南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (5): 859-867.doi: 10.12122/j.issn.1673-4254.2023.05.23

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奇异变形杆菌modABC基因缺失株的构建及其生物学特性

黄 伊，丁 信，黄 楠，陈灿雄，邓小燕   

  1. 广州市传染性疾病临床快速诊断与预警重点实验室，广东 广州 510180；广州医科大学金域检验学院，广东 广州 510180
  • 出版日期:2023-05-20 发布日期:2023-06-12

Construction and biological characterization of a Proteus mirabilis strain with modABC gene deletion

HUANG Yi, DING Xin, HUANG Nan, CHEN Canxiong, DENG Xiaoyan   

  1. Guangzhou Key Laboratory for Clinical Rapid Diagnosis and Early Warning of Infectious Diseases, Guangzhou 510180, China; KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510180, China
  • Online:2023-05-20 Published:2023-06-12

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摘要： 目的 基于同源重组方法构建奇异变形杆菌钼酸盐转运蛋白编码基因modABC基因敲除株，并采用电感耦合等离子质谱法检测胞内钼含量，探究modABC基因对奇异变形杆菌生物学特性的影响。方法 利用自杀载体pCVD442通过同源重组方法构建modABC基因敲除株ΔmodABC，并通过PCR及Sanger测序鉴定；采用电感耦合等离子质谱法测定亲本株PMI 和敲除株ΔmodABC 胞内钼酸盐含量，比较亲本株PMI 和敲除株ΔmodABC 在体外有氧和厌氧条件下的生存能力。结果 利用融合PCR成功将目的基因替换为卡那霉素抗性基因Kn，与自杀载体pCVD442重组并转导到奇异变形杆菌亲本株PMI，随后发生同源重组，modABC 基因被敲除，通过 PCR 及 Sanger 测序分析证实基因组目的基因已缺失，成功构建了 modABC 基因敲除株ΔmodABC；敲除株ΔmodABC 胞内钼含量约为1.22 mg/kg，明显低于亲本株PMI（1.46 mg/kg，P<0.001）；亲本株PMI和敲除株ΔmodABC在 LB液体培养基中具有相似的有氧生长曲线，二者在厌氧条件下细菌的增殖速率均明显下降，并且在硝酸盐存在的LB培养基中，敲除株ΔmodABC的厌氧生长受到明显抑制（P<0.005）。结论 基于自杀载体 pCVD442 的同源重组可成功用于奇异变形杆菌modABC基因敲除，modABC基因参与奇异变形杆菌钼酸盐的摄取并与细菌厌氧硝酸盐生长相关。

关键词: 钼酸盐转运蛋白；同源重组；自杀载体；奇异变形杆菌

Abstract: Objective To construct a modABC gene knockout strain of Proteus mirabilis and explore the effect of modABC gene deletion on biological characteristics of Proteus mirabilis. Methods Fusion PCR was used to obtain the fusion gene of modABC and the kanamycin-resistant gene Kn, which was ligated with the suicide vector pCVD442 and transduced into Proteus mirabilis. The modABC gene knockout strain of Proteus mirabilis was obtained after homologous recombination with the suicide vector. PCR and Sanger sequencing were used to identify genomic deletion of modABC gene in the genetically modified strain. The concentration of molybdate in the wild-type and gene knockout strains was determined using inductively coupled plasma mass spectrometry (ICP-MS), and their survival ability in LB medium was compared under both aerobic and anaerobic conditions. Results PCR and sanger sequencing confirmed genomic deletion of modABC gene in the obtained Proteus mirabilis strain. The concentration of intracellular molybdenum in the modABC gene knockout strain was 1.22 mg/kg, significantly lower than that in the wild- type strain (1.46 mg/kg, P<0.001). Under the aerobic condition, the modABC gene knockout strain grown in LB medium showed no significant changes in survival ability compared with the wild-type strain, but its proliferation rate decreased significantly under the anaerobic condition and also when cultured in nitrate-containing LB medium under anaerobic condition. Conclusion Homologous recombination with the suicide vector can be used for modABC gene knockout in Proteus mirabilis. modABC gene participates in molybdate uptake and is associated with anaerobic growth of Proteus mirabilis in the presence of nitrate.

Key words: molybdate transporter; homologous recombination; suicide vector; Proteus mirabilis