DNA duplication-mediated activation of a two-component regulatory system serves as a bet-hedging strategy for .

Unlabelled: strain E264 ( E264) and close relatives stochastically duplicate a 208.6 kb region of chromosome I via RecA-dependent recombination between two nearly identical insertion sequence elements. Because homologous recombination occurs at a constant, low level, populations of E264 are always heterogeneous, but cells containing two or more copies of the region (Dup+) have an advantage, and hence predominate, during biofilm growth, while those with a single copy (Dup-) are favored during planktonic growth. Moreover, only Dup+ bacteria form 'efficient' biofilms within 24 hours in liquid medium. We determined that duplicate copies of a subregion containing genes encoding an archaic chaperone-usher pilus ( ) and a two-component regulatory system ( ) are necessary and sufficient for generating efficient biofilms and for conferring a selective advantage during biofilm growth. BubSR functionality is required, as deletion of either or , or a mutation predicted to abrogate phosphorylation of BubR, abrogates biofilm formation. However, duplicate copies of the genes are not required. Instead, we found that BubSR controls expression of and by activating a promoter upstream of during biofilm growth or when the 208.6 kb region, or just , are duplicated. Single cell analyses showed that duplication of the 208.6 kb region is sufficient to activate BubSR in 75% of bacteria during planktonic (BubSR 'OFF') growth conditions. Together, our data indicate that the combination of deterministic two-component signal transduction and stochastic, duplication-mediated activation of that TCS form a bet-hedging strategy that allows E264 to survive when conditions shift rapidly from those favoring planktonic growth to those requiring biofilm formation, such as may be encountered in the soils of Southeast Asia and Northern Australia. Our data highlight the positive impact that transposable elements can have on the evolution of bacterial populations.

Author Summary: Transposable elements naturally accumulate within genomes in all kingdoms of life. When present in the same orientation, a pair of homologous elements can act as substrates for DNA recombination reactions that can duplicate and delete intervening sequences - giving rise to genetically heterogenous populations. We showed here that strain E264 uses this mechanism to amplify genes encoding a two-component regulatory system and an archaic chaperone usher pilus, priming the cells for rapid biofilm formation. The formation of a small subpopulation of biofilm-ready bacteria serves as a bet- hedging strategy, ensuring overall population survival should conditions change rapidly from those in which planktonic growth is optimal to those in which adherence and biofilm formation is required.