Investigating the regulation of recA in the emerging pathogen Acinetobacter baumannii

Acinetobacter baumannii (Ab) is an emerging multidrug‐resistant, opportunistic pathogen. Ab survives desiccation, remaining on hospital surfaces affecting immunocompromised individuals. Ab antibiotic resistance acquisition has been linked to its DNA damage response (DDR). DDR genes, including error‐prone DNA polymerases, are involved in DNA damage and desiccation induced mutagenesis. In Escherichia coli , RecA, the cells' main recombinase, binds single stranded DNA, the signal of DNA damage, forming the nucleoprotein filament (RecA*), which is endowed with co‐protease activity. RecA* promotes cleavage and inactivation of LexA, the global DDR repressor, resulting in expression of DDR genes. In Ab , this well‐known circuitry does not exist in part because Ab lacks a functional LexA. We have shown that conserved DDR genes in Ab form two phenotypic subpopulations in response to DNA damage: one with low and another with high expression. The DDR in Ab is dependent on RecA. Thus, we are focused on understanding the mechanism underlying recA regulation to begin to understand the regulatory network that underlies this bimodality. We have found that the recA gene contains a cis ‐acting element in its 5′ untranslated region (UTR). In‐vitro transcription demonstrated that this 5′UTR contains secondary structure. A plasmid‐borne reporter was constructed that contains the cis ‐regulatory element driving a fluorescent reporter. Mutants of the cis ‐regulatory element that deletes putative regulatory sequences or predicted in silico secondary structure were constructed using site‐directed mutagenesis. Using fluorescent microscopy of the reporter strains, we have shown that the cis ‐acting regulatory element and its structure are important for bimodal expression of recA, while the promoter contains elements for DNA Damage sensing. Furthermore, we found that the recA 5′UTR modulates transcript levels and subsequently RecA in the cell. Disrupting the structure of the 5′UTR results in a significant decrease of UV survival and antibiotic resistance acquisition. This provides evidence that in Ab, two levels of regulation mediate recA . Remarkably, the Ab promoter and UTR sequence is capable of responding not only in Ab but also in other bacterial species as observed through similar fluorescent reporters induced with DNA damaging agents. Bimodal DDR gene expression may provide a strategy for survival and plasticity in an ever‐changing environment. These findings may also provide insight into the evolution of other highly antibiotic resistant bacteria lacking conserved DDR gene networks Support or Funding Information NIH Grant (GM088230) to VG