[Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome ofPyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties ofPyprol encoded byPYCH_07700in vitro, aiming to find a new prolidase. [Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeonThermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. [Results] Pyprol showed the best performance at 100 ℃ and pH 6.0.Pyprol binding to Co²⁺ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L.Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidasePfprol fromP.furiosus.Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 ℃ under 40 MPa, compared with at the atmospheric pressure. [Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase ofP.yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.

[Objective] Listeria monocytogenes (Lm) is a ubiquitous foodborne pathogen causing listeriosis.Lm can grow at low temperatures and thus may cause safety problems of refrigerated food and threaten the public health. The growth ofLm at low temperatures involves the inhibition of flagellar gene expression, which restricts flagellar biosynthesis. MogR is a transcriptional repressor which represses the expression of flagellar genes during intracellular infection and during extracellular growth ofLm at 37 ℃, resulting in no biosynthesis of flagella. Whereas MogR is deprived of repression function and the bacteria produce flagella during growth at 20–30 ℃. Our studies demonstrated thatLm significantly reduced the flagellar production at 4 ℃, but the molecular mechanism of which remained unclear. This study aims to reveal the relationship between the reduction of flagella and MogR repression at 4 ℃. [Methods] We constructed themogR-deleted mutant ΔmogR and the flagellin geneflaA-deleted mutant ΔflaA (as the control strain with no flagella), and their complementary strains cΔmogR and cΔflaA with theLm strain ATCC 19115 as the parental strain. Then, we analyzed the swarming motility, flagellar biosynthesis, and transcriptional levels of flagellar genes in above five strains at 4 ℃, 28 ℃, and 37 ℃, respectively. The growth curves of these strains were determined at 4 ℃, 28 ℃, and 37 ℃, respectively. [Results] Compared with the parental strain, ΔmogR showed significantly increased in motility, flagellar biosynthesis, and transcriptional levels of flagellar genes (P < 0.01, 0.001, and 0.001, respectively) at 4 ℃. The growth of ΔmogR markedly decreased compared with the parental strain (P < 0.05) at 4 ℃. The data of motility, flagellar biosynthesis, and transcriptional levels of flagellar genes in cΔmogR had no significant differences compared with the parental strain. [Conclusion] The reduction in flagellar biosynthesis was associated with the repression function of MogR inLm at 4 ℃. The reduction in flagellar biosynthesis was of benefit toLm proliferation at low temperatures. This study enriched our understanding of the mechanism ofLm growth at low temperatures.

[Objective] To identify efficient aerobic denitrifying bacteria in the phyllosphere and provide new ideas for the prevention and control of air pollution by phytoremediation. [Methods] We used the enrichment culture method combined with the Griess reagent and bromothymol blue to isolate aerobic denitrifying bacteria from the phyllosphere and analyzed the phylogenetic relationship of the isolates based on the 16S rRNA gene sequences. Efficient aerobic denitrifying bacteria were screened out and their denitrification conditions were optimized. [Results] Thirteen strains were isolated from the phyllosphere of six landscape plant species:Photinia serratifolia,Ligustrumlucidum,Osmanthus fragrans,Camphora officinarum,Euonymus japonicus, andMagnoliagrandiflora. According to the 16S rRNA gene sequences, the 13 strains were classified into 7 genera, 7 families of 4 phyla. Specifically, 4, 3, and 2 strains belonged toEnterobacter,Achromobacter, andPseudomonas, respectively, and the remaining 4 strains belonged toAcinetobacter,Sphingobacterium,Microbacterium, andPseudarthrobacter. The comparative analysis revealed that strain SF outperformed the other strains in denitrification. The factors (carbon source, temperature, initial pH, C/N ratio, and rotational speed) influencing the denitrification performance of SF were optimized by single factor tests and response surface methodology. The denitrification conditions of SF were optimized as follows: glucose as the carbon source, initial pH 7.5. C/N ratio of 9.7, rotational at 180 r/min, and temperature of 33.5 ℃. Under the optimized conditions, the total nitrogen removal rate reached 93.3% in 72 h when the initial nitrate concentration was 361 mg/L. [Conclusion] The rich culturable aerobic denitrifying bacteria in the phyllosphere of landscape plants laid a foundation for phytoremediation. It is feasible to synergistically reduce nitrogen oxide pollution in the air by screening efficient denitrifying strains and improve the denitrifying performance by optimizing the culture conditions.

Heyndrickxia coagulans characterized by low nutrient requirements, high titers of fermentation products, and thermal tolerance has become a major microbial species for lactic acid fermentation. We have demonstrated that phosphate stimulates the gene expression of L-lactate dehydrogenase inH.coagulans to increase L-lactic acid production, the mechanism of which, however, remains unknown. [Objective] To primarily investigated the mechanism of phosphate in stimulating the gene transcription of lactate dehydrogenase inH.coagulans. [Methods] RT-PCR was employed to analyze the transcriptional level changes of lactate dehydrogenase inH.coagulans after phosphate addition. The core promotor region responsive to phosphate stimulation was identified by conventional methods of molecular biology. [Results] The key element responsive to phosphate was located in the upstream promoter region of the L-lactate dehydrogenase gene. The core region of the promoter was responsible for phosphate stimulation. The identified promoter was employed to promote D-lactate production with diammonium phosphate addition. [Conclusion] This study reported a new phosphate-responsive gene element, providing a theoretical basis for improving the synthesis efficiency of other biochemicals.

[Objective] We investigated the diversity, composition, and functions of soil bacterial communities of wildGlycyrrhiza uralensis Fisch., a typical halophyte and medicinal plant, in the habitats with different degrees of salinization. The study can help to reveal the linkage between soil salinity and microbiome related to the growth, development, and quality formation ofG.uralensis, being essential for improving the quality of cultivatedG.uralensis. [Methods] Soil samples were collected from six main habitats of wildG.uralensis. High-throughput sequencing was employed to compare the diversity, composition, and functions of soil bacterial communities among the habitats of un-salinization (US), light salinization (LS), moderate salinization (MS), and heavy salinization (HS) and to excavate the dominant bacteria in different habitats. [Results] The soil bacterial richness and diversity of wildG.uralensis were higher in LS and MS habitats than in US and HS habitats and were the lowest in the HS group. The principal component analysis (PCA) revealed differences in the composition and functions of soil bacterial communities between groups with different degrees of salinization (P < 0.05). The redundancy analysis (RDA) showed that total salinity (TS) was an important factor influencing the composition and functions of the soil bacterial community in the native habitat. The dominant bacterial genera in the US and LS groups were the same, all of which were plant-beneficial bacteria, includingNocardioides,Streptomyces, andMarmoricola. The significantly dominant genera in the MS group included both the beneficial bacteria unidentified_Acidobacteria and the halophilic bacteriaHalomonas andMarinobacter. The soil bacteria in the HS group were dominated by salinophilic or salinity-tolerant bacteria, includingHalomonas,Marinobacter,Truepera,Alifodinibius,Salinimicrobium, andSalegentibacter. The PICRUSt prediction results underlined the potential of soil bacteria in interactions with plants in US, LS, and MS groups, suggesting that beneficial bacteria in the three habitats influenced the growth, development, and quality formation of wildG.uralensis. Moreover, the prediction results emphasized that the soil bacteria endowed the HS group with the potential of self-repairing for adaptation to highly saline environments and improving the salt tolerance of wildG.uralensis. This result suggested that halophilic and salt-tolerant bacteria in the HS habitat played a key role in the salt tolerance of wildG.uralensis. The MS habitat had both kinds of bacteria, being worthy of attention. [Conclusion] The soil bacterial diversity and richness of wildG.uralensis were remarkably higher in LS and MS habitats than in US and HS habitats. The composition and functions of soil bacterial communities in US and LS habitats were similar and differed significantly from those of the HS habitat, and the MS habitat had characteristics of both the above.

The conventional rescue system of newcastle disease virus (NDV) contains a cDNA clone plasmid and three helper plasmids expressing NDV: nucleocapsid protein (NP), phosphoprotein (P) and polymerase protein (L), respectively. These four plasmids have to be transfected into the same host cell simultaneously to complete the assembly of the virus, which is relatively inefficient. [Objective] To improve the rescue efficiency of NDV, this study aims to establish a two-plasmid rescue system. [Methods] The expression cassettes of NP, P, and L genes were constructed and sequentially cloned into the eukaryotic expression vector pCI to generate a single-helper plasmid, named pCI-NPL, capable of co-expressing NP, P, and L proteins. During this process, the genomic cDNA of the NDV LaSota strain was incorporated downstream of the CMV promoter in the pCI expression vector. Simultaneously, the reporter gene EGFP was inserted into the genome between the P and M genes, accompanied by the introduction of a hammerhead ribozyme sequence at the 5′ end and a hepatitis delta virus ribozyme sequence at the 3′ end. This culminated in the development of the full-genome transcriptional plasmid termed pCI-LaSota-EGFP. The two plasmids pCI-LaSota-EGFP and pCI-NPL were co-transfected as a two-plasmid system into BHK-21 cells to rescue the recombinant virus rLaSota-EGFP. The biological characteristics of the virus were then examined. [Results] RT-PCR, fluorescence microscopy, Western blotting, and growth characterization confirmed that rLaSota-EGFP was correctly constructed and expressed the foreign gene. The rescued recombinant virus rLaSota-EGFP had similar biological characteristics to wild-type (WT) LaSota. [Conclusion] A novel two-plasmid rescue system for NDV based on the CMV promoter was successfully established, laying a foundation for the efficient rescue of recombinant NDV and other paramyxoviruses.

Fusarium head blight (FHB) caused byFusarium graminearum is one of the major diseases limiting wheat production. Biocontrol has been considered an effective and sustainable method for the control of crop diseases. [Objective] To screen out endophytic strains with inhibitory effects onF.graminearum from wheat grains and evaluate their biocontrol potential, providing strain resources and theoretical support for the development and utilization of biocontrol agents against FHB in wheat. [Methods] Plate confrontation, spore germination, and cell-free supernatant (CFS) inhibition experiments were carried out to screen out the endophytic strains with antagonistic activity againstF.graminearum from wheat grains. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were employed to observe the conidial morphology, membrane integrity, and mycelial reactive oxygen species ofF.graminearum treated with the CFS. Pot experiments were performed to verify the biocontrol effects of the endophytic bacteria on FHB. Illumina HiSeq was used for whole genome sequencing. [Results] A highly efficient endophytic strain JB7 inhibiting the growth ofF.graminearum was isolated from wheat grains. The CFS of strain JB7 in the decline stage showed the inhibition rate of 85.23% on the spore germination ofF.graminearum. Moreover, it led to concavity on spore surface, cell membrane damage, leakage of nucleic acids and proteins, and accumulation of reactive oxygen species in the mycelia ofF.graminearum. The CFS of strain JB7 significantly increased the content of soluble protein and malondialdehyde. Strain JB7 could produce protease, cellulase, glucanase, and siderophores. Pot experiments showed that strain JB7 decreased the disease index of FHB (P < 0.05) in wheat. The strain was identified asBacillus methylotrophic JB7 by whole genome sequencing, and it carried 12 gene clusters for the synthesis of secondary metabolites with antimicrobial functions. [Conclusion] Strain JB7 could inhibit the growth ofF.graminearum and demonstrated a strong control effect on FHB, serving as a candidate strain for the biocontrol of FHB in wheat.

[Objective] To analyze the polysaccharide hydrolysis activity and genomic characteristics of a Gram-negative bacterial strain FZY0027 isolated from intertidal seawater. [Methods] The strain FZY0027 was identified based on the morphological characteristics, 16S rRNA gene sequence, and the whole genome sequence determined by Illumina NovaSeq and Oxford Nanopore PromethION. Bioinformatics tools such as dbCAN, EasyCGTree, BRIG, and Easyfig were used to compare the strain FZY0027 withSaccharophagus degradans 2-40^T. The 3, 5-dinitrosalicylic acid (DNS) method was employed to measure the polysaccharide hydrolysis activity of strain FZY0027. [Results] The 16S rRNA gene sequence showed the similarity of 99.9% between strain FZY0027 andS.degradans 2-40^T, and thus strain FZY0027 was preliminarily identified asS.degradans FZY0027. The highest levels of reducing sugars (2.28, 1.75, and 1.10 mg/mL, respectively) were produced by FZY0027 through the hydrolysis of starch, xylan, and mannose. The genome of strain FZY0027 was 5 178 381 bp, encoding a total of 4 156 genes, with the G+C content of 45.8%. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between strain FZY0027 andS.degradans 2-40^T were 96.5%, 96.7%, and 70.0%, respectively. A total of 303 genes were annotated in the Carbohydrate-Active Enzyme database, and there was a significant difference in the number (137 and 130, respectively) of genes encoding glycoside hydrolases (GHs) between strain FZY0027 andS.degradans 2-40^T. Strain FZY0027 carried multiple genes involved in the hydrolysis of starch and xylan, which was corresponding to its strong ability to hydrolyse starch and xylan. However, compared withS.degradans 2-40^T, strain FZY0027 could only hydrolyse a few polysaccharides under the experimental conditions in this study, which suggested that this strain may require specific culture conditions to fully exert its polysaccharide hydrolysis ability. [Conclusion] Strain FZY0027 is a versatile polysaccharide-hydrolyzing bacterium with the potential for bioresource utilization.

[Objective] The microecological effects of transgenic plants withBt andBar genes are an important aspect of environmental safety assessment. However, few studies concern the impacts of rice genotypic alterations induced byBt andBar transformation on the microbial community composition and potential functions in different tissues of rice plants. [Methods] High-throughput sequencing of bacterial 16S rRNA gene and fungal ITS was performed to analyze the microbial community structure and potential functions in the rhizosphere soil, roots, stems, and leaves ofBt andBar transgenic rice T1C-1 and its parent Minghui63 (control) at the heading stage. [Results] The bacterial and fungal diversity varied among different tissues in rice plants, being significantly higher in the underground niches (rhizosphere soil and roots) than in the aboveground parts (leaves and stems). T1C-1 significantly affected the Shannon index and Simpson index of endophytic fungi in leaves but had no significant effect on the microbial diversity in the stems, roots, or rhizosphere soil. The endophytic fungiAspergillus andTalaromyces showed increased relative abundance in the leaves of T1C-1, which suggested their involvement in processes such as carbon metabolism, energy metabolism, and transcription. The average clustering coefficient and average degree of the microbial communities in T1C-1 were significantly higher than those in Minghui63, indicating that T1C-1 increased the complexity of the microbial community network. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) was employed to predict the functional enzyme genes of endophytic fungi in the leaves, which showed that T1C-1 significantly altered the pathways such as carbon metabolism, lipid metabolism, and energy metabolism compared with Minghui63. [Conclusion] The community composition and potential functions of endophytic fungi in leaves were more sensitive to T1C-1 than those in the rhizosphere soil, while T1C-1 did not decrease the diversity of endophytic fungi in leaves. More attention should be paid to the diversity changes of endophytic microorganisms in different ecological niches of plant tissues in the evaluation of the microecological effects of transgenic plants.

[Objective] To investigate the diversity of bacteria and fungi in the pathosphere ofPlasmopara viticola and screen out the strains with potential biocontrol effects on grape downy mildew. [Methods] The leaves infected byP.viticola were collected from seven representative grape-producing regions in northern and southern China in two consecutive years. The collected leaves were cultured in a humid environment, and the newly growing downy mildew was aseptically picked by forceps to prepare the sporangial suspensions ofP.viticola. The strains were isolated by the conventional culture method and identified based on the morphological characteristics, BOX-PCR fingerprints, and molecular sequences. Furthermore, the clustering analysis of different strains was conducted. Sporangial inhibition was tested with equal volumes of strain suspension or fermentation mixed with the sporangial suspension ofP.viticola, and the control effects of isolates and their sterile fermentation against grape downy mildew were tested on detached grape leaves. [Results] A total of 90 bacterial strains and 110 fungal strains were isolated, belonging to eight bacterial genera and 14 fungal genera, respectively. The pathosphere ofP.viticola in the same province and the same year exhibited similar microbial community composition. Notably, strains ofPseudomonas spp. andCladosporium spp. exhibited stable populations on grape cultivars collected from different provinces. A majority (over 80.0%) of strains with stable populations in two consecutive years demonstrated significant biocontrol effects against grape downy mildew. SixAcremonium strains with ubiquitous distribution demonstrated the biocontrol effect up to 100.0%. Sterile fermentation of the fungal strainsAspergillus niger NX2F,Thecaphora amaranthi BJ1G, andRhizopus stolonifer BM1L showed the control effects of 100.0% against grape downy mildew. [Conclusion] The culturable bacterial and fungal communities in the pathosphere ofP.viticola were mainly affected by geographical factors in different provinces, and most of the culturable microorganisms presented stable and strong biocontrol effects on grape downy mildew. To the best of our knowledge, it is the first comprehensive report thatAcremonium spp. were epibiotic fungi and consistently associated withP.viticola, providing rich and valuable biocontrol resources for grape downy mildew.