[Objective] Pseudoalteromonas, an endemic and dominant genus in the ocean, possesses multiple methyl-accepting chemotaxis proteins (MCPs), the functions of which remain unclear. [Methods] The chemotaxis ofPseudoalteromonas arabiensis N1230-9 isolated from surface seawater of the Pacific Ocean towards 23 carbon sources was analyzed by a plate-based assay for swimming motility. Two sCache domain-containing genes (woc28264 andwoc27036) encoding MCPs were deleted by homologous recombination. The chemotaxis of the two mutants to 10 carbon sources was then analyzed. [Results] Strain N1230-9 showed obvious chemotaxis to 10 carbon sources: trehalose, maltose, sucrose, N-acetylglucosamine, L-malic acid, sodium acetate, sodium propionate, sodium pyruvate, citric acid, and succinic acid. WOC28264 was a specific chemotactic receptor for L-malic acid and sucrose, while WOC27036 was a specific chemotactic receptor for citric acid and succinic acid. Both WOC28264 and WOC27036 were chemotactic receptors for N-acetylglucosamine and trehalose. [Conclusion] WOC28264 and WOC27036 have overlapping carbon source effectors.

[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.

[Objective] This study aims to investigate the soil fungal community structure in the tea plantations of Heshan and explore the relationship between fungal community and soil physicochemical properties. [Methods] We employed high-throughput sequencing to analyze the soil fungal community composition of 49 rhizosphere soil samples collected from healthy and mismanaging tea plantations in Heshan. The redundancy analysis (RDA) was performed to analyze the effects of soil physicochemical properties on the fungal community structure. The pairwise Wilcoxon signed-rank test was performed to compare the fungal taxa between the two kinds of tea plantations. FUNGuild was used to predict the soil fungal functions. [Results] Basidiomycota,Mortierellomycota, andAscomycota were the three dominant phyla in the rhizosphere soil of tea plantations in Heshan. The mismanagement of tea plantations increased the relative abundance ofBasidiomycota, but significantly decreased the relative abundance ofMortierellomycota. The fungal richness, Chao1 index, and ACE index were significantly lower in the mismanaging tea plantation than in the healthy tea plantation. The total nitrogen, total phosphorus, available phosphorus, organic matter, and available nitrogen were the primary driving factors for the differences of soil fungal β diversity. The soil pH, total phosphorus, exchangeable Ca²⁺ and Mg²⁺, available phosphorus, and available potassium were significantly correlated with the fungal taxa. Compared with the healthy tea plantation, the mismanaging tea plantation showed decreased relative abundance of core fungal taxa but increased relative abundance of medium and rare taxa. Ten of OTUs was common between healthy and mismanaging tea plantations. The pathogens causing tea gray blight, i.e.,Pseudopestalotiopsis theae andFusarium keratoplasticum, were identified in the healthy tea plantation. The relative abundance of biocontrol fungi,Trichoderma spirale andT.atroviride, significantly increased in the mismanaging tea plantation. The mismanaging tea plantation demonstrated increased relative abundance of pathotrophic, pathotrophic- saprotrophic-symbiotrophic, and symbiotrophic fungi but decreased relative abundance of saprotrophic-symbiotrophic fungi. [Conclusion] We elucidated the relationship of management mode with fungal community composition and soil physicochemical properties, providing the insights into the prevention and control of pathogens infecting black tea and the screening of biocontrol fungi in Heshan.

Pathogenic mycoplasma has the ability to invade host cells, which is a key mechanism to their pathogenicity. Functional proteins that mediate the entry of mycoplasma may serve as potential drug or vaccine targets. [Objective] To clone and express the LRR5 protein encoded byMBOVPG45_0564 inMycoplasmabovis and to explore its role in the invasion of host cells byM.bovis. [Methods] The NCBI database was used for the homology analysis ofMBOVPG45_0564, and the structure of LRR5 protein was predicted by the Discovery Studio Client system. After prokaryotic expression of LRR5 protein, the mouse polyclonal antibody was prepared, and the subcellular localization of LRR5 protein was observed by immunoelectron microscopy. The invasion ofM.bovis into embryonic bovine lung (EBL) cells after LRR5 antibody blocking was observed by plate counting and laser confocal microscopy. After conjugation of LRR5 protein to the surface of fluorescent microspheres, the entry of the microspheres into EBL cells was observed by laser confocal microscopy and a high-content live-cell imaging system. [Results] MBOVPG45_0564 was a conserved sequence inM.bovis and encoded LRR5, a membrane-associated protein with a typical crescent-like spatial conformation. Multiple repeating leucine motifs were assembled in a supercoiled manner to form a solenoid protein structural unit. After LRR5 antibody blocking, the invasion rate ofM.bovis to EBL cells reduced (P<0.05), and the fluorescent microspheres conjugated with LRR5 protein could successfully enter EBL cells. [Conclusion] The LRR5 protein encoded byMBOVPG45_0564 is localized on theM.bovis membrane and plays a role in the invasion ofM.bovis into host cells.

[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.

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.

Persulfidation plays a role in protein functioning and signaling, maintaining the physiological and metabolic balance of cells, protecting cells from oxidative stress, and regulating sulfur homeostasis. This article summarized the internal relationship of hydrogen sulfide, reactive sulfur species, and cysteine metabolism, expounded the mechanism of sulfur homeostasis regulation, and introduced the role of persulfidation in microbial sulfur homeostasis, providing new thoughts for the future research.

[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] Streptococcus thermophilus is widely used in the dairy industry as a common starter for fermented dairy products such as yogurt and cheese. Most strains ofS.thermophilus are galactose-negative (Gal⁻) and unable to metabolize galactose and excrete it extracellularly, which results in an increase in the galactose content in fermented milk.S.thermophilus can be treated by chemical mutagenesis to metabolize galactose and then used to develop the fermented milk products with low galactose content. [Methods] We used nitrosoguanidine (NTG) to induce the mutation ofS.thermophilus IMAU80846. Furthermore, we measured the activities of β-galactosidase (β-Gal), galactokinase (GalK), pyruvate kinase (PK), and glucokinase (GK) in the wild-type and mutant strains ofS.thermophilus IMAU80846 and analyzed the amino acid sequences encoding these enzymes.S.thermophilus IMAU80846Y that could metabolize galactose was obtained. We performed whole genome sequencing of the mutant strain and measured the content of lactic acid, lactose, galactose, and glucose in the fermented milk products produced with the wild-type strain and the mutant strain. We then compounded the wild-type strain and the mutant strain withLactobacillus delbrueckii subsp.bulgaricus IMAU20450, respectively, and characterized the two groups of fermented milk products during fermentation and storage. Finally, we prepared a fermented milk product with low galactose content. [Results] S.thermophiles IMAU80846Y had higher activities of β-Gal and GalK and lower activities of PK and GK than the wild-type strain. The amino acid sequences and whole genome sequences showed that the mutant strain had mutations in the genes involved in carbohydrate metabolism. The HPLC results showed that the fermented milk produced with the mutant strain had lower content of lactose and galactose and higher content of lactic acid and glucose than that produced with the wild-type strain. Compared with the compound group with the wild-type strain, the compound group with the mutant strain improved the titration acidity, viable cell count, viscosity, and water holding capacity of fermented milk. [Conclusion] The mutagenesis with NTG changed the ability ofS.thermophilus IMAU80846 to metabolize galactose, and the mutant strain could be used to produce the fermented milk with low galactose content.

[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.