Abstract:Esterases have been gradually developed and used in medicine, chemistry, and food industry since their discovery. Only a few esterases applied in industrial production are from animals and plants, and most of novel esterases are discovered from microorganisms, a vast enzyme resource pool in the nature. However, a large number of new microbial esterases do not meet the requirements of industrial application due to low activity and poor stability. Meanwhile, the mining of new microbial esterases is limited by low versatility and high cost of the screening and activity evaluation methods. This paper summarizes the recent research advances in the classification, discovery, structural and catalytic properties, modification, optimization, and application of microbial esterases, aiming to facilitate the discovery and industrial application of esterases.

Abstract:Nitrate is more stable in natural water than ammonia and thus is more difficult to be removed. Conventionally, the removal of nitrate relies on anaerobic denitrification, which is hard in natural water containing high concentration of dissolved oxygen. In this sense, aerobic denitrification is of particular importance for nitrate removal in natural water. This study reviewed the isolation, purification, microbial metabolism, and environmental influencing factors of aerobic denitrifiers. Moreover, the application of aerobic denitrification in remediation of micro-polluted drinking water sources is also introduced. Similar to their anaerobic counterparts, aerobic denitrifiers affiliate to a wide range of genera, such as Pseudomoas, Alcaligenes, Paracoccus, and Bacillus. Most of the aerobic denitrifiers have a satisfactory nitrate removal efficiency under suitable conditions (25-37 ℃, DO (dissolved oxygen)=3-5 mg/L, pH 7-8, C/N ratio=5-10). However, aerobic denitrification still has drawbacks of unstable removal efficiency and the loss of microbes. Moreover, little efforts are made in relevant study on pilot-scale and full-scale application and thus further investigation is needed.

Abstract:Natural transformation, a mechanism of horizontal gene transfer, plays an important role in the generation of genetic diversity, the repair of DNA damage, and the spread of drug resistance genes and virulence genes. Riemerella anatipestifer (RA) is the first bacterium of Weeksellaceae found to be able to undergo natural transformation. We established a variety of gene editing methods based on this property, which accelerated the research on the genetic diversity and pathogenic mechanism of RA. In addition, we identified the essential nutrients for natural transformation of RA through systematically screening the factors affecting the natural transformation. Subsequently, we identified the essential genes involved in natural transformation by screening the mutant library. Finally, we discovered a unique system of natural transformation in RA. Reviewing the studies of natural transformation in other bacteria, we introduced the research progress in the natural transformation of RA, aiming to deeply understand the natural transformation mechanism of this bacterium and provide references for further deciphering the mechanisms of drug resistance and virulence gene acquisition of this bacterium.

Abstract:The rare rod-shaped Gordonia was discovered by Tsukamura in 1971. A total of 41 valid species have been isolated and purified, which are widely distributed in mangrove rhizosphere, oil wells, hydrocarbon-contaminated soil, wastewater, and diseased humans. Owing to the functions of biotransformation, biodegradation, and active substance synthesis, Gordonia has great potential for drug development and environmental remediation. This study reviews the establishment, taxonomic characteristics, bioactive substances, and biodegradation function of Gordonia, which is expected to lay a foundation for the high-quality development of the resources of Gordonia.

Abstract:Electroactive microorganisms, with the unique ability of extracellular electron transfer, play a key role in geochemical cycle and environmental remediation. Cytochrome c participates in not only direct but also mediated extracellular electron transfer of electroactive microorganisms. The cytochrome c-mediated electron transfer is essential in the circulation of iron, manganese, carbon and other elements in the earth’s environment. Furthermore, it demonstrates good application potential in many fields such as energy production, wastewater treatment, and bioremediation. Taking the two model genera (Shewanella and Geobacter) of electroactive microorganisms as examples, this paper introduces the intracellular-to- extracellular electron transfer pathways of electroactive microorganisms, expounds the important role of cytochrome c in extracellular electron transfer, summarizes the analytical methods of cytochrome c-mediated electron transfer, and finally puts forward the future research direction of microbial extracellular electron transfer.

Abstract:Halophilic archaea are an important group in the domain archaea. They are of great value in the fields of genetics and metabolism, evolution and adaptation, technology frontier and synthetic biology. Haloarchaeal promoter is indispensable for both fundamental and applied research on halophilic archaea. In this paper, we first described the structure and functions of archaeal promoter in transcription. Then, we summarized and discussed the research achievements in haloarchaeal promoter from the following three aspects: the methods for studying promoter, the constitutive and inducible haloarchaeal promoters reported so far, and their applications. The challenges and future research focusing on mining, designing, developing, and optimizing haloarchaeal promoter were also prospected.

Abstract:Electroactive microorganisms can exchange electrons in their extracellular environment with electrodes or other cells through direct contact, conductive pilli, or redox mediators, in which extracellular polymeric substances (EPS) play a crucial part. EPS are a mixture of cell secretions, cell lysates, and other substances attached on the surface of microbial cells to protect them from external environment, comprising proteins, polysaccharides, humics, among others. Thus, the composition and characteristics of EPS from electroactive microorganisms influence the electroactive properties of the EPS and the extracellular electron transfer of the microorganisms, allowing them to be applied in the environmental bioremediations. For a clear understanding of the electroactive properties of EPS from electroactive microorganisms and the influence on extracellular electron transfer of the microorganisms, we introduced the methods for characterizing the electroactive properties of the EPS and summarized the influence of the composition, chemical properties, physical properties, and spatial distribution of the substances on their electroactive properties. In addition, we descried the applications of the EPS in decolorization of dye wastewater, adsorption of heavy metals, biotransformation of organic pollutants, leachate management, etc. Finally, the future research directions in the characterization method, the scale of experiments, and the interaction mechanisms were summed up.

Abstract:Epigenetic control plays important roles in regulation of eukaryotic gene expression, which includes DNA methylation, histone modification, and chromatin remodeling. Among them, chromatin remodelers function by affecting the binding of histone-modifying enzymes and transcription factors. The chromatin remodeling complex INO80 is evolutionarily conserved and it promotes nucleosome sliding and eviction by using the energy obtained from ATP hydrolysis. In addition to the role in DNA replication and repair, INO80 regulates the gene expression in budding yeast Saccharomyces cerevisiae by altering DNA accessibility. This review summarized the classification and composition of chromatin remodeling complexes in S. cerevisiae and introduced the roles (including expulsion of RNA polymerase Ⅱ, response to signaling pathways, and alteration of gene expression levels) of the multi-subunit complex INO80 in the dynamic regulation of gene expression. Furthermore, the research progress in the role of INO80 in the response of S. cerevisiae to environmental stress was reviewed. Studying the functions of INO80 benefits understanding the delicate metabolic regulation mechanisms in eukaryotes, which provides a basis for developing microbial metabolic engineering and synthetic biology tools employing chromatin remodelers and other epigenetic regulators to improve stress tolerance and fermentation efficiency.

Abstract:Plague caused by Yersinia pestis is a fulminating infectious disease, resulting in nearly 200 million deaths in human history. It is listed as a category A infectious disease in China. As a highly infectious pathogen, Y. pestis is considered one of the most potential biological weapons and biothreat agents. Prophylactic vaccination is a powerful measure for counteracting this disease. In the development of plague vaccines, live-attenuated vaccine is an important research direction. This review introduces the recent progress, current challenges, as well as the future prospects on the development of live-attenuated plague vaccines.

Abstract:[Objective] To explore the metabolite composition and metabolite synthesis potential of Brevibacterium casei G20 in response to saline-alkali stress, and to provide a reference for the mining of potential functional molecules and the rapidly and stably responsive genetic logic gate line induced by saline-alkali. [Methods] We used LC-MS to detect the metabolites of G20 at four growth phases in saline-alkali and normal environments, and focused on the analysis of the lag and logarithmic phases rich in metabolites with high differential fold changes. [Results] B. casei G20 grew well in the environment with pH 10.0 and 9% NaCl, and the pH of the environment gradually decreased with the growth. According to the detection result in positive ion mode and negative ion mode, the numbers of differential metabolites of B. casei G20 during growth in the saline-alkali environment were 0.69, 0.75, and 0.81 times that in the normal environment, respectively. The metabolites that differed between the lag and logarithmic phases under saline-alkali stress were mainly benzenoids, organic acids and their derivatives, and organic heterocyclic compounds. Among them, the MS signal intensity of the organic acids such as indole-3-acetic acid, kynurenic acid, and gluconic acid that were up-regulated in the logarithmic phase was lower than that in the lag phase. The possible osmoprotectants in the strain were l-citrulline, l-proline, N-acetylornithine, l-carnitine, etc. The differential compounds with larger fold changes or higher MS signal intensity in the lag phase were pilocarpine, phytosphingosine, and citrate, and in the logarithmic phase were histamine, l-proline, and thiamine. The differential metabolic pathways of the strain were mainly the amino acid metabolism and carbohydrate metabolism. There were also structural analogs of betaine and trigonelline in the metabolites of the strain. In addition, for metabolites with large changes induced by saline-alkali stress, such as histamine, l-proline, and choline, we could infer their synthetic and metabolic pathways through the coupling of metabolomic and genomic data. [Conclusion] The strain secreted organic acids such as indole-3-acetic acid, kynurenic acid, and Gluconic acid to reduce the pH value of the environment, and accumulated osmoprotectants such as l-citrulline and l-proline to maintain osmotic balance. In addition, the strain may also have the ability to synthesize structural analogs of betaine, trigonelline, and pilocarpine, allowing the construction of a new synthetic pathway based on prokaryotic bacteria. Moreover, the enzyme-coding genes and upstream and downstream element sequences in the synthetic and metabolic pathways of compounds such as histamine, l-proline, and choline in the strain can serve as a reference for sorting out the logic gate gene circuits induced by saline-alkali stress.

Abstract:[Objective] To rescue the recombinant influenza A/WSN/33 (H1N1) virus expressing the tandem affinity purification TAP-tag. [Methods] The PA sequence of influenza A/WSN/33 (H1N1) virus was modified and then inserted into the TAP sequence to construct a recombinant plasmid pHW-PA-TAP-WSN. The recombinant influenza virus WSN PA-TAP expressing exogenous TAP-tag was rescued by reverse genetics and then identified by plaque assay, RNA sliver staining, etc. [Results] The recombinant influenza viruses WSN PA-TAP were successfully rescued. The sequencing results showed that the sequence of the recombinant virus genome was correct, and the eight fragments of the rescued virus were observed by RNA silver staining. The growth curve of WSN PA-TAP was established with MDCK cells. The recombinant WSN PA-TAP had weaker replication ability than the wild-type WSN. Western blotting showed that the PA-TAP fusion protein had a molecular weight of 96 kDa. [Conclusion] The recombinant influenza virus WSN PA-TAP capable of expressing the exogenous TAP-tag was successfully rescued, which provided a new method for the screening of host proteins related to influenza A virus polymerase. Furthermore, the recombinant influenza virus provides a basis for the exploration of influenza A virus carrying foreign genes.

Abstract:[Objective] We isolated and screened microbes with growth-promoting function and salt tolerance from the rhizosphere soil of halophytes in dry and saline habitats and studied their growth-promoting ability, hoping to provide high-quality bacterial resources and a reference for alleviating soil salinization in arid areas. [Methods] Strains with salt tolerance and phosphate-solubilizing and potassium-releasing abilities were screened with selective media. Then, the ability of the strains to produce indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophore, and exopolysaccharide was tested. Through antagonism test, strains with excellent functions and no antagonism to each other were selected to prepare the compound bacterial mixture. The suspension of each of the strains and that of the mixture of strains without antagonism to each other were respectively used to infect seeds of radish and maize, and the influence on seed germination rate under salt stress and plant growth under both drought and salt stresses was observed. Finally, 16S rRNA gene sequencing was performed to identify the strains. [Results] Strains yl923, hs032, and hy127 with salt tolerance and growth-promoting ability were screened out. yl923 can solubilize phosphate (46.29 mg/L), help release potassium (58.07 mg/L), and produce IAA (29.23 mg/L), ACC deaminase (13.83 U/mg), and siderophores (SU=0.43). hs032 was outstanding in producing IAA (61.18 mg/L) and siderophore (SU=0.23) and hy127 excelled in producing ACC deaminase (15.29 U/mg). Through 16S rRNA gene sequencing, yl923 and hs032 were identified as Bacillus subtilis and hy127 was determined to be Priestia megaterium. The three showed no antagonism to each other and thus they can be used together as compound bacterial mixture. The 2% mixture suspension can improve the seed germination rate (77%) under salt stress. Moreover, it can increase the root length, plant height, dry weight, and chlorophyll production (P<0.05), and significantly reduce the malondialdehyde (MDA) content (60%) of maize under drought and salt stresses. [Conclusion] Strains yl923, hs032 and hy127 show excellent salt tolerance and growth-promoting function. The compound mixture prepared with the three can remarkably promote plant growth under drought and salt stresses and those strains have the potential to improve salinized soil in arid regions.

Abstract:[Objective] To explore the effect of ferrous sulfate (FeSO₄) on the intestinal health of iron deficiency anemia (IDA) mice. [Methods] A total of 45 24-day-old male Kunming mice with initial body weight of (16.0±1.2) g were randomized into 3 groups (15 in each): control group (normal diet, distilled water), IDA group (low-iron diet for 2 weeks for modeling, deionized water), and IDA-Fe²⁺ group (after modeling, gavage with FeSO₄ for 3 weeks, deionized water). Sampling performed at the end of the experiment. [Results] Anemia indexes of mice in IDA-Fe²⁺ group, such as erythrocyte, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), returned to normal levels compared with those in the IDA group (P>0.05), indicating that FeSO₄ can alleviate IDA. Compared with the control group, IDA and IDA-Fe²⁺ groups had high levels of oxidative stress indexes and tumor necrosis factor α (P<0.01) and low level of Occludin (P<0.001). The indexes, except for the total antioxidant capacity (TAC), were insignificantly different between the IDA-Fe²⁺ and IDA groups (P>0.05). As for the hematoxylin and eosin (HE) staining results of colon tissue, the IDA group showed atrophy of colonic mucosa, defective arrangement of epithelial cells in mucosa, and atrophy of lamina propria. After intragastric administration of FeSO₄ for 3 weeks, IDA-Fe²⁺ group still demontrated defective arrangement of epithelial cells in the mucosa, necrotic and defective acini of mucosa, and atrophy of lamina propria. According to the results of Illumina MiSeq high-throughput sequencing of fecal microbiota, IDA could lead to the disorder of intestinal microflora and the variation of diversity indexes (Shannon, Chao1, Ace, and Simpson indexes) of intestinal flora (P<0.05). IDA-Fe²⁺ group showed significant increase in diversity indexes of the flora and improvement of the overall structure of intestinal flora as compared with the IDA group (P<0.05). To be specific, at the phylum level, the abudance of Bacteroidetes decreased (P<0.05) and that of Verrucomicrobia increased (P<0.01), both returning to the normal levels. At the genus level, the abundance of the dominant genera changed. The abundance of the beneficial Akkermansia and Coprobacter increased (P<0.05) and that of the pathogenic Parabacteroides decreased (P<0.01). [Conclusion] FeSO₄ can alleviate IDA symptoms by improving the blood indexes, but failes to improve the oxidative stress indexes, inflammation indexes, and colon tissue pathological changes of IDA mice. However, FeSO₄ can increase the diversity of intestinal flora and the abundance of main dominant flora in IDA mice, raising the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria.

Abstract:[Objective] To explore the functional differences of different acetic acid bacteria in the vinegar pei of Zhenjiang aromatic vinegar via comparative genomics. [Methods] The taxonomic status of acetic acid bacteria were identified by culture-dependent technology and 16S rRNA gene full-length sequencing. The growth and metabolism of different acetic acid bacteria were compared by comparative genomic analysis combined with fermentation performance tests. [Results] Acetobacter pasteurianus and Komagataeibacter europaeus were the main acetic acid bacteria in the vinegar pei of Zhenjiang aromatic vinegar. K. europaeus had higher GC content and larger genome than A. pasteurianus. Functional annotation showed that the number and types of the genes involved in carbohydrate and amino acid metabolism varied greatly between A. pasteurianus and K. europaeus, and the number of carbohydrate-active enzymes was higer in K. europaeus. The functionally differential genes in K. europaeus compared with A. pasteurianus were mainly involved in the pentose phosphate pathway, fatty acid biosynthesis, and fructose and mannose metabolism. The verification experiment showed that K. europaeus increased the conversion rate of alcohol by producing more alcohol dehydrogenase, acetaldehyde dehydrogenase, and ATP and changing the fatty acid composition of the cell membrane. [Conclusion] This study revealed the genetic differences between A. pasteurianus and K. europaeus in vinegar pei. K. europaeus can improve the intracellular microenvironment to adapt to the high acid environment by massively producing energy and the enzymes involved in ethanol conversion and changing the composition of fatty acids in cell membrane. The results of this study can deepen the understanding of the acid tolerance mechanisms of different acetic acid bacteria.

Abstract:[Objective] Listeria monocytogenes is an important zoonotic foodborne intracellular pathogen. Here, we aimed to explore the biological functions of cytochrome aa3 terminal oxidase subunit QoxB in L. monocytogenes during bacterial growth and infection. [Methods] We constructed a qoxB deletion mutant ΔqoxB based on the wild-type strain EGD-e via homologous recombination. The differences regarding bacterial growth and intracellular infection were analyzed between the wild-type and the mutant strains. In addition, changes in the transcription of flagella-associated genes induced by the deletion of qoxB were determined by real-time quantitative reverse-transcription PCR (qRT-PCR). [Results] The deletion of qoxB did not affect the bacterial growth ability in vitro. However, the motility of ΔqoxB was significantly decreased compared with that of EGD-e. After incubation at 30 ℃ for 24 h and 48 h, the diameter of motility halos of ΔqoxB decreased by 35.86% and 34.2%, respectively, compared with that of EGD-e. The expression abundance of 22 flagella-associated genes reduced significantly in ΔqoxB. Moreover, compared with EGD-e, ΔqoxB showed significantly reduced abilities of cell adhesion, invasion, proliferation, and intracellular spread. [Conclusion] We demonstrated for the first time that QoxB played a critical role in the motility and infection of L. monocytogenes. The findings lay an important basis for elucidating the role of cytochrome terminal oxidase in the pathogenesis of bacterial pathogens.

Abstract:[Objective] To isolate and screen bacteria against Enterococcus pernyi (the pathogen of Antheraea pernyi empty-gut disease) from the oak garden soil of Sericultural Research Institute of Liaoning Province and thus to lay a foundation for the biocontrol of the disease. [Methods] Bacteria were isolated from soil by spread plate technique and the antagonistic strain was screened out with the inhibition zone method. The strain was identified according to morphological features, physiological and biochemical characteristics, and molecular biology. The antagonistic strain was labeled with green fluorescent protein with natural transformation method, and the colonization of the antagonistic strain in oak leaves and gut of A. pernyi was determined. The control of empty-gut disease of A. pernyi was identified by indoor and field experiments. [Results] A total of 87 bacterial strains were isolated from soil. Among them, BF-49 was found to have significant antagonistic activity against E. pernyi. The identification results revealed that BF-49 had the closest genetic relationship with Bacillus velezensis. Therefore, it was identified as B. velezensis and named B. velezensis BF-49. The fluorescence-labeled strain BF-49-GFP could colonize in the gut of A. pernyi for 5 days, and the count of BF-49-GFP reached 1.25×10⁴ CFU/g 20 days after inoculation on the oak leaves. The indoor and field control of empty-gut disease by the 10-fold diluted BF-49 fermentation broth was 78.25% and 74.42%, respectively, which were significantly higher than those of the control agents. [Conclusion] The B. velezensis BF-49 has significant control effect on empty-gut disease of A. pernyi, being a candidate strain for developing the biological control agent against empty-gut disease.

Abstract:[Objective] To investigate the phylogenetic position of the siderophore-producing halophilic bacterium JSM 104105 in the family Halomonadaceae. [Methods] Molecular phylogenetic analysis of strain JSM 104105 was performed by means of multilocus sequence analysis (MLSA) based on 3 housekeeping genes, i.e., 16S rRNA gene, DNA gyrase B subunit gene (gyrB) and RNA polymerase sigma factor RpoD gene (rpoD), and then its exact systematic position was investigated by comparative genomics analysis based on whole-genome sequences, including comparisons of G+C content, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) values, and phylogenomic analysis. [Results] The MLSA results showed that the data both from single-gene (16S rRNA, gyrB, or rpoD) sequence comparisons and concatenated 16S rRNA-gyrB-rpoD sequence comparisons were consistent, and could indicate the phylogeny of strain JSM 104105 and related taxa of Halomonadaceae, and that strain JSM 104105 belonged to the genus Halomonas, being in closestly genetic relationship with Halomonas gudaonensis CGMCC 1.16133^T (16S rRNA gene similarity, 98.9%), Halomonas azerbaijanica TBZ202^T (98.6%) and Halomonas lysinitropha 3(2)^T (97.3%), and strain JSM 104105 formed a coherent branch in the phylogenetic trees which were generated based on either single-gene sequence or concatenated-gene sequence comparisons. MLSA data also suggested that strain JSM 104105 could represent a different phylogenic subline separated from recognized Halomonas species. The results of comparative genomics analysis supported the conclusion from MLSA data. Either ANI (78.9%-91.6%) or dDDH values (22.1%-43.7%) were all well below the standard criteria (ANI, 95%-96%; dDDH≥70%) for delineation of prokaryotic species. The results of phylogenomic analysis also showed that strain JSM 104105 clearly represented an independent subclade in Halomonas. [Conclusion] From the perspective of molecular phylogeny, the combination of MLSA and comparative genomics analysis demonstrated definitely that strain JSM 104105 belongs to Halomonas, being phylogenetically closely related to H. gudaonensis, H. azerbaijanica and H. lysinitropha, and that strain JSM 104105 cannot be assigned to any recognized species but represents a novel genospecies of the genus Halomonas.

Abstract:[Objective] To investigate the biological roles of the disulfide bond formation protein DsbG in the acid tolerance and flagella-mediated motility of Listeria monocytogenes by constructing the dsbG-deleted and complemented strains. [Methods] The dsbG-deleted and complemented strains were constructed via the homologous recombination method. The growth and survival rates of the strains in the media at different pH gradients were analyzed. The transcriptional levels of the genes involved in acid tolerance were determined by real-time quantitative PCR and compared between the wild-type and mutant strains exposed to acid stress. The motility, transcriptional levels of flagella-associated genes, and flagellar morphology were compared between wild-type and mutant strains by semi-solid culture, real-time quantitative PCR, and transmission electron microscopy, respectively. [Results] The deletion of dsbG did not affect the growth of the bacteria in a medium with neutral pH. However, it significantly reduced the survival rate of bacteria in the media with hydrochloric acid and citric acid (pH 3.5). It down-regulated the transcriptional levels of the genes (argD and argF) in the arginine pathway by 2.4 and 3.7 times, respectively. Moreover, the lack of dsbG impaired the flagellum formation and bacterial motility and down-regulated the transcriptional levels of flagella-associated genes flaA, flgB, and flgD by 29.7, 6.7, and 6.9 times, respectively, compared with the wild-type strain. [Conclusion] This study for the first time, demonstrated that DsbG could sense low pH stress, play a role in the development of acid tolerance, and more importantly, contribute to the flagella-mediated motility of L. monocytogenes via regulating the transcription of flagella-associated genes. This study helps to understand the roles of the disulfide bond formation protein family in the environmental adaptation of L. monocytogenes and provides a theoretical basis for the prevention and control of foodborne pathogens.

Abstract:[Objective] To investigate the effects of deletion of an Aco-like autoregulator synthase gene, scyA1, on the physiological metabolism and regulatory network of cells, and thus to reveal the global roles of ScyA1 in cellular activities. [Methods] Dry cell weight was measured to determine the effect of scyA1 deletion on the growth of cells in liquid fermentation. Comparative RNA-Seq analysis between the scyA1 mutant and the wild type NMWT1 was performed to explore the genome-wide differentially expressed genes after 3 and 6 days of fermentation. [Results] The deletion of scyA1 did not affect the biomass of cells in liquid fermentation. After the deletion of scyA1, the expression of the genes involved in glycolysis and tricarboxylic acid cycle showed significant bidirectional differences, and the expression levels of the genes involved in the pathway from l-galactose to UDP-glucose, the pentose phosphate pathway, amino acid (l-valine, l-isoleucine, and l-tryptophan) biosynthesis, and purine nucleotide degradation were significantly upregulated. Moreover, the expression levels of a variety of gene clusters for the biosynthesis of secondary metabolites and the genes encoding conserved transcriptional regulators and mycelium structural proteins were significantly downregulated, while those of a few were significantly upregulated. [Conclusion] ScyA1 affected primary metabolism, secondary metabolism, and expression of conserved regulatory factors and mycelium structure-related genes of the test strain. The results of this study demonstrated new details of the global function of the Aco-like autoregulator synthase in Streptomyces.

Abstract:[Objective] To validate expression and sequence of the previously identified nce-miR-34537, predict the target gene of nce-miR-34537 and the molecular characteristics of the gene, determine the expression profiles of nce-miR-34537 and its target gene during the Nosema ceranae infection in Apis mellifera ligustica workers, so as to provide a foundation for revealing the role of nce-miR-34537 in N. ceranae infection. [Methods] Stem-loop-RT-PCR and Sanger sequencing were performed to verify the expression and sequence of nce-miR-34537. The molecular properties and conserved motifs of the target gene of nce-miR-34537, PIP5KI (encoding type I phosphatidylinositol 4-phosphate 5-kinase), were predicted via bioinformatics tools. The phylogenetic tree was constructed based on amino acid sequences. The expression profiles of nce-miR-34537 and its target genes were determined via RT-qPCR. [Results] The existence and expression of nce-miR-34537 were confirmed in N. ceranae spores. nce-miR-34537 can target 151 genes including PIP5KI. The deduced PIP5KI protein was predicted to have the molecular formula of C₈₈₂H_{1 364}N₂₂₆O₂₅₁S₇ and the molecular weight of 19.37 kDa and contain 160 residues, 17 phosphorylation sites, and no classical signal peptide. PIP5KI can simultaneously locate in the nucleus, mitochondrion, cytoplasm, and secretory vesicles. N. ceranae PIP5KI (AAJ76_2700017870) contained four conserved motifs, while the other PIP5KI (EEQ82436.1) contained five motifs. PIP5KI proteins in N. ceranae, N. apis, and Spraguea lophii were grouped into one clade in the phylogenetic tree, and N. ceranae PIP5KI (EEQ82436) and N. apis PIP5KI (EQB60549.1) had the closest evolutionary distance. Compared with that 1 dpi (day post inoculation), the expression level of nce-miR-34537 was up-regulated at 2 dpi (P>0.05) and down-regulated at 4, 6, and 8 dpi (P<0.05), and that of PIP5KI was up-regulated at 2 dpi (P<0.05) and down-regulated at 4, 6, and 8 dpi (P<0.05).[Conclusion] nce-miR-34537 is expressed in N. ceranae spores. N. ceranae PIP5KI (EEQ82436) and N. apis PIP5KI (EQB60549.1) have the closest genetic relationship. The expression of both nce-miR-34537 and the target gene PIP5KI is first up-regulated and then down-regulated during N. ceranae infection in A. m. ligustica workers. The results indicate that nce-miR-34537 may participate in the infection process via positive regulation of PIP5KI expression.

Abstract:[Objective] To study the role of Streptomyces rochei D74 in promoting sunflower growth and inhibiting Orobanche cumana parasitism, and the effect on rhizosphere microorganisms. [Methods] We developed a sunflower-O. cumana-S. rochei D74 co-culture system to observe the effect of D74 on the parasitism of O. cumana. Through field experiment, we observed the influence of D74 on physiological indexes of sunflower and the number of epigaeous O. cumana, and measured the yield indexes of sunflower to evaluate the influence of D74 on quality and yield of sunflower kernels. High-throughput sequencing of 16S rRNA genes and traditional microbial culture method were used to explore the effect of D74 on microbial community structure in sunflower rhizosphere. [Results] D74 promoted the growth of sunflower, as manifested by the increase (P<0.05) in stem weight (by 37%-40%) and disk weight (by 21%-37%), and inhibited (P<0.05) the seedling emergence of 28%-46% O. cumana. In addition, it raised the crude protein content in sunflower kernels by 5%-9%, percentage of large kernels by 66% (P<0.05), and 100-grain weight by 8%-18%. In the field, D74 elevated the yield by about 30% (P<0.05), which has far-reaching significance for improving farmers’ economic returns. The sunflower rhizosphere microbial community was significantly affected by D74 which regulated the number of beneficial microorganisms. [Conclusion] D74 can significantly reduce the number of O. cumana in sunflower rhizosphere, promote the growth of flower discs, and increase the large kernels, which should be further applied and promoted.

Abstract:[Objective] Cyanophages are ubiquitous in natural water ecosystems and have an important ecological status and function. They play a role in controlling cyanobacterial populations and community structure by infecting cyanobacteria, which indicates the great application potential of cyanophages in controlling cyanobacterial blooms. This study aims to explore the diversity and phylogenetic status of cyanophage psbA gene in the plateau lakes in Yunnan Province, so as to lay a theoretical foundation for further understanding the ecological role of cyanophages in plateau lakes and exploiting cyanophage resources. [Methods] The psbA gene was used as a molecular target to study the genetic diversity of cyanophages in Dianchi Lake, Fuxian Lake, and Xingyun Lake in Yunnan Province. [Results] A total of 100 psbA sequences of cyanophages were obtained from different lakes. The phylogenetic analysis showed that the psbA gene from the lakes had close genetic distance to those in the East Lake in China, the paddy water in northeast China, and the Japanese paddy floodwater, but were more distantly related to those in the marine waters. The diversity of cyanophage psbA gene in Fuxian Lake was higher than that in Dianchi Lake, Xingyun Lake, and Yilong Lake. The novel psbA assemblies of cyanophage existed in the plateau lakes in Yunnan Province. The genetic diversity of psbA gene showed no significant difference between autumn and winter. [Conclusion] The psbA gene of cyanophages in the main plateau lakes in Yunnan Province has high genetic diversity and is closely related to the psbA gene of cyanophage in freshwater environment. Moreover, there are novel psbA assemblies of cyanophages in the plateau lakes.

Abstract:[Objective] To decipher the functional mechanism of Tlp1 which is the only soluble chemoreceptor containing PAS domain in Sinorhizobium alkalisoli YIC4027. [Methods] We constructed the gene-deleted mutant strain based on the Red homologous recombination system and triparental conjugation. The growth rate, chemotaxis, aerotaxis, cell flocculation, biofilm formation, exopolysaccharide (EPS) production, colonization, and competitive nodulation of the wild type and the mutant strain were studied. [Results] Compared with the wild type, the mutant strain showed unaffected growth, weakened chemotaxis, aerotaxis, colonization, and competitive nodulation, and increased cell coagulation, biofilm formation, and EPS production. [Conclusion] This study confirmed that the soluble chemoreceptor Tlp1 in YIC4027 affected the chemotactic movement of the cells for the first time.

Abstract:[Objective] Pseudomonas boreopolis GO2 synthesizes bioflocculant with lignocellulosic biomass. In this study, the whole genome sequencing and comparative genome analysis of GO2 were performed to explore its genome characteristics and analyze genes related to lignocellulose degradation and polysaccharide bioflocculant synthesis. [Methods] Illumina NovaSeq was used for genome sequencing, and SMRT and other software for genome assembly, phylogenetic analysis, and gene prediction and annotation. Moreover, the genome was respectively compared with that of four similar model strains. [Results] The genome of GO2 was 4 498 896 bp, with guanine-cytosine (GC) content of 69.5% and 3 906 coding genes. It showed the highest 16S rRNA gene similarity (99.93%), DNA-DNA hybridization (DDH) (88.00%), and average nucleotide identity (ANI) (98.36%) to P. boreopolis JCM 13306. Therefore, this strain was named as P. boreopolis GO2. GO2 shared 2 348 core orthologous genes with the four model strains, and these genes were mainly involved in the pathways of carbohydrate metabolism, amino acid metabolism, and energy metabolism. The 307 specific genes of GO2 were mainly involved in the pathways of transcription, replication, repair, and cell wall/membrane biogenesis. GO2 contained 226 carbohydrate-active enzymes (CAZymes) genes, accounting for 5.79% of the total number of genes. Among the CAZymes genes, 82 belonged to the glycoside hydrolases (GHs) family which was related to plant cell wall degradation and 3 clusters of polysaccharide synthesis genes were formed by glycosyltransferases (GTs) family genes. [Conclusion] Based on the comparative analysis of genome sequences, GO2 is named Pseudomonas boreopolis GO2, and its genome contains rich plant cell wall degrading enzyme genes and three polysaccharide synthesis gene clusters, which may play an important role in the synthesis of bioflocculants by with lignocellulosic biomass in GO2.

Abstract:[Objective] To explore the distribution of ferric reducing bacteria (FeRB) and anoxygenic phototrophic bacteria (AnPB) in the soil along the Yellow River. [Methods] Soil samples were collected from the beach and paddy fields at the Yuanyang section of the Yellow River. High-throughput sequencing of 16S rRNA gene and quantitative real-time PCR were combined with statistical analysis to reveal the structure and abundance of FeRB and AnPB and the main environmental factors affecting the bacteria. [Results] The dominant FeRB families (genera) were Hydrogenophilaceae (Thiobacillus), Bacillaceae (Bacillus), Clostridiaceae, Rhodobactereace (Rhodobacter) and Geobacteraceae (Geobacter). The dominant AnPB families (genera) were Rhodobactereace (Rhodobacter), Chloroflexaceae (Chloronema) and Acetobacteraceae (Roseomonas). The relative abundance of Rhodobacteraceae (AnPB) was negatively correlated with that of Bacillaceae and Clostridiaceae (FeRB). The relative abundance of Sphingomonadaceae (AnPB) was negatively correlated with that of Hydrogenophilaceae and Clostridiaceae (FeRB). Soil nitrate nitrogen (NO₃^--N) was negatively correlated with the relative abundance of Rhodobacteraceae but positively correlated with that of Geobacteraceae. Ferrous ions (Fe²⁺) explained 13.5% and 41.8% of the community variations of FeRB and AnPB, respectively; pH explained 65.7% and 42.8%, respectively. The number of total bacteria (BAC), Geo(thermo)bacter (GEO) and phototrophic purple bacteria (PPB) in the Yellow River beach was 2.52 (±3.43)×10⁹, 5.21 (±7.58)×10⁷ and 2.9 (±3.70)× 10⁷ copies/g dry soil, respectively, and that in the paddy soil was 3.82 (±1.29)×10¹⁰, 3.05 (±2.44)×10⁸ and 4.31 (±0.90)×10⁸ copies/g dry soil, respectively. Moreover, the PPB in the upmost soil layer (0-1 cm) were significantly more than those in the 1-2 cm and 2-3 cm soil layers. Fe²⁺ explained 81.5% variations in the absolute abundance of BAC, GEO and PPB. [Conclusion] The potential community of FeRB and AnPB and the abundance of GEO and PPB varied between different soil types. Overall, Fe²⁺ played a key role in shaping the distribution pattern of FeRB and AnPB.

Abstract:[Objective] We screened out hydrogenotrophic microorganisms using inorganic carbon sources for high efficiency in denitrification under weak acidic conditions, to investigate the effects of different inorganic carbon sources on the denitrification capacity of the system, and to explore the variation in water quality parameters, microbial community structure, and denitrification cycle in the reactor during long-term cultivation. [Methods] We developed a sequential batch reactor for continuous cultivation of microorganisms by supplying hydrogen, inorganic carbon source, nutrient solution and nitrate in time, and the apparatus have the advantages of low cost, excellent air tightness, and hydrogen utilization rate calculation. [Results] The cultivated microorganisms showed higher efficiency in denitrifying nitrates with the mixture of NaHCO₃ and CO₂ as inorganic carbon sources than that of NaHCO₃ as solo carbon source. Under the conditions of ambient temperature of 20 ℃, pH of 6.3-7.0, and initial nitrate nitrogen dosage of 15 mg-N/L, the highest reaction rate of NO₃^--N was 1.374 mg-N/(L·h) and the highest utilization rate of hydrogen was 43.4%. In addition, the denitrification cycle was 16 h, with no nitrite accumulation detected. The cultivated microorganisms were mainly Acidovorax, accounting for 84.4%. [Conclusion] It is feasible and efficient to use this apparatus and method to cultivate microorganisms. Hydrogenotrophic microorganisms could be screened for denitrification by inorganic carbon source under weak acidic condition. The findings lay a theoretical basis for the bioremediation of groundwater polluted by nitrate and a foundation for the simultaneous denitrification and uranium fixation of hydrogenotrophic microorganisms in acidic environment in the near future.

Abstract:[Objective] To investigate the antibacterial effect of the phage endolysin Lysin1902 combined with ε-poly-lysine (ε-PL) on Escherichia coli O157:H7. [Methods] We employed Mega to construct the phylogenetic tree and used online tools to analyze the amino acid sequence of Lysin1902. After prokaryotic expression, Lysin1902 was purified. We then detected the lytic activity of Lysin1902 against autoclaved E. coli O157:H7 by plate lysis assay and detected the lysis activity of Lysin1902 or ε-PL on live E. coli O157:H7 in 96-well plates. Further, we adopted checkerboard assay to measure the bactericidal activity of Lysin1902 combined with ε-PL. [Results] We successfully expressed and purified Lysin1902. Lysin1902 showed lytic activity against autoclaved E. coli O157:H7 but cannot effectively lyse live E. coli strains. After the addition of Lysin1902, the concentration of ε-PL that could completely control the proliferation of E. coli O157:H7 decreased from 0.7 mg/mL to 0.1 mg/mL. [Conclusion] Lysin1902 was successfully expressed and purified. Lysin1902 alone has no lytic activity against E. coli O157:H7, while the combined use of Lysin1902 with ε-PL significantly increased the lytic activity of ε-PL against E. coli O157:H7.

Abstract:[Objective] Indole-3-acetic acid is crucial for plant growth and development and physiological activities. Indole-3-acetic acid N-acetyltransferase YsnE plays an important part in indole-3-acetic acid synthesis. This study aims to analyze the metabolic pathways of YsnE involved in indole-3-acetic acid synthesis of Bacillus amyloliquefaciens. [Methods] Through deletion and overexpression of ysnE, the role of ysnE in indole-3-acetic acid synthesis was elucidated. Combined with the addition of indole-3-acetic acid synthesis intermediates (indole pyruvic acid (IPA), indole-3-acetamide (IAM), tryptamine (TAM), indole-3-acetonitrile (IAN)) and in vitro enzymatic conversion experiments, the metabolic pathways of ysnE involved in indole-3-acetic acid synthesis were clarified. [Results] YsnE played an important role in indole-3-acetic acid synthesis in B. amyloliquefaciens HZ-12. The amount of IPA, IAM, and IAN consumed in ysnE deletion strain was significantly reduced, and YsnE functioned as indolepyruvate decarboxylase YclB, and indoleacetamide hydrolase/nitrilase/nitrile hydratase YhcX, and affected indole-3-acetic acid synthesis by participating in IPA, IAM, and IAN pathways. [Conclusion] YsnE influences the IPA, IAM, and IAN pathways to involve in indole-3-acetic acid synthesis, which lays a foundation for analysis of indole-3-acetic acid synthesis pathway and breeding strains with high yield of indole-3-acetic acid by metabolic engineering.

Abstract:As the core protein of a multifunctional protein complex Spt-Ada-Gcn5- acetyltransferase (SAGA) in yeast, Spt7 is not only responsible for maintaining the stability of the SAGA complex, but also responsible for the transcription of more than 10% of genes. However, there were few studies on the functions of Spt7 in filamentous fungi. [Objective] To investigate the effects of Spt7 on Aspergillus niger CGMCC 1062. [Methods] In this study, the plasmid with spt7 gene knockout was transferred into Aspergillus niger CGMCC 1062 by Agrobacterium tumefaciens transformation method. The colony morphology of Δspt7 strain and control group was observed, which grew on CM medium, different carbon sources, and H₂O₂-containing mediums. The relative transcription levels of glycolysis key genes and sporin-producing related genes were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). [Results] The Δspt7 strain was successfully obtained. It was found that the growth of Δspt7 strain was slow, the colony became white, and the sporulation was delayed. The knockout of the spt7 gene significantly affected the use of different carbon sources by the strain. However, Δspt7 strain and the control group grew normally on the plate with 20 mmol/L H₂O₂. The transcriptional levels of fbp, pfk, trk, pks, fda, and gsdA genes in the Δspt7 strain were 2.65 times, 4.46 times, 6.05 times, 4.90 times, 3.20 times, and 3.20 times lower than those in the control group, respectively. The transcriptional levels of wetA, abaA, and brlA were down-regulated by 529.93 times, 172.40 times, and 9.61 times, respectively, as compared with the control group. [Conclusion] The deletion of spt7 gene affects the normal growth, colony morphology, and conidial production of the strain.