Δ) was constructed based on the genetic homologous recombination. The daughter cells produced by the mother cell of the PMT1-deleted yeast strain (pmt1Δ) treated with tunicamycin (inducing endoplasmic reticulum stress) were counted under a light microscope, and the replicative lifespan of the strain was examined. A microplate reader was used to measure the alkaline phosphatase activity of the pho8Δ60 pmt1Δ strain in the SD-N medium (for inducing autophagy). Western blotting was employed to determine the expression level of the autophagy marker Atg8 in the presence of tunicamycin. The transcript levels of autophagy-related genes ATG1 and ATG8 in the pmt1Δ strain treated with tunicamycin were determined by RT-qPCR. [Results] The replicative lifespan of the pmt1Δ strain was shortened by 38.7%, while the alkaline phosphatase activity of pmt1Δ strain was increased compared with those of the wild type in the presence of tunicamycin. The expression levels of GFP-Atg8 fusion protein and free GFP in the pmt1Δ strain were up-regulated with the increase in the concentration of tunicamycin. The transcript levels of ATG1 and ATG8 were up-regulated in the pmt1Δ strain treated with tunicamycin. [Conclusion] Endoplasmic reticulum stress impairs the replicative lifespan and enhances the autophagy of PMT1-deleted yeast cells.

Abstract:[Objective] To study the transcriptome regulation mechanism of Fusarium graminearum under different pH stress conditions, analyze the changes in gene expression levels, explore the metabolic pathways involved in the responses of F. graminearum cells to acidic or alkaline conditions, and reveal how F. graminearum regulates intracellular metabolism and synthesis to adapt to the changes in extracellular pH. [Methods] F. graminearum was cultured in the PDB media with initial pH 4.5, 6.5, and 8.0 for 48 h, and the total RNA of the strains was extracted to construct the cDNA library. Transcriptome sequencing and bioinformatics analysis were used to identify the differentially expressed genes (DEGs), and the metabolic pathways involved were analyzed. The expression levels of target genes were determined by RT-qPCR. [Results] Under acidic conditions, a total of 4 283 DEGs were identified, including 2 032 genes with up-regulated expression and 2 251 genes with down-regulated expression. Under alkaline conditions, a total of 498 DEGs were identified, including 269 genes with up-regulated expression and 229 genes with down-regulated expression. Gene ontology (GO) enrichment analysis revealed 211 GO terms for the up-regulated genes and 72 GO terms for the down-regulated genes under acidic conditions. Under alkaline conditions, GO analysis yielded 33 GO terms for the up-regulated genes and 40 GO terms for the down-regulated genes. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed 22 up-regulated pathways and 32 down-regulated pathways under acidic conditions as well as 8 up-regulated pathways and 13 down-regulated pathways under alkaline conditions. The expression of the genes associated with membrane transporters and hydrolysis of carbohydrates was up-regulated, and that of the genes related to protein metabolism was down-regulated, which assisted F. graminearum cells to adapt to changes in the external environment. At the same time, F. graminearum maintained the internal environment balance by reducing secondary metabolism and amino acid metabolism under acidic and alkaline conditions, respectively, to resist extracellular pH stress. [Conclusion] In the acidic environment, F. graminearum adapts to the changes in the extracellular environment by promoting the production of ribonucleoprotein complexes and secondary metabolism. In an alkaline environment, F. graminearum senses and responds to external stresses via amino acid metabolism. The analysis of the metabolic pathways of F. graminearum cells provides gene expression data for studying the responses of F. graminearum to different pH environments. The findings of this study are helpful to understand the pathogenic mechanism of F. graminearum.

Abstract:[Objective] To select the elite strains among the microorganisms isolated from the rhizosphere of Brassica oleracea var. capitata and evaluate the secondary metabolism potential of the strains, laying a solid foundation for mining the microbial resources in the rhizosphere of this plant. [Methods] The roots of B. oleracea var. capitata were collected from Xiannüshan Town in Wulong District of Chongqing. The conventional methods were then used to isolate microbial strains, and one strain of Serratia marcescens was selected for genome sequencing (PacBio RS II and Illumina HiSeq) and bioinformatic analysis. The antiSMASH was used for the detection and comparison of biosynthetic gene clusters (BGCs) encoding secondary metabolites. Red/ET recombination was employed to capture the BGC for prodigiosin. [Results] One prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The genome of this strain contained 11 putative BGCs (1-11) for secondary metabolites, of which 9 BGCs displayed low similarities to the BGCs encoding known compounds. This result suggested that the strain had great potential of producing novel secondary metabolites. Prodigiosin was identified by HPLC and high-resolution mass spectrometry (HRMS). Heterologous expression of BGC4 resulted in the production of prodigiosin in Escherichia coli. Four common promoters were used to drive the expression of BGC in E. coli, and the highest prodigiosin production was observed with the rpoS promoter. [Conclusion] A prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The BGC for prodigiosin was captured from the chromosome of this strain and expressed successfully in the surrogate host E. coli BAP1.

Abstract:[Objective] In view of the enhanced cell-to-cell spread ability of the dsbA-deleted strain (ΔdsbA) of Listeria monocytogenes, this study aims to elucidate the mechanism that how the disulfide bond formation protein DsbA mediates this biological process. [Methods] The mRNA and protein levels of virulence factors in the wild type and ΔdsbA were compared by RT-qPCR and Western blotting, respectively. The immunofluorescence co-localization analysis method was employed to observe the impact of DsbA deficiency on the actin recruitment by the virulence factor ActA in the cell-to-cell spread of L. monocytogenes (analyzing the length and quantity of the comet tails formed on one side of the bacteria by co-localization of ActA and actin). The presence or absence of interaction between DsbA and ActA was determined by isothermal titration calorimetry (ITC). [Results] Compared with the wild type, ΔdsbA showed no significant changes in the mRNA levels of virulence factors, downregulated protein levels of InlA, InlB, PlcA, and PlcB, and upregulated protein levels of ActA and LLO. In addition, ΔdsbA showed increased number and average length of comet tails, which indicated that the actin recruitment of ΔdsbA was enhanced. The ITC results revealed that DsbA bound to ActA, which gradually showed endothermic reactions, suggesting the presence of interaction between DsbA and ActA. [Conclusion] This study proved for that DsbA attenuated the recruitment ability of actin by regulating virulence proteins, thus affecting the cell-to-cell spread of L. monocytogenes. The findings help to further dissect the virulence regulatory mechanisms of L. monocytogenes during host infection, which is of great importance for controlling the contamination of zoonotic intracellular pathogens threatening public health.

Abstract:Plastic pollution is an environmental problem that has aroused global concern, and plastics degradation by insect gut microbiota is a new initiative to solve this problem. Despite the important role of insect gut microbiota in the degradation of plastics, little is known about the composition and dynamics of insect gut microbiota. [Objective] To study the effects of feeding plastics on the physiological indices and the composition and dynamics of gut microbiota in the larvae of four insect species. [Methods] Polystyrene (PS), polyethylene (PE), and wheat bran (control) were used as the sole carbon source respectively to feed the larvae of Zophobas atratus Fab., Tenebrio molitor L., Tenebrio obscurus F., and Galleria mellonella L. The dynamics of gut microbiota in the larvae of the four insect species were investigated by fluorescence in situ hybridization, and the correlations between the physiological indices and gut microbiota were analyzed. [Results] All four insect species fed with PS and PE had significantly lower body weight gain and body length increase than those in the control. The survival rates of Z. atratus and T. molitor larvae fed with PS were 25.33% and 11.75%, respectively, higher than those fed with PE. The dominant microbial taxa of the gut microbiota in the four insect species were Firmicutes (relative abundance of 16.98%-54.93%), Betaproteobacteria (5.91%-39.34%), Gammaproteobacteria (4.62%-30.86%) of Proteobacteria, and Euryarchaeota (9.99%-58.05%). [Conclusion] In addition to Firmicutes and Proteobacteria, archaea were also dominant in the gut microbiota in the larvae of the four species fed with PE and PS. The relative abundance of the main microbial taxa in the insect gut varied dynamically over time and was influenced by the types of plastics as well as the insect species. The body weights and body lengths of Z. atratus and T. molitor were correlated with their gut microbiota.

Abstract:[Objective] The Yellow River estuary located at the confluence of the Yellow River, land, and ocean is an area with mixed freshwater and seawater and a diverse and productive estuary ecosystem. This study aims to characterize the bacterial communities in freshwater and seawater of the Yellow River estuary. [Methods] High-throughput absolute abundance quantification was adopted to measure the absolute abundance of bacterial communities. The dominant taxa, α and β diversity, co-occurrence network, assembly mechanisms, and potential functions were compared between the bacterial communities in freshwater and seawater. The correlations between dominant taxa and environmental factors were explored. [Results] The absolute abundance of bacteria in freshwater was 2.61×10⁶ copies/mL, which was 1.8 times of that in seawater. The common dominant phyla in freshwater and seawater were Actinomycetota, Pseudomonadota, Cyanobacteriota, and Bacteroidota, with significant differences in absolute abundance. The abundance of Actinomycetota ranked first in freshwater, which was approximately equal to the sum of all dominant phyla in seawater. The abundance of Pseudomonadota was the highest in seawater. The alpha diversity of bacteria in freshwater was higher than that in seawater. There were significant differences in the bacterial community structure between freshwater and seawater, mainly due to the differences in the abundance of the dominant taxa. The bacterial co-occurrence network in freshwater was more complex and stable than that in seawater, and stochastic processes dominated the bacterial community assembly in both freshwater and seawater. The bacterial communities in freshwater and seawater presented different functions, while they shared some common functions. Metabolism was the most abundant function, with higher relative abundance in freshwater than in seawater. Five environmental factors ((pH, oxidation-reduction potential (ORP), electrical conductivity (EC), total organic carbon (TOC), and total nitrogen (TN)) correlated with the dominant bacterial taxa to different extent. There were collinear relationships among the four environmental factors except EC. The dominant genera showing positive correlations with pH, TOC and TN were all negatively correlated with ORP, and vice versa. Actinomycetota and Pseudomonadota were positively and negatively correlated with pH, respectively. [Conclusion] The bacterial communities showed great differences between freshwater and seawater in the Yellow River estuary. The differences were mainly reflected in the abundance, diversity, functional structure, and co-occurrence network. The bacterial communities in freshwater and seawater had similar dominant taxa and assembly mechanisms. The results provide data support for studying the microbial ecology and exploiting microbial resources in the Yellow River estuary.

Abstract:[Objective] To study the sulfur oxidation characteristics of Halothiobacillus diazotrophicus LS2 under different oxygen levels and to decipher the mechanism of strain LS2 adapting to low-oxygen environments. [Methods] The concentrations of S₂O₃^2- and SO₄^2- were measured by ion chromatography. Bacterial growth was determined by plate dilution coating method. The differentially expressed genes and related metabolic pathways were identified and analyzed by transcriptome sequencing and bioinformatics technology. [Results] Strain LS2 oxidized reduced sulfur compounds and grew under 0.2%-21.0% oxygen, and it maintained high sulfur oxidation activity under the oxygen level above 1.6%. Comparative transcriptomic analysis screened out 851 differentially expressed genes that might be related to the adaptation to low oxygen, including 464 up-regulated genes and 387 down-regulated genes. In sulfur metabolism, thiosulfate sulfurtransferase, sulfur oxidase/reductase, and sulfide:quinone oxidoreductase were up-regulated, while the Sox enzyme system was down-regulated, which indicated that strain LS2 might change the sulfur oxidation pathway to adapt to low-oxygen environment. In the low-oxygen group, the cbb₃-type cytochrome c oxidase was up-regulated to increase the O₂-binding efficiency. Meanwhile, since less electron could be received by O₂, the nitrogenase genes nifDKH and Fix complex genes fixA, fixB, fixC, fixX were up-regulated, making N₂ and CO₂ the alternative electron accepters to maintain redox balance, which explained the higher maximum bacterial growth in low-oxygen environments. [Conclusion] Strain LS2 is a sulfur-oxidizing bacterium that can maintain high sulfur oxidation activity in the low-oxygen environment. Sulfide:quinone oxidoreductase, high-oxygen-affinity terminal oxidases, and nitrogenase play a role in the adaptation to the low-oxygen environment. This study is of positive significance for deciphering the mechanism of sulfur oxidation under low oxygen and provides a theoretical basis for optimizing the treatment process of sulfur-containing wastewater.

Abstract:[Objective] To realize the de novo biosynthesis of caffeic acid from glucose by reconstruction of its biosynthetic pathway in Escherichia coli. Fine-tuning gene expression allows us to improve caffeic acid production, which paves a way for the high production of caffeic acid and its derivatives in E. coli. [Methods] The biosynthetic pathway of caffeic acid was reconstructed based on FjTAL and EchpaBC, which encoded the tyrosine ammonia lyase in Flavobacterium johnsoniaeu and the 4-hydroxyphenylacetate 3-hydroxylase complex in E. coli, respectively. The reconstructed pathway was then introduced into commonly used E. coli strains. we improved the expression levels of FjTAL and EchpaBC by screening constitutive promoters, utilizing an intermediate-based biosensor, and increasing the copy number of the key gene. Thus, a total of fourteen recombinant strains were obtained, and the production of caffeic acid and the intermediate p-coumaric acid in these strains was quantified by HPLC. Moreover, the effects of different nitrogen sources and substrate concentrations on the production of caffeic acid were investigated. [Results] We realized de novo biosynthesis of caffeic acid from glucose in E. coli. The use of constitutive promoters other than the commonly used T7 promoter contributed to the yield increase of caffeic acid. When glucose was used as the substrate, the yield of caffeic acid was increased from 1.40 mg/L to 96.40 mg/L. When tyrosine was used as the substrate, the yield of caffeic acid was increased from 1.78 mg/L to 123.31 mg/L. Furthermore, the yield of caffeic acid reached 162.73 mg/L when a p-coumaric acid biosensor instead of a constitutive promoter was used to drive the expression of EchpaBC. Moreover, the yield of caffeic acid was improved to 185.15 mg/L in the case of introducing an extra copy of EchpaBC. [Conclusion] We constructed the strains with high production of caffeic acid by promoter engineering, using an intermediate-base biosensor, and increasing copy number of the key gene. Our study laid a solid foundation for the high production of caffeic acid.

Abstract:[Objective] To investigate the antibiotic resistance profiles, resistance genes, and bla_NDM transmission characteristics of bacterial isolates from six turtle farms in two regions (three in Lanxi (LX) and three in Xiaoshan (XS)) of Zhejiang Province. [Methods] Bacterial strains were identified by 16S rRNA gene sequencing. The Kirby-Bauer method was employed to examine the antibiotic susceptibility of isolated bacteria. PCR was employed to detect resistance genes carried by the isolated bacteria. Conjugation transfer experiments were conducted to obtain bla_NDM-carrying plasmids, and the conjugation frequency was analyzed to assess the transfer capabilities of bacterial donors. Additionally, the modified Carba NP test, plasmid replicon typing, and minimum inhibitory concentration determination were carried out to evaluate the transmission of bla_NDM-carrying plasmids among bacteria. [Results] A total of 244 bacterial strains were isolated, with similar distribution between the two regions and Enterobacteriaceae being dominant. PCR amplification of integrons revealed various resistance genes, including those conferring resistance to aminoglycosides and trimethoprim. We identified numerous resistant bacteria capable of integrating additional exogenous resistance genes. All the isolated strains exhibited resistance to at least one antibiotic, with some displaying resistance to carbapenems. PCR detection of resistance genes indicated the dissemination of carbapenem resistance genes within some farms. This study confirmed the presence of bla_NDM on plasmids capable of inter-bacterial transmission. Moreover, the farms in LX exhibited significantly higher conjugation frequency of bla_NDM-carrying plasmids than the farms in XS. [Conclusion] Antibiotic resistance of bacteria in aquaculture environments is becoming increasingly serious. The carbapenem resistance gene bla_NDM has spread within turtle farms.

Abstract:In Pseudomonas aeruginosa, tryptophan can be converted into anthranilate via the KynABU pathway, and anthranilate as a substrate is further converted into alkyl quinolones (AQs), including 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ), under the action of pqsABCDE, a synthetic gene cluster of AQs. At the same time, anthranilate can be degraded into the tricarboxylic acid cycle under the catalysis of the anthranilate dioxygenase complex AntABC, while the biological effect of AntABC on P. aeruginosa remains unclear. [Objective] To construct and characterize the phenotype of the antABC-deleted mutant of P. aeruginosa. [Methods] With P. aeruginosa PAO1 as the starting strain, we constructed the antABC-deleted mutant by homologous recombination to study the effects of the operon on tryptophan degradation, biofilm formation, pyocyanin synthesis, motility, and virulence of P. aeruginosa. [Results] The deletion of antABC or kynU completely inhibited the growth of P. aeruginosa with tryptophan as the sole carbon source, while ΔpqsA did not present this phenotype, indicating that antABC was essential for the degradation of tryptophan by P. aeruginosa, and KynABU-AntABC pathway was the only way for the degradation of tryptophan by the bacterium under the culture conditions in this study. In addition to affecting tryptophan degradation in P. aeruginosa, the deletion of antABC promoted the biofilm formation of P. aeruginosa by inducing the expression of the extracellular polysaccharide synthesis operon pel, and it promoted the synthesis of pyocyanin by inducing the expression of the pyocyanin synthesis operons phz1 and phz2. In addition, the deletion of antABC enhanced the swarming motility and twitching motility of P. aeruginosa. Interestingly, further deletion of pqsA completely reversed the physiological phenotypes of ΔantABC. Therefore, the regulation of antABC on these physiological phenotypes depended on AQs. The deletion of antABC increased the HHQ accumulation while inhibiting the synthesis of PQS in P. aeruginosa. These results indicated that the regulation of these physiological phenotypes by antABC mainly depended on HHQ. In addition, the deletion of antABC enhanced the virulence of P. aeruginosa to Chinese cabbage and Galleria mellonella larvae, while further deletion of pqsA only partially reversed this virulence phenotype. Moreover, the deletion of antABC caused increased accumulation of anthranilate in P. aeruginosa. Therefore, the enhancement of antABC deletion on the virulence of P. aeruginosa was mediated by HHQ and anthranilate together. Finally, bioinformatics analysis revealed that the missense mutations of antABC operon occurred in more than 90% of clinical isolates of P. aeruginosa. Therefore, antABC was expected to be used as a biomarker to determine whether clinical isolates of P. aeruginosa were highly virulent. [Conclusion] AntABC plays an important role in the tryptophan degradation, biofilm formation, pyocyanin synthesis, motility, and virulence of P. aeruginosa. This finding lays a foundation for the clinical diagnosis and antimicrobial development of P. aeruginosa infection.

Abstract:[Objective] To establish a methodology assessing the existence and active state of biofilms on mudflat. [Methods] We sampled the biofilms on mudflat and the surface sediments in the nearby regions, and compared the dominant algal species and their abundance between the biofilms and their nearby regions by 18S rRNA gene sequencing. Furthermore, we employed chlorophyll a (Chl-a) assay and flow cytometry to compare the concentration of Chl-a and the amount of Chl-a-containing cells between the biofilms and their nearby regions. [Results] The mudflat biofilms mainly harbored Diatomea, Dinoflagellata, Ochrophyta, Chlorophyta, Cryptophyceae, and Phragmoplastophyta, the relative abundance of which, however, varied significantly in different seasons or geographic locations. There was a significant difference in the concentration of Chl-a between biofilms and their nearby regions. The amount of Chl-a-containing cells in biofilms was significantly higher than that in the nearby regions. A method for assessing biofilms on mudflat was established based on the relative abundance of algae, the concentration of Chl-a, and the amount of Chl-a-containing cells. In brief, a sample is classified as a biofilm in the case of the relative abundance of algae higher than 40%, the Chl-a concentration higher than 500 mg/m³, and the amount of Chl-a-containing cells more than 500 cells/μL. Otherwise, the sample is classified as inexistence of biofilm. Additionally, the amount of Chl-a-containing cells more than 1 500 cells/μL indicates that the biofilm is in the vigorous growth phase, and that between 500 cells/μL and 1 500 cells/μL suggests that the biofilm is in the colonization or recession phase. [Conclusion] We compared the dominant algal species and their relative abundance, the concentration of Chl-a, and the amount of Chl-a-containing cells between biofilms on mudflat and their nearby regions, and established a methodology assessing the existence and active state of biofilms on mudflat based on the above indicators. The findings enriched the knowledge of biofilms on mudflat and provided a theoretical basis for understanding the microbial carbon sequestration capacity of biofilms on mudflat.

Abstract:[Objective] Due to the short half-life of mRNA transcript, their anti-degradation ability and ability to recruit ribosomes to initiate translation have significant effects on the gene expression in prokaryotes. However, the multiple functional regions in the 5′ end, of mRNA can affect the half-life and thus the expression of target genes, including the Shine-Dalgarno (SD) sequence and its upstream and downstream translational standby sites (TSSs) and the N-terminal coding sequence (NCS). The own and cross-regional structural differences will affect gene expression. Therefore, it is important to analyze the structure-activity relationship of each functional region. [Methods] We employed primer extension sequencing (SHAPE-seq) to analyze the structural characteristics of seven different 5′mRNAs in Escherichia coli and collected the mRNA abundance and protein level information under their regulation. [Results] Under the regulation of unstructured NCS, the mRNA abundance and protein level were increased by 10 and 19 times, respectively. The formation of secondary structure TSS with a stem length of 10 nt increased the mRNA abundance. When the SD sequence was wrapped to form a secondary structure, the efficiency of translation initiation mediated by the SD sequence was affected (protein level downregulation by 10%). The combination of TSS and NCS significantly increased the mRNA abundance and protein level by 11 and 60 folds, respectively. [Conclusion] We characterized the structure of each region of 5′mRNA conducive to prokaryotic gene expression and revealed the structure-activity relationship of each functional region, providing a new regulatory element for target gene expression in industrial microorganisms.

Abstract:[Objective] Riemerella anatipestifer (RA) is a Gram-negative bacterium that mainly infects domesticated birds such as ducks and geese. The clinical isolates of RA are multi-drug resistant and increasing year by year. However, the transmission ways of antibiotic resistance genes in RA have not been identified. This study aims to identify the transmission ways and distribution of antibiotic resistance genes in the clinical isolates of RA. [Methods] The drug resistance phenotypes of the reference strain RA ATCC 11845 and the clinical isolates RA CH-1 and RA CH-2 to 28 antibiotics belonging to 10 categories were determined. The antibiotic resistance genes were identified by genome analysis and construction of gene deletion strains. The transmission ways of antibiotic resistance genes were identified by natural transformation. The distribution of these resistance genes in different clinical isolates was detected by PCR. [Results] RA CH-1 and RA CH-2 were resistant to β-lactams, tetracyclines, macrolides, lincosamides, and amide alcohols, while RA ATCC 11845 was sensitive to the above antibiotics. The resistant strains became sensitive to the corresponding antibiotics after the deletion of 13 resistance genes, respectively, indicating that these genes were involved in antibiotic resistance. All the resistant genes can be transferred to the sensitive strain RA ATCC 11845 by natural transformation. The detection rates of resistance genes in 100 clinical isolates from 2017 to 2023 varied within the range of 3% to 89%. [Conclusion] Antibiotic resistance genes can be transmitted in RA through natural transformation, and different antibiotic resistance genes presented varied distribution in clinical isolates, among which tetX (B739_0030) and bla_OXA (G148_1768) were carried by the most and fewest strains, respectively.