Chen Shih-Jie, Shu Hung-Yu, Lin Guang-Huey
Master Program in Microbiology and Immunology, School of Medicine, Tzu Chi University, Hualien 97004, Taiwan.
Department of Bioscience Technology, Chang Jung Christian University, Tainan 71101, Taiwan.
Microorganisms. 2021 Mar 18;9(3):629. doi: 10.3390/microorganisms9030629.
In this study, we show that ATCC 19606 harbors two sets of genes, respectively encoded in chromosomal DNA and a pMAC plasmid. We found no significant difference in organic hydroperoxide (OHP) resistance between strains with or without pMAC. However, a disk diffusion assay conducted by exposing wild-type, , represented gene on chromosome, or single mutants, or -- double mutants to -butyl hydroperoxide (BHP) found that the genes, represented genes on pMAC plasmid, may be able to complement the function of their chromosomal counterparts. Interestingly, single mutants generated in ATCC 17978, which does not harbor pMAC, demonstrated delayed exponential growth and loss of viability following exposure to 135 μg of BHP. In a survival assay conducted with larvae, these mutants demonstrated almost complete loss of virulence. Via an electrophoretic mobility shift assay (EMSA), we found that OhrR-C was able to bind to the promoter regions of both chromosomal and pMAC - genes, but with varying affinity. A gain-of-function assay conducted in showed that OhrR-C was not only capable of suppressing transformed genes but may also repress endogenous enzymes. Taken together, our findings suggest that chromosomal genes act as the major system in protecting ATCC 19606 from OHP stresses, but the genes on pMAC can provide a supplementary protective effect, and the interaction between these genes may affect other aspects of bacterial viability, such as growth and virulence.
在本研究中，我们发现ATCC 19606含有两组基因，分别编码于染色体DNA和一个pMAC质粒中。我们发现，有无pMAC的菌株在对有机氢过氧化物（OHP）的抗性方面没有显著差异。然而，通过将野生型、染色体上代表基因的、单个突变体或双突变体暴露于叔丁基氢过氧化物（BHP）进行的纸片扩散试验发现，pMAC质粒上代表基因的基因可能能够补充其染色体对应基因的功能。有趣的是，在不含有pMAC的ATCC 17978中产生的单个突变体，在暴露于135μg BHP后，表现出指数生长延迟和活力丧失。在用幼虫进行的存活试验中，这些突变体表现出几乎完全丧失毒力。通过电泳迁移率变动分析（EMSA），我们发现OhrR-C能够结合染色体和pMAC基因的启动子区域，但亲和力不同。在中进行的功能获得试验表明，OhrR-C不仅能够抑制转化的基因，还可能抑制内源性酶。综上所述，我们的研究结果表明，染色体基因是保护ATCC 19606免受OHP胁迫的主要系统，但pMAC上的基因可以提供补充保护作用，并且这些基因之间的相互作用可能会影响细菌活力的其他方面，如生长和毒力。
