遵义医科大学学报2025，Vol.48Issue(7)：665-673,9.
肺炎克雷伯菌wzb基因缺失菌株表型及转录组分析
Phenotype and transcriptome analysis of wzb gene deletion strains of klebsiella pneumoniae
摘要
Abstract
Objective To investigate the effect of wzb gene deletion on the phenotype and gene expression level of klebsiella pneumoniae.Methods Using the wild-type strain W14 as the background strain,the wzb gene was knocked out using the temperature-sensitive homologous recombination system pKO3.The wzb gene knockout strains were verified by PCR technology.The △wzb deletion strains were complemented using pGEM-Teasy.The size and morphology of the colonies were observed on the solid plate of luria-bertani(LB),and the growth curve of the strains was determined in the LB liquid medium.The influence of the deletion of the wzb gene on capsule formation was explored through transmission electron microscopy and the determination of uronic acid content.The effect of wzb deletion on the phage sensitivity of W14 was explored through the plaque formation experiment.The influence of wzb deletion on the gene expression level of K.pneumoniae was clarified through transcriptome a-nalysis.Results The PCR results indicated that the △wzb deletion strain and the wzb/△wzb complementary strain were successfully constructed.The colonies of the △wzb deficient strain observed on the LB plate(1.33±0.21)were significantly smaller than those of the wild-type strain(2.04±0.16,P＜0.000 1),the colony size of the wzb/△wzb replenishment strain(2.14±0.20)returned to the wild-type level(P=0.286 8).The results of the growth curves showed that the growth rates of the three strains from high to low were wzb/△wzb replenishment strain,wild-type strain and △wzb deletion strain(P=0.012).The results of transmission electron microscopy in-dicated that the deletion of the wzb gene led to the loss of the ability of the strain to produce capsules,and the wzb/△wzb complementary strain could restore the ability of the strain to produce capsules.The test results of uronic acid content indicated that the uronic acid content of △wzb deficient strains(91.15±5.51,70.98±4.37)mg/L was much lower than that of wild-type strains(1 395.32±36.55,371.94±28.91)mg/L(P＜0.001);The wzb/△wzb complementary strain(1 571.29±219.10,427.93±20.06)mg/L showed no signifi-cant difference from the wild-type strain(P＞0.05).The results of the plaque formation experiment showed that the deletion of the wzb gene reduced the sensitivity to phiW14,and the wzb/△wzb complementary strain could re-store the sensitivity of the strain to phages.Analysis of the RNA-seq results revealed that the deletion of the wzb gene led to changes in various genes related to biological processes,among which the changes in genes related to membrane transport and metabolism were significant.Conclusion The deletion of the wzb gene makes the strain lose the ability to produce capsules and reduces the sensitivity of the strain to phiW14 bacteriophage.Meanwhile,the deletion of the wzb gene leads to significant changes in the expression of genes related to membrane transport and metabolism.关键词
肺炎克雷伯菌/wzb/基因敲除/荚膜/噬菌体敏感性Key words
klebsiella pneumoniae/wzb/gene knockout/capsule/phage sensitivity分类
医药卫生引用本文复制引用
付彤彤,范政,陈雨晨,刘洪伯,李周霏,袁静..肺炎克雷伯菌wzb基因缺失菌株表型及转录组分析[J].遵义医科大学学报,2025,48(7):665-673,9.基金项目
北京市自然科学基金资助项目(NO:5244032). （NO:5244032）
