摘要
目的构建变异链球菌UA159 vicK ATP缺失突变株,研究vicK基因ATP结合位点对变异链球菌生长、产酸耐酸、细胞外多糖(exopolysaccharides,EPS)和生物膜形成等生物学功能的影响。方法quick change法构建vicK ATP缺失突变质粒,同时引入Sma lI,Sma lI插入kan基因盒(lox71-kan-lox66),建立vicK ATP缺失同源重组载体。所获载体转化变异链球菌,卡那霉素筛选阳性菌。pCrePA质粒转化阳性菌,30℃移除kan基因,37℃去除pCrePA,同源重组法获得无标记基因组vicK ATP缺失突变株。表达纯化VicK ATP缺失蛋白,并验证其ATP激酶活性。结果成功构建UA159 vicK ATP缺失突变株和补偿株。vicK ATP突变株生长速率和pH值下降均较野生株缓慢;实验株随致死性酸处理时间延长,生存率呈下降趋势,但突变株生存率较野生株高,即耐酸性增强;突变株EPS和生物膜形成较野生株明显减少。结论vicK基因ATP结合位点对变异链球菌的生长、产酸、酸耐受、EPS和生物膜形成起着重要调控作用。
To construct a vicK ATP-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 Sma lI site.The plasmids were digested with Sma lI,and a kanamycin gene flanked by two lox P 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 vicK ATP-deletion mutant strain.The VicK ATP-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 vicK ATP-deletion mutant strains and complemented strains were successfully constructed.Compared with S.mutans UA159,the vicK ATP 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 vicK ATP 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.
作者
黄珊珊
宋秀宇
徐巧丽
饶慧华
张加勤
HUANG Shan-shan;SONG Xiu-yu;XU Qiao-li;RAO Hui-hua;ZHANG Jia-qin(Department of Pathology,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China;Xiamen Central Blood Service Station,Xiamen 361003,China;Department of Clinical Laboratory,Zhangzhou Affiliated Hospital of Fujian Medical University,Zhangzhou 363000,China;Jiangxi Maternal and Child Health Hospital,Nanchang 330006,China;Department of Clinical Laboratory,the First Affiliated Hospital of Xiamen University,Xiamen 361003,China;Xiamen Key Laboratory of Genetic Testing,Xiamen 361003,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2021年第5期435-443,共9页
Chinese Journal of Zoonoses
基金
福建省卫生计生中青年骨干人才培养项目(No.2017-ZQN-84)。
