Regulation of the mobile genetic element ICEBs1 by a conserved repressor and anti-repressor

Abstract

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-Phrl sensory system activate ICEBsl gene expression, excision, and transfer by inactivating the ICEBsl repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBslencoded anti-repressor ImmA (YdcM). Under ICEBsl-inducing conditions, ImmA cleaves ImmR at a specific site to induce the element. We found that changing the amount or the specific activity of ImmA can cause derepression of ICEBs1 without activation by RecA or RapI. We isolated and characterized mutations in immA (immAh) that cause derepression of ICEBsl gene expression in the absence of inducing signals. However, we also found that ImmA levels did not significantly change during activation by RapI, indicating that RapI-mediated induction is likely due to increased activity of ImmA. Therefore, we propose that RapI and RecA induce ICEBs1 by increasing its specific activity. Along with earlier observations, some ImmAh mutants highlighted the importance of ImmA's C-terminal sequence for regulation of ImmA protein levels. We demonstrated that GFP tagged with C-terminal residues of ImmA was less abundant in vivo than untagged GFP. We screened cells with mutations of ATP-dependent proteases for effects on ICEBsl expression, and found that ClpXP might play a role in regulating ImmA stability and ICEBs1 gene expression.

(cont.) To learn more about the repressor, ImmR, we isolated and characterized mutants of immR (immR(ind-)) that attenuate induction of ICEBs1 gene expression under the normally inducing conditions of treatment with DNA damaging reagent and overproduction of RapI. All four identified immR(ind-) mutations fall within a stretch of 10 residues flanking the cleavage site, emphasizing the importance of this sequence for ImmR proteolysis and ICEBsl induction. To further characterize the C-terminal portion of ImmR, we demonstrated that it interacts with ImmA and with itself in yeast two-hybrid assays, indicating that this part of the protein likely functions in ImmR oligomerization and recognition of ImmR by ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mode of regulation by ImmA and ImmR may be conserved in various systems.