The influence of vicK gene ATP binding site on the biological function of Streptococcus mutans
HUANG Shan-shan1, SONG Xiu-yu2, XU Qiao-li3, RAO Hui-hua4, ZHANG Jia-qin5,6
1. Department of Pathology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005,China; 2. Xiamen Central Blood Service Station, Xiamen 361003,China; 3. Department of Clinical Laboratory, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, China; 4. Jiangxi Maternal and Child Health Hospital, Nanchang 330006, China; 5. Department of Clinical Laboratory, the First Affiliated Hospital of Xiamen University, Xiamen 361003,China; 6. Xiamen Key Laboratory of Genetic Testing, Xiamen 361003,China
Abstract:To construct a vicKATP-deletion mutant and research its roles in growth, acid production, acid tolerance, and formation of EPS and biofilms in Streptococcus mutans UA159, we constructed plasmids for expression of a vicK ATP binding site deletion mutant through site-directed mutagenesis simultaneously introducing a SmalI site. The plasmids were digested with SmalI, and a kanamycin gene flanked by two loxP sites was introduced, thus yielding the homologous recombination vector. The vector was integrated into the chromosome of S. mutans UA159. The homologous recombination strain was selected on the basis of resistance to kanamycin. The selected mutant strains were transformed with the thermosensitive plasmid pCrePA to excise the kan gene at 30 ℃ and cultured at 37 ℃ to excise the pCrePA, thus yielding an unmarked vicKATP-deletion mutant strain. The VicKATP-deficient protein was expressed and purified, and its ATP kinase activity was verified. The growth conditions, acid production, acid tolerance, and formation of EPS and biofilms were measured The vicKATP-deletion mutant strains and complemented strains were successfully constructed. Compared with S. mutans UA159, the vicKATP mutant strains showed slower growth and less acid production. All test strains displayed decreased growth at nonpermissive pH over time, but unexpectedly, loss of the vicK ATP binding site enhanced the survival rate. The formation of EPS and biofilm in vicKATP mutant strains was significantly less than that in wild-type strains. The ATP binding site in the vicK gene is therefore important for regulating growth, acid production, acid tolerance, and the formation of EPS and biofilm in S. mutans.
黄珊珊, 宋秀宇, 徐巧丽, 饶慧华, 张加勤. vicK基因ATP结合位点对变异链球菌生物学功能的影响[J]. 中国人兽共患病学报, 2021, 37(5): 435-443.
HUANG Shan-shan, SONG Xiu-yu, XU Qiao-li, RAO Hui-hua, ZHANG Jia-qin. The influence of vicK gene ATP binding site on the biological function of Streptococcus mutans. Chinese Journal of Zoonoses, 2021, 37(5): 435-443.
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