| Summary: | In this study, we show that <i>Acinetobacter baumannii</i> ATCC 19606 harbors two sets of <i>ohrR-ohr</i> 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, <i>∆ohrR-C</i>, <i>C</i> represented gene on chromosome, or <i>∆ohr-C</i> single mutants, or <i>∆ohrR</i>-<i>C∆ohr</i>-<i>C</i> double mutants to <i>tert</i>-butyl hydroperoxide (<i>t</i>BHP) found that the <i>ohrR-p-ohr-p</i> genes, <i>p</i> represented genes on pMAC plasmid, may be able to complement the function of their chromosomal counterparts. Interestingly, <i>∆ohr-C</i> single mutants generated in <i>A. baumannii</i> ATCC 17978, which does not harbor pMAC, demonstrated delayed exponential growth and loss of viability following exposure to 135 μg of <i>t</i>BHP. In a survival assay conducted with <i>Galleria mellonella</i> 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 <i>ohr</i>-<i>p</i> genes, but with varying affinity. A gain-of-function assay conducted in <i>Escherichia coli</i> showed that OhrR-C was not only capable of suppressing transformed <i>ohr-C</i> genes but may also repress endogenous enzymes. Taken together, our findings suggest that chromosomal <i>ohrR-C-ohr-C</i> genes act as the major system in protecting <i>A. baumannii</i> ATCC 19606 from OHP stresses, but the <i>ohrR-p-ohr-p</i> 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.
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