Abstract:Recently, the emergence and global prevalence of many new antibiotic genes have led to a worldwide public health crisis. CRISPR-Cas9 (an adaptive immune system of bacteria) that can be used to cleave specific targets of resistant bacteria and against exogenous nucleic acid, serves a revolutionary gene editing tool to control antimicrobial resistance. Our previous study has established a plasmid-mediated CRISPR-Cas9 system targeting the mcr-1 gene, to efficiently and specifically eliminate mcr-1 gene and resensitize colistin resistant E. coli. Moreover, it is also necessary to optimize delivery methods of the CRISPR-Cas9 system for clinical treatment. This review focuses on the process of CRISPR-Cas9 technology in the prevention and control of antibiotic resistance in recent years, including the discovery process, mechanism, delivery method, advances in the experimental results in vitro and the existing problems, to provide new insights to the prevention and control of antibiotic resistance.

Abstract:The CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins) system is a bacterial acquired immune defense system, which can be used by bacteria to prevent the invasion of viruses, plasmids and other exogenous nucleic acids. With the further research, the CRISPR-Cas system has been successfully developed into an important tool to edit genes, and applied into gene modification in animals, plants and microorganisms. However, the CRISPR-Cas gene-editing tool sometimes exists in gene off-target effect, which limits its wide application. Recently, by combining a newly discovered anti-CRISPR protein (ACP) with CRISPR-Cas system, a gene-editing tool that can control the efficiency of gene off-target has been successfully developed. In this review, the CRISPR-Cas system and ACP were firstly introduced in brief, then the application of CRISPR-Cas gene editing tool and ACP in microbial gene modification was summarized, and the prospect of ACP-CRISPR-Cas applied in microbial gene editing was discussed.

Abstract:Chitin is the second most abundant carbohydrate polymer in nature, but often discarded as wastes. N-acetylglucosamine (GlcNAc), the final hydrolysate of chitin, is an important functional amino sugar compound that can be used in medicine, healthcare, and skin care products with a great demand. Therefore, it is of great significance to convert chitin to GlcNAc with high value added. Chitinase can specifically hydrolyze chitin to produce high value-added N-acetylglucosamine, to replace chemical processing strategy, thus reducing environmental pollution and improving product quality. This review briefly introduces specific features and classification of microbial chitinases. Then, the roles, mode and yield of endochitinase, chitobiosidase, and β-N-acetylglucosidase in the production of GlcNAc from chitin in recent years are elaborated, to provide references for enzymatic production of GlcNAc.

Abstract:Burkholderia spp. is a class of Gram-negative bacteria with a wide geographical and niche distribution. In recent years, with the increasing research on plant associated Burkholderia, more and more evidences suggest that Burkholderia is an important plant-associated beneficial microorganism. Burkholderia could promote plant growth through biological nitrogen fixation, phosphorus solubilizing, to increase nitrogen and phosphorus absorption, production of phytohormones like IAA and antifungal metabolites to inhibit the growth of phytopathogenic microorganisms. All of these make Burkholderia with great application potential in promoting plant growth and health. This article reviews the research progresses of the plant-associated beneficial Burkholderia and discusses its application prospects in agriculture.

Abstract:Iron-reducing bacteria are typical dissimilatory metal-reducing bacteria that are widespread in nature, including marine sediments and deep formations. These bacteria can reduce the Fe(III) of iron oxides to Fe(II) and play an important role in the biogeochemical iron cycle of iron and carbon. Aside from Fe(III), other high valence state metals and organic pollutants can also serve as the terminal electron acceptors of iron-reducing bacteria; therefore, these bacteria can be utilized for the pollution remediation of soil/groundwater and toxicity reduction. In microbial electrochemical systems, the electrons produced by the oxidation of organic matter by iron-reducing bacteria can be directly transferred to the electrode to generate electricity. Many microbial electrochemical technologies have been developed and are commonly used in wastewater regeneration and energy conversion fields, including microbial fuel cells, microbial electrolysis cells, microbial desalination cells, microbial fuel cells coupled with Fenton processes, and photocatalytic microbial fuel cells, which are utilized for microbial power generation, biosensors, biological hydrogen production, directional fermentation, seawater desalination, pollutant decomposition, and mineralization. Here, we summarize research progress on iron-reducing bacteria, including metabolic mechanisms, micro-ecological functions, environmental remediation, water regeneration, and energy conversion. We highlight outstanding questions in the study of iron-reducing bacteria and point out directions meriting future research. Generally, this review will aid both theoretical and applied studies of iron-reducing bacteria.

Abstract:Eukaryotic cells stimulated with external stresses such as heat shock, oxidative stress, nutrient deficiency or viral infections will induce a series of cellular responses, including stress granules (SGs), to facilitate the survival in the condition of environment stresses. As an aggregation product of translation initiation complex in cytoplasm, SGs play an important role in gene expression and homeostasis. Virus infection is one of the conditions that induces the production of SG. After the viruses invade the host cells, the host's translation system were hijacked by the viruses to fulfill its life cycle. Thus, host cells suspend the translation system and form the SGs to antagonize the invasion of viruses. This paper reviews the production and function of SG, the interaction between viruses and SGs, and the relationship between SGs and virus-induced innate immunity, in order to provide a direction for further research on antiviral targets.

Abstract:Extremophiles thrive in environments considered extreme for human life. To adapt to the extreme environments, a distinct abundant group of microbial bioactive molecules was produced in the extremophiles. Ectoine, as a compatible solute, was discovered originally in the halophilic bacterium and enable the bacteria to survive in salty environments. In addition to be functional as an osmotic counterweight, ectoine was able to protect proteins, nucleic acids, cell membranes and even the whole cells against various stressors such as heating, drying, freezing and radiation. Therefore, ectoines exhibit great potential for the application in the fields of biomolecule protection, biomedicine and biotechnology. With the development of synthetic biology and metabolic engineering, the traditional approach for ectoine production in halophilic bacteria was replaced by heterologous production of ectoines in the non-halophilic bacteria. In this paper, we overviewed the research progress on the microbial production and application of ectoines, which provides an important reference for the future development and commercial application of ectoines.

Abstract:Streptococci are the most common oral bacteria that play an important role in oral microbial ecology and pathogenicity. Most of oral streptococci can enter a specific physiological state known as competence, during which the bacteria can take up DNA from the environment and integrate it into their genome to gain new phenotypes. Many studies have shown that the competence regulatory pathways of oral streptococci are closely associated with their phenotypes including biofilm formation, bacteriocin production, acid resistance, oxidative stress, cell autolysis and drug resistance. Studies on the interplay among different phenotypes can not only advance the understanding of microbial interactions in the oral cavity, but also promote the management of oral infectious diseases. This review article summarizes the research progress on the relationship between competence and phenotypic traits of four typical oral streptococci, including Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguis and Streptococcus pneumoniae.

Abstract:Lipoic acid is a derivative of octanoic acid with two sulfur atoms at carbon atoms 6 and 8. The strong antioxidant activity of lipoic acid makes it have a good application prospect in the fields of health food, cosmetics and medicine. Lipoic acid as an important cofactor is required for several key enzymes complexes, like α-ketoacid dehydrogenase, and it also involves in energy generation and intermediate metabolisms. The protein lipoylation pathways have been well studied in the model organism Escherichia coli, including the lipoic acid de novo synthetic pathway dependent on LipB-LipA and the lipoic acid salvage pathway dependent on LplA. However, the lipoylation pathways in different bacteria show a highly diversity. The GcvH protein in certain bacteria also participates in protein lipoylation eventhough the related enzymes are different. In this review, we comprehensively discussed the current research progress on lipoic acid dependent multiple enzyme complexes, the lipoyl domain, GcvH proteins, as well as the protein lipoylation pathways in differen bacteria, aiming to provide theoretical support for further understanding the protein lipoylation in bacteria, developing the targeted antibacterial drugs, and efficient lipoic acid production with biological methods.

Abstract:The genus Lentzea, established by Yassin et al in 1995, is a typical group of filamentous rare actinomycetes. This genus currently contains 23 valid species, and its typical taxonomic characteristics includes: meso-diaminopimelic acid in the cell wall; MK-9(H₄) as the dominant menaquinone; the principal phosphate lipids include phosphatidylethanolamine, diphosphatidylglycerol, phosphotidylinositol and phosphatidylglycerol; the G+C content of genomic DNA is 68.6 mol%–79.6 mol%. Metabolites from strains of the genus Lentzea have significant diversity of biological activities, including human immunodeficiency virus type 1 integrase inhibitory activity, anti-tumor activity, anti-tuberculosis activity and so on, gradually showing the potential application value in biopharmaceutical research. Our research group isolated several strains of the genus Lentzea during the study on actinobacterial biodiversity of limestone crust in Guizhou province, and Lentzea xinjiangensis DHS C013^T as well as Lentzea pudingi DHS C021^T were proposed as novel species of the genus using the polyphasic taxonomy methods. On the basis of our study, the relevant literatures and the latest research findings, this paper reviews the establishment and taxonomic characteristics of the genus Lentzea, ecological diversity, functional genome and application of natural active metabolites.

Abstract:The colonization of carbapenem-resistant Enterobacteriaceae (CRE) in the intestinal lumen usually precedes or coexists with CRE infection. The intestinal flora and host normally interact with the colonized CRE to achieve a stable equilibrium state. When dysbacteriosis happens, the intestinal flora losses its resistance to the colonized CRE, increasing the risk of CRE infection. A large number of studies suggest that the resistance of intestinal symbiotic flora to the colonization of CRE can not only prevent infection, but also reduce the risk of mutual transmission among patients exposed to the medical environment. To provide a new idea and method for prevention and control of CRE infection, we studied the prevalence of CRE，the colonization mechanisms of Enterobacteriaceae, and the mechanisms of colonizing resistance to CRE of intestinal flora.

Abstract:Streptococcus pneumoniae is surrounded by capsular polysaccharide (CPS), which is a critical virulent factor of vbacteria, an antigen for host antibodies, but also a basis for serotyping. Some serotypes causes serious in vasive diseases to human, therefore, their CPSs are made into vaccines that have greatly reduced the infection from pneumococci. Many CPSs have complicated chemical structure and are usually decorated by O-acetyl groups. Capsular O-acetylation is easily changed and plays an important biological role. This review focuses on the research progress of pneumococcal O-acetylation. We introduce the genetic basis, biosynthesis and serological characterization of CPS. We make a summary for O-acetylated CPS structures and related O-acetyltransferases as well as the identification and biological function of O-acetylation. Meanwhile, we also discuss the effects of O-acetylation to pneumococcal microevolution and polysaccharide-vaccines. The prospects for future studies are finally proposed. This review is expected to lay the foundation for mechanism investigation of O-acetylation in pneumococcal invasion and provide a guide for designing polysaccharide-vaccines.

Abstract:[Objective] To screen antagonistic strain resources of Cotton Fusarium Wilt (Fusarium oxysporum f. sp. vasinfectum), endophytic bacteria were isolated from the root of Rehmannia glutinosa, a medical herb, and the excellent strains’ characteristics of plant growth promotion and salt and alkali tolerance were analyzed, so as to explore the excellent strain resources and provide reference value for the research and development of biological control agents of F. oxysporum f. sp. vasinfectum. [Methods] The antagonistic test of F. oxysporum f. sp. vasinfectum was performed by plate confrontation method, and the changes in mycelia were studied through fluorescence microscopy. Such indexes as indoleacetic acid (IAA) production and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were determined by spectrophotometry. Phosphate solubilization was assayed by plate culture method. Bacteriostasis of volatile substances was examined by separation culture method. Salt and alkali tolerance was measured by turbidimetric method. The physiological and biochemical characteristics of the strains were determined, 16S rRNA gene were sequenced and analyzed for phylogenetic status analysis, and disease-control effects were demonstrated by greenhouse inoculation test. [Results] The endophytic bacteria of R. glutinosa exerted antagonistic effects on F. oxysporum f. sp. vasinfectum, among which the strains DH9, DH66 and DH92 exhibited obvious antagonistic effects. Compared with the control group, the hyphae in the treatment group showed knotting, bending and breaking, the end branches of hyphae increased significantly, and most of the edges were coral branches, and were obviously embedded by the bacteria. The volatile substances produced by strains DH83, DH66, DH92, DH9 and DH56 exerted inhibitory effects on F. oxysporum f. sp. vasinfectum, but such effects were not salient. IAA contents of seven strains (DH92, DH30, DH71, DH83, DH93, DH9 and DH56) were higher than 1.32mg/L.DH92 had the highest yield, which amounted to 34.696 mg/L. The activities of ACC deaminase produced by DH92 and DH30 were 118.612 μmol/(mg·h) and 103.795μmol/(mg·h), respectively. DH92 had the highest ability to dissolve inorganic phosphorus, with diameter of dissolving circle/colony (D/d) being 1.51, DH71 had the highest ability to dissolve organic phosphorus, and D/d was 4.50. The strains DH9 and DH56 could tolerate 7% and 3% NaCl concentration respectively, and could grow at pH 8–10, with certain resistance. Combined with the culture characteristics, physiological and biochemical characteristics and 16S rDNA sequencing and phylogenetic analysis, results showed that DH30 exhibited the highest similarity with Bacillus safensis, DH9 showed the highest similarity with Bacillus subtilis, and DH92 had the highest similarity with pantoa ananatis. Control effect of treatment group with DH92 was 77.29%, and those of others were above 63%. Therefore, these strains can be used as biocontrol resources against F. oxysporum f. sp. vasinfectum.[Conclusion] There are antagonistic strains of F. oxysporum f. sp. vasinfectum in the root of R. glutinosa, among them. The isolation of Pantoa ananatis DH92 from R. glutinosa root has not been reported before. The excellent endophytic bacteria of R. glutinosa have the characteristics of plant growth promotion and certain salt and alkali tolerance, which provide theoretical basis for the control of F. oxysporum f. sp. vasinfectum and the development of biological control agents.

Abstract:[Objective] Analyze the characteristics of the pan-resistant genome of Salmonella. [Methods] In this study, the pan-resistome analysis pipeline developed in our previous researches was used to identify the pan-resistant genomic structure and to analyze the correlation between drug-resistant genes and serotype, sequence type and isolation information, etc. based on the sequencing of 16,365 Salmonella strains downloaded from the EnteroBase database.[Results] All drug-resistant genes were categorized into 104 alleles, among which 18 alleles belonged to the core resistome and 86 alleles belonged to the accessory resistome. It had an open pan-resistant genome. The same serotype (or sequence type) had similar drug-resistant genes profile, however, the distribution of drug-resistant genes in different serotypes (or sequence types) is significantly different (P<0.05). Moreover, there was a certain correlation between drug-resistant genes and sample sources, separation countries and isolated years. The proportion of 23 acquired drug-resistant genes and their subtypes revealed that 10 (43.48%) of them increased yearly, and 10 (73.91%) of them had only one dominant type. [Conclusion] All the results obtained in this study have revealed the distribution and trend of Salmonella drug-resistant genes in recent years, in further to provide new ideas to study the drug-resistance of pathogens.

Abstract:[Objective] This study aimed to analyze the genetic characteristics of Lactobacillus (L.) plantarum P9 and Lp-6, to provide information for future identification of L. plantarum. [Methods] Whole genome sequencing of L. plantarum strains P9 an Lp-6 was performed with PacBio SMRT sequencing and comparatively analyzed together with 110 genome sequences to identify characteristics usable in differentiating between L. plantarum strains. [Results] The genome length of P9 and Lp-6 strains was 3314.2 kb and 3482.5 kb, respectively. The GC content (%) of Lp-6 and P9 strains was 44.38% and 44.32%, and they contained 8 and 9 plasmids, respectively. Phylogenetic analysis showed a close genetic distance between L. plantarum P9 and ATCC 14917, and Lp-6 was closer to the ancestral branch. Comparing with L. plantarum WCSF1, the 22 kb-genome fragment of xerS was inverted in Lp-6 and was missing in P9 strain. A 13 kb-genomic fragment with the tagF gene was inserted in the Lp-6 genome; a 14.4 kb-gene fragment (containing the gpmA gene) was inserted in the P9 genome. [Conclusion] Through comparative genomic analysis of P9 and Lp-6 together with other L. plantarum, some specific genomic features were identified, which could be used for differentiation between strains.

Abstract:[Objective] Aspergillus fumigatus is a cellulolytic fungus. The research of its genome will facilitate the mining and development of enzyme resources associated with cellulose degradation. [Methods] A. fumigatus HZ1 was isolated from the gut of Cyrtotrachelus buqueti by CMC selective medium and Congo red staining method. Illumina PE50 platform was used to conduct genome sequence and the relative bioinformatics analysis was performed subsequently. Furthermore, the cellulase activities of this strain were determined by DNS method. [Results] The genome size of A. fumigatus HZ1 was 27.45 Mb with a GC content of 49.43%. The results from NR, KOG, GO, Swissprot, eggNOG, KEGG and Pfam database annotations reveal that the genome contained 9473 genes, and the carbohydrate active enzyme (CAZyme) annotation shows that the genome contained 534 CAZyme genes and coincided with the distribution of CAZyme in the other four A. fumigatus genomes. A variety of cellulase genes, hemicellulase genes and ligninase genes related to lignocellulose degradation were identified. In addition, cellulase activity showed an increasing trend and high activity in CMC medium. [Conclusion] Our findings provide reference for the application of A. fumigatus.

Abstract:[Objective] The aim of our study is to express Antrodia camphorata immunomodulatory protein (ACA) in Escherichia coli efficiently.[Methods] We synthesized ACA gene with E. coli codon bias using DNAWorks 3.2.4 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pET-32a and pGEX-4t-2, transformed them into E. coli and named them pET-32a-ACA/BL21 and pGEX-4t-2-ACA/BL21 respectively after sequencing. We investigated the effects of codon bias, vectors, mediums and the induction conditions on the expression of recombinant ACA (rACA), and further optimized the protein expression using response surface methodology (RSM) subsequently. To evaluate the bioactivity of rACA, we determined the proliferation of RAW264.7 cells, analyzed the phagocytosis of macrophages, observed the cell morphology, detected the secretion of cytokine such as nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) after treated RAW264.7 cells with purified rACA. [Results] pET-32a-ACA/BL21 was more suitable for the expression of ACA. The rACA was expressed mainly in soluble form with a yield of 187.122 μg/mL, when the pET-32a-ACA/BL21 was cultured in optimized SOB medium (8.92 g/L yeast extract) and induced with 0.42 mmol/L IPTG at 25 ℃ for 7 h. This was the highest expression report for fungal immunomodulatory protein in E. coli so far. Moreover, the purified rACA promoted the proliferation of RAW264.7, enhanced the phagocytosis activity, changed the cell morphology as well as induced NO, TNF-a and IL-1β production within murine macrophages. [Conclusion] The highly soluble expression, and the very similar biological activities to native ACA make it a good candidate of native ACA for potential application as a food supplement or immunomodulatory agent in pharmaceuticals and even medical studies.

Abstract:[Objective] Ginkgo biloba extract plays an important role in the prevention and treatment of cardiovascular and nervous system diseases. In consideration of the fact that gut microbiota has been identified as a new drug target, it is of great significance to study the interactions between ginkgetin and ginkgolide B with human gut microbiota, which will lay a foundation for further understanding the function and mechanism of Ginkgo biloba extract. [Methods] In this study, batch fermentation, bacterial amount detection, 16S rDNA high-throughput sequencing, GC and HPLC measurement were used to study the interactions between ginkgetin and/or ginkgolide B with human gut microbiota in vitro. [Results] Neither ginkgetin nor ginkgolide B alone had a significant effect on the total amount of gut microbiota, the composition of intestinal flora and the production of short chain fatty acids by the gut microbiota. However, when ginkgetin and ginkgolide B were added in combination, the proportion of bacterial represented by the family Coriobacteriaeae and genus Cupriavidus increased significantly, while the proportion represented by the genus Gemella decreased significantly. Functional gene prediction analysis found that genes encoding K00076, K12143, K07716, and K00220 significantly enriched in the presence of ginkgetin and ginkgolide B. Moreover, K00076 and K00220 are oxidoreductases that catalyze the transfer of electrons from the CH-OH donor group and may be involved in the metabolism and modification of ginkgetin and ginkgolide B. The degradation and modification rates of ginkgetin and ginkgolide B by human gut microbiota in vitro were ~70% and ~35%, respectively. [Conclusion] The relative percentage of some intestinal bacteria was significantly changed by the combined addition of ginkgetin and ginkgolide B in vitro. Meanwhile, human gut microbiota has the function on metabolism or modifying ginkgetin and ginkgolide B.

Abstract:[Objective] In this paper, an anaerobic electrical membrane bioreactor (E-MBR) coupled the anaerobic membrane bioreactor (MBR) and microbial fuel cell (MFC) was applied in the industrial coking wastewater treatment. [Methods] The culture conditions including nutrients and metal elements of the influent of the reactor were optimized by orthogonal experiments as 14.3 mg/L PO₄^3–, 0.2 mg/L Fe²⁺, 0.1mg/L Fe³⁺, 0.1mg/L Co²⁺, 0.2 mg/L Mn²⁺. Under this conditions, the removal efficiency of COD, the sludge characteristics, electrical performance, extracellular polymers (EPS), microbial community structure and membrane fouling of anaerobic sludge in the reactor were investigated. [Results] The results showed the removal rate of COD was increased by 23% compared with that without optimization. Meanwhile, sludge concentration (MLSS), specific gravity and the settling speed increased, while the sludge volume index (SVI) decreased, indicating that the enhancement of the anaerobic sludge granulation and settling performance. Each component of the EPS of the sludge such as the ratio of protein to polysaccharide (P/C) soluble microbial products (SMP), loose EPS (LB-EPS) and tightly bound EPS (TB-EPS) decreased by 0.12, 0.25 and 0.16, respectively, which indicated that sludge was more easily degraded. The electrical performance of the anaerobic sludge was enhanced. The results of high-throughput molecular sequencing manifested that the community structure of sludge in the reactor had changed significantly, and the dominant bacteria group was prominent. The results of scanning electron microscopy (SEM) showed that the pollution of the cathode membrane of the reactor was also reduced. [Conclusion] The optimization of influent culture conditions could improve the efficiency of wastewater treatment, shorten the cleaning cycle and make the operation more stable, which provided a theoretical basis for energy saving and environmental protection of industrial wastewater treatment technology.

Abstract:[Objective] The biosynthetic pathway of NADPH in Corynebacterium glutamicum was modified to block NADPH production, thus constructing an NADPH-auxotrophic C. glutamicum recombinant.[Methods] To block NADPH production in cell, we firstly inactivated the glucose-6-phosphate dehydrogenase (Zwf) and malic enzyme (MalE) in an L-lysine high-producing strain C. glutamicum Lys-χ, and replaced the native NADP⁺-dependent isocitrate dehydrogenase (NADP⁺-Icd_Cg) with NAD⁺-Icd_Sm from Streptococcus mutans. Then, we introduced the proton-pumping nicotinamide nucleotide transhydrogenase (PntAB) from Escherichia coli with different expression level controlled by different strength promoters into NADPH-auxotrophic C. glutamicum recombinant. Lastly, we analyzed the changes of intracellular redox level and production intensity of L-lysine in the recombinant strains with different PntAB expression levels. [Results] There was no detectable NADPH in the recombinant strain C. glutamicum Lys-χ ΔZMI_Cg::I_Sm (i.e., Lys-χ1), indicating that strain Lys-χ1 was an NADPH-auxotrophic C. glutamicum. Strain Lys-χ1 grew well and accumulated L-lysine in the basic medium with gluconolactone as carbone source, whereas it could not grow with glucose, pyruvate, α-ketoglutaric acid and oxalacetic acid as carbon source. In addition, overexpression of PntAB in strain Lys-χ1 replenished the intracellular NADPH level, but the NADPH level in different recombinant strains was different because there was the different expression level of PntAB under the different intensity of promoters. And these affected the cell growth and L-lysine production.[Conclusion] The strain Lys-χ1 could be used as a chassis cell for the capacity of strategies to regenerate NADPH in vivo, thus obtaining recombinant strains with different intracellular NADPH levels. Therefoere, this study provided a basis for investigating the regulatory mechanism of NADPH on physiology and metabolism of microbial cells.

Abstract:[Objective] To characterize a type I restriction-modification (RM) system C5423-5425 in uropathogenic Escherichia coli CFT073 and to determine its function and physiological significance. [Methods] Lambda Red recombination system was used to construct methyltransferase gene deletion mutant Δc5424 in CFT073. The modification sites and recognition motif of C5424 methyltransferase were obtained by single molecule real-time sequencing analysis. A transformation efficiency assay was used to test that the RM system C5423-5425 can block transformation of exogenous DNA. Genes regulated by C5424 were identified by transcriptome sequencing and real-time quantitative PCR. The physiological function of c5424 was studied by soft agar plate motility assay. [Results] A type I RM system was identified by bioinformatics methods. A c5424 deletion mutant was constructed. We found that the C5424 recognition motif was G^mAGNNNNNNNGTCA/TG^mACNNNNNNNCTC, and its distribution in the genome was presented. C5424 contributed to reducing transformation of foreign DNA. Deletion of c5424 significantly affected the expression of 17 genes, including motB and rclR. Lacking c5424 significantly affected bacterial motility and the resistance to hypochlorous acid in CFT073. [Conclusion] In this paper, the type I RM system C5423-5425 was characterized in detail, and it is physiologically important for uropathogenic E. coli CFT073. Thus, our data provided valuable information for bacterial epigenetics studies.

Abstract:[Objective] We investigated the effects of the nitrate-specific activator NirA1 on the growth and stress resistance of Beauveria bassiana. [Methods] The expression of NirA1 was detected by RT-PCR in different culture mediums. Several NirA1 strains, including gene knockout (ΔNirA1), complementary (ComNirA1), and over-expression (OENirA1), were constructed and the functions of NirA1 involved in fungal developmental differentiation, stress reactions and virulence were analyzed. [Results] Expression level of NirA1 was higher in CZB medium than that in PDB or SDB. The growth of ΔNirA1 in mediums with different nitrogen sources was significantly slower compared to wild type. The expression of nitrate transporter NrtA was down-regulated in NirA1 deletion mutant. Compared to wild type, conidial yield in CZA, PDA and 1/4 DAY mediums decreased by 21.6%, 16.2% and 25.6% respectively. The growth inhibition rate of ΔNirA1 in 32 ℃, 1 mol/L NaCl and H₂O₂mediums was reduced by 29.0%, 25.2% and 49.0%, but increased by 34.1% and 96.2% in SDS and Congo red mediums respectively. Bioassay of fungal strains were performed using 3^rd-instar larvae of G. mellonella. Compared to wild type, deletion of NirA1 increased fungal virulence, decreasing mean lethal time (LT50) by 17.4%. [Conclusion] These results indicated NirA1 is involved in the utilization of nitrogen sources and play crucial roles in colony growth, conidiation, stress responses, and pathogenesis of B. bassiana.

Abstract:[Objective] Ste50 is an important adaptor protein in fungi, which plays important roles in signal connection and transmission in multiple MAPK pathways. In this study, Aflste50 protein of Aspergillus flavus was identified and its effects on growth, conidiation, pathogenicity and response to osmotic stress were found. [Methods] Firstly, the ste50 gene was identified in A. flavus NRRL 3357 by bioinformatics method. Then, the effect of gene knockout on growth, secondary metabolite synthesis and stress response of A. flavus were tested. [Results] Compared with the wild type, the growth rate and AFB₁ synthesis of △Aflste50 strain were decreased. And sclerotia formation of A. flavus was blocked when ste50 was absent. At the same time, the pathogenicity to peanut and maize seeds was impaired in △Aflste50. Also, AflSte50 regulated the phosphorylation of MAP kinase under osmotic stress, but did not respond to cell wall stress.[Conclusion] Ste50 (AFLA_ 002340) is an adaptor protein of A. flavus, which affects the growth and development, as well as the synthesis of AFB₁ in this fungus. It can respond to osmotic stress and play a role in the HOG pathway.

Abstract:Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen, which often exists in the intestines of ruminants, such as cow and sheep, as normal flora.[Objective] In this study, we isolated and identified Shiga toxin-producing Escherichia coli isolates from cow and sheep feces samples, and analyzed their pathogenic potentials Totally 11 Shiga toxin-producing Escherichia coli strains from sheeps and 1 isolate from cattle were isolated from Jiangsu, Yunnan, and Hebei, and another 10 isolates of STEC from cattle were donated by Tong Pan Group of Xinjiang Agricultural University. [Methods] We conducted the separation and identification through bacterial selective culture and detection of specific genes stx1 and stx2. We analyzed the pathogenic potential of the Shiga toxin-producing Escherichia coli isolates by Vero cytotoxicity test, hemolytic activity test, and toxin factor detection. [Results] In this study, the separation and identification result revealed that 11 isolates of sheep origin were isolated with an isolation rate of 17.5% (11/63); 1 isolate from cow origin was isolated with an isolation rate of 0.7% (1/134). The pathogenic potential results showed that, among the 11 sheep-derived isolates, 5 of them had strong toxicity to Vero cells, and 3 had hemolytic activity. Among the 11 cow-derived isolates, 5 had strong toxicity to Vero cells, and 3 had hemolytic activity. The eae gene carrying rate of the 11 sheep-derived Shiga toxin-producing Escherichia coli isolates was 63.6% (7/11), while the eae gene carrying rate of the cow-derived 11 Shiga toxin-producing Escherichia coli isolates was only 9.0% (1/11). [Conclusion] The results indicated that the isolation rate and pathogenic potential of the Shiga toxin-producing Escherichia coli strains derived from sheep were higher than the strains of cow origin. Therefore, sheep, as the host of Shiga toxin-producing Escherichia coli strains, should be paid higher attention than cow.

Abstract:[Objective] Ensifer alkalisoli YIC4027 is a new type of high-efficiency nitrogen-fixing bacteria isolated from the root nodules of the host plant Sesbania cannabina. This study compared the chemoreceptors genes and related proteins of E. alkalisoli with other well-researched species. [Methods] NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of E. alkalisoli. HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein(MCP). [Results] There were two major chemotaxis clusters and 13 MCP homologs containing diverse signal-sensing architectures in E. alkalisoli. In addition, except that one of the cytoplasmic domains of these MCPs was composed of 40 heptad repeats, the rest were composed of 36 heptad repeats. [Conclusion] Despite the high similarity presented between the chemoreceptors of E. alkalisoli and those of well-studied species, E. alkalisoli shows its own unique characteristics. The classification of these chemotactic pathways by gene comparison and related protein analysis enables us to better understand how E. alkalisoli responds to changes in environment via exquisite signal transductions in chemotaxis system.

Abstract:Using high-property terminators enable significant enhancement of transcription termination, mRNA stability, heterologous gene expression in classic microorganisms. However, design of complex gene circuits was limited by the lack of terminators specifically adapted to Bacillus subtilis. [Objective] Exploiting new high-performance terminators from Bacillus subtilis and further redesigning to enrich the artificial terminators suitable for this chassis. [Methods] The terminators from Bacillus subtilis and Bacillus subtilis phage were respectively constructed into the terminator measurement plasmid to determine the termination efficiency (TE). Multiple tandem terminators were constructed with combinatorial patterns of strong-weak, strong-strong, and weak-weak and subsequently TEs of those tandem terminators were individually determined. The tandem terminators with high TE were harnessed to verify the function of heterologous expression of L-aspartate amino lyase (AspA) and β-glucuronidase (GusA) in B. subtilis. [Results] The TE of terminator TB5 is 98%, which is strongest among the natural terminators in B. subtilis. Meanwhile, expression level of GFP harboring TB5 was up-regulated by 2.2 folds, and the expression level of RFP was down-regulated by 27 folds. For the dual-tandem terminators, the expression levels of RFP regulated by TH1.5b-TB5 (TE=97%) and TB5-TB5 (TE=98%) tandem terminators were decreased by 30 folds. For the triple-tandem terminators, TB2-TB5-TB5 combination no longer increase the expression level of GFP compared with the corresponding dual combination. Nevertheless, the expression of RFP is only 1/300 of the control. Finally, heterologous expression of AspA and GusA confirmed that two tandem terminators, TH1.5b-TB5 and TB10-TB5, significantly improved the gene expression in B. subtilis. [Conclusion] Strong synthetic terminators are able to reinforce the heterologous gene expression in surrogate host. Importantly, the efficiency of termination is further improved by rational combination of natural terminators in tandem manner. These artificially redesigned terminators could be conveniently and portably constructed into bacterial gene circuits to augment and stabilize gene expression.

Abstract:[Objective] To study the regulatory effect of TetR family transcription factor Ms0606 on the drug resistance of Mycobacterium smegmatis. [Methods] Firstly, the growth curve was measured to detect the regulatory role of Ms0606 in mycobacterial resistance; electrophoretic mobility shift assay and DNase I footprinting assay were used to identify the conservative sequence recognized by the transcription factor Ms0606, and then explore its potential target genes. Secondly, reverse transcription-qPCR and β-galactosidase activity experiments were used to detect the regulatory effect of Ms0606 on the target gene Ms0608. The potential roles of Ms0606 in the regulation of drug resistance are further discussed. [Results] Overexpressing Ms0606 made mycobacteria more sensitive than the wild-type strain in response to isoniazid, whereas disrupting Ms0606 is resistant to isoniazid; Ms0606 could recognize a conserved 22 bp palindromic motif within the upstream region of its self-operon, using the palindrome sequence to search M. smegmatis genome, it is found that Ms0606 may regulate 5 potential target genes; Reverse transcription-qPCR and β-galactosidase activity experiments revealed Ms0606 acts as a repressor and negatively regulates Ms0608 expression, which may affect mycobacterial antibiotic resistance. [Conclusion] I have identified a new TetR family transcriptional regulator of Mycobacterium smegmatis encoded by Ms0606, which regulates mycobacterial sensitivity to isoniazid, and further characterized its target genes and its regulatory mechanism for mycobacterial resistance.

Abstract:Food safety issues caused by food-borne pathogens have become a critical factor threatening human health. Therefore, studying the infection mechanism of food-borne pathogens is of great significance to control the harm caused by pathogens. [Objective] Taking the common food-borne pathogenic bacteria Salmonella typhimurium as the research object, we studied the genetic manipulation methods for knocking out its pathogenicity associated transcriptional regulator coding gene SlyA by systematically compared the technical differences between CRISPR/Cas9-guided-Cytidine Base Editor (CBE) and λ-Red homologous recombination methods. The work present here not only provides detailed data for the application of gene editing technology for S. typhimurium, but also provides a useful reference for the development of gene editing technology for other pathogen species. [Methods] The PCR, Golden Gate, and Sanger sequencing were used to construct CBE and λ-Red gene editing systems and verify the gene edit results. Editor-R was used to analysis base edit efficiency of CBE system. Western blot was used to verify the results of gene editing at the protein expression level. In addition, phenotypic identification was used to verify the knockout results. [Results] The PCR product sequencing, Western blot verification results and hemolysin activity identification showed that the CBE gene editing system has been successfully applied to S. typhimurium. The SlyA null mutant of S. typhimurium was constructed based on the two above-mentioned technology, respectively. [Conclusion] Although the CRISPR/Cas9-guided Cytidine Base Editor has the advantages of simpler operation and more efficient editing than the λ-Red system, the system still has several shortcomings, such as the need to establish a specific gRNA sequence and PAM locus which are not so common in non-model strains. In addition, the CBE system has the problem of instability during gene editing. Nevertheless, CBE was successfully applied to Salmonella typhimurium for the first time, which will provide a basis for optimizing and improving the gene editing of this strain.

Abstract:[Objective] The purpose of this study was to reveal the biological function of rpfG gene in walnut bacterial black spot pathogen (Xanthomonas arboricola pv. juglandis) XajDW3F3, so as to provide a target for the development of prevention and treatment agents for walnut bacterial black spot. [Methods] The rpfG gene of Xanthomonas campestris pv.campestris 8004 and Xanthomonas campestris pv.campestris PXO99^A were used as template sequences to search the genome sequence of DW3F3, a wild-type strain of Xaj. With homologous recombination technology to knock out XajrpfG, the virulence factors and stress resistance of the mutant strain were detected by biochemical methods. [Results] Homologous gene was identified in XajDW3F3 genome through sequence homology analysis. The deletion mutant of rpfG was successfully obtained. Compared with the wild type, the mutant reduced the production of biofilm and extracellular polysaccharides, the ability of biofilm formation mutant strains ΔrpfG was only about 44.58% of wild type, the yield of exopolysaccharide decreased from 8.47 mg/mL in wild type to 5.23 mg/mL in ΔrpfG; but ΔrpfG increased the motility and flocculation activity, motility experiments showed that the motility diameter of ΔrpfG increased 12.38% than the wild type. The secretion of extracellular enzymes also changed at different degrees: the ability to secrete cellulase was significantly decreased and amylase activity was increased, but the ability to secrete protease was not changed. What’s more, after the deletion of rpfG, the resistance of Xaj to adversity (NaCl, pH, SDS, CuSO₄) was decreased. [Conclusion] The results showed that rpfG is an important pathogenic gene of walnut bacterial black spot pathogen, and endows bacteria with a certain degree of resistance to adversity.