Construction and biological characterization of a Proteus mirabilis strain with modABC gene deletion.
10.12122/j.issn.1673-4254.2023.05.23
- Author:
Yi HUANG
1
;
Xin DING
1
;
Nan HUANG
1
;
Canxiong CHEN
1
;
Xiaoyan DENG
1
Author Information
1. Guangzhou Key Laboratory for Clinical Rapid Diagnosis and Early Warning of Infectious Diseases, Guangzhou 510180, China.
- Publication Type:Journal Article
- Keywords:
Proteus mirabilis;
homologous recombination;
molybdate transporter;
suicide vector
- MeSH:
Humans;
Gene Deletion;
Nitrates;
Proteus mirabilis/genetics*;
Gene Knockout Techniques
- From:
Journal of Southern Medical University
2023;43(5):859-867
- CountryChina
- Language:Chinese
-
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.
