Activation of phenotypic subpopulations in response to ciprofloxacin treatment in Acinetobacter baumannii

Abstract

Summary The multidrug-resistant, opportunistic pathogen, Acinetobacter baumannii, has spread swiftly through hospitals worldwide. Previously, we demonstrated that A. baumannii regulates the expression of various genes in response to DNA damage. Some of these regulated genes, especially those encoding the multiple error-prone DNA polymerases, can be implicated in induced mutagenesis, leading to antibiotic resistance. Here, we further explore the DNA damage-inducible system at the single cell level using chromosomal transcriptional reporters for selected DNA damage response genes. We found the genes examined respond in a bimodal fashion to ciprofloxacin treatment, forming two phenotypic subpopulations: induced and uninduced. This bimodal response to ciprofloxacin treatment in A. baumannii is unique and quite different than the Escherichia coli paradigm. The subpopulations are not genetically different, with each subpopulation returning to a starting state and differentiating with repeated treatment. We then identified a palindromic motif upstream of certain DNA damage response genes, and have shown alterations to this sequence to diminish the bimodal induction in response to DNA damaging treatment. Lastly, we are able to show a biological advantage for a bimodal response, finding that one subpopulation survives ciprofloxacin treatment better than the other.

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

“…5D). As mentioned previously, bimodal recA expression may serve as a bet-hedging strategy to increase fitness (30,47,48). It will be interesting to investigate the broader role for RNA-mediated cis regulation within the whole genome of A. baumannii, given the example of recA regulation that we have presented.…”

“…The insert and vector were then ligated using T4 ligase (Promega) at 4°C for 18 h. All plasmids were confirmed with sequencing using primers PNLACF and PNLACR (Table S2) to confirm correct assembly after transformation into competent E. coli and selection for Tet r . All plasmids were introduced into A. baumannii cells by electroporation (30). Transformants were selected for on LB agar supplemented with Tet.…”

“…For normal distributions, the percent difference between the mean values should be low, whereas for nonuniform (e.g., bimodal) distributions, these values would be higher (39). To set the DAG value limits for our experiments, we determined the DAG in a unimodally distributed system (30), in this case an E. coli strain with a P recA -gfp plasmid reporter (Fig. 4D).…”

“…In previous work we found that for A. baumannii ATCC 17978, the DDR is bimodal, unlike in E. coli (30). That is, in A. baumannii, when expression of a number of conserved DDR genes, including recA, is measured, we find DDR Low (low expression of DDR genes) and DDR High (high expression of DDR genes) phenotypic subpopulations.…”

The landscape of intrinsic and evolved fluoroquinolone resistance inAcinetobacter baumanniiincludes suppression of drug-induced prophage replication

“…In certain cases, the SOS response also induces mobile genetic elements that carry antibiotic resistance or toxin genes, potentially influencing the spread of resistance or virulence traits (25). The A. baumannii SOS repair response is non-canonical, lacking clear orthologs of many major players in other systems (26) and is characterized by a phenotypically variable response within cell populations (27). Inactivation of RecA, a central protein mediating DNA recombinational repair and SOS induction, or the RecBCD Exonuclease V complex responsible for double-strand break repair, greatly raises fluoroquinolone sensitivity in A. baumannii (28, 29).…”