Abstract
LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD50), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was strongerthan that of the othertwo strains. The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.
概 要
目 的
通过 kp05372 基因的敲除来研究其对林麝肺炎克雷伯氏菌生物表型的影响.
创新点
解析了 LysR 家族新基因 kp05372 对林麝肺炎克雷伯氏菌生物表型的影响.
方 法
通过基因敲除和回补技术构建了 kp05372 的基因缺失株和基因回补株, 比较了 kp05372 基因缺失前后, 林麝肺炎克雷伯氏菌在生长曲线、 药敏试验、 生物被膜、 抗逆性、 细菌半数致死量、 定殖能力、 病理形态学等生物学特性所表现出的差异.
结 论
成功构建了 kp05372 基因的缺失株及回补株. kp05372 基因的缺失导致林麝肺炎克雷伯氏菌耐药性和耐酸性发生了变化, 毒力、 生物膜和定殖能力降低, 并且调控了上游临近区域的基因表达.
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Acknowledgments
We thank the Animal Quarantine Laboratory of the College of Veterinary Medicine of Sichuan Agricultural University (Wenjiang, China) for providing experimental conditions for the experiment. We also thank Guangzhou KnoGen Biotech Co., Ltd. for providing help for the experiment. We extend our thanks to the hard work of the experimental contributors.
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Wei YANG, Wu-you WANG, and Yan LUO designed the experiment. Wei YANG and Wu-you WANG wrote the paper. Wu-you WANG carried out gene knockout and complementation experiments and other bacterial in vitro experiments. Wei YANG and Wei ZHAO performed animal experiments and quantitative RT-PCR analysis, and Dong YU provided assistance for the experiment. Yan LUO, Jian-guo CHENG, Yin WANG, Xue-ping YAO, and Ze-xiao YANG gave assistance for the experiment and made suggestions on the content of the manuscript. All authors have full access to all data in the study and are responsible for the integrity and security of the data.
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Wei YANG, Wu-you WANG, Wei ZHAO, Jian-guo CHENG, Yin WANG, Xue-ping YAO, Ze-xiao YANG, Dong YU, and Yan LUO declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed and animal experiments were approved by the National Institute of Animal Health Care and Use Committee at Sichuan Agricultural University (approval number SYXK2019-187).
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Project supported by the Sichuan Province Academic and Technical Leadership Development Funding Project and the Science & Technology Achievements Transfer Project of Sichuan (No. 2017YSZH0008), China
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Yang, W., Wang, Wy., Zhao, W. et al. Preliminary study on the role of novel LysR family gene kp05372 in Klebsiella pneumoniae of forest musk deer. J. Zhejiang Univ. Sci. B 21, 137–154 (2020). https://doi.org/10.1631/jzus.B1900440
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DOI: https://doi.org/10.1631/jzus.B1900440
Keywords
- Moschus berezovskii
- Klebsiella pneumoniae
- LysR transcription factor
- kp05372 gene
- Biological characteristics
- Upstream gene