The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile / 生物工程学报

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Group Ⅱ introns and the application in biotechnology: a review / 生物工程学报

Group Ⅱ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg2+ concentration. Finally, we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group Ⅱ introns in biotechnology.

A temperature-inducible Targetron system for efficient gene inactivation in Escherichia coli / 生物工程学报

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.