Abstract
In this study, we show that Acinetobacter baumannii ATCC 19606 harbors two sets of ohrR-ohr 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, ∆ohrR-C, C represented gene on chromosome, or ∆ohr-C single mutants, or ∆ohrR-C∆ohr-C double mutants to tert-butyl hydroperoxide (tBHP) found that the ohrR-p-ohr-p genes, p represented genes on pMAC plasmid, may be able to complement the function of their chromosomal counterparts. Interestingly, ∆ohr-C single mutants generated in A. baumannii ATCC 17978, which does not harbor pMAC, demonstrated delayed exponential growth and loss of viability following exposure to 135 μg of tBHP. In a survival assay conducted with Galleria mellonella 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 ohr-p genes, but with varying affinity. A gain-of-function assay conducted in Escherichia coli showed that OhrR-C was not only capable of suppressing transformed ohr-C genes but may also repress endogenous enzymes. Taken together, our findings suggest that chromosomal ohrR-C-ohr-C genes act as the major system in protecting A. baumannii ATCC 19606 from OHP stresses, but the ohrR-p-ohr-p 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.
Highlights
Bacteria are often exposed to reactive oxygen species (ROS) and other organic hydroperoxides (OHPs) produced by host phagocytic cells [1] as part of the immune response [2,3]
Ohr is a thiol peroxidase that is central to the bacterial response against OHPs [4,5], and it acts to neutralize OHPs via a redox-active disulfide bond formed by two conserved cysteines at the catalytic site [6]
We identified the presence of another ohrR-C-ohr-C gene cluster in the chromosomal DNA of A. baumannii ATCC 19606 through in silico analysis, and subsequently investigated the function and significance of this newly-discovered set of ohr-c genes
Summary
Bacteria are often exposed to reactive oxygen species (ROS) and other organic hydroperoxides (OHPs) produced by host phagocytic cells [1] as part of the immune response [2,3]. Bacteria have evolved several strategies to protect themselves against oxidative stress. One such strategy is to produce enzymes that can directly detoxify OHPs by transforming them into unreactive alcohols. Ohr (organic hydroperoxide resistance protein) is a thiol peroxidase that is central to the bacterial response against OHPs [4,5], and it acts to neutralize OHPs via a redox-active disulfide bond formed by two conserved cysteines at the catalytic site [6].
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