DNA AMPLIFICATION-MEDIATED PHASE VARIATION AND TWO-COMPONENT REGULATORY SIGNALING COORDINATE AS A BET-HEDGING STRATEGY IN BURKHOLDERIA THAILANDENSIS

When I joined the Cotter lab in 2019, we knew that a 208.6 kb region of the B. thailandensis strain E264 was present at multiple copies within populations. The initial model involved the formation of a large extrachromosomal, circular copy of the region, termed a ‘megacircle’, caused by activity of one of the transposable elements bounding the region and the BcpA toxin received from a sibling bacterium. The presence of megacircles altered phenotypes, leading populations to bind Congo-red, form biofilms, and produce a golden pigment during colony biofilm formation. As a graduate student, I proved, using pulsed-field gel electrophoresis, that the 208.6 kb region is amplified as tandem duplications on the chromosome, and not megacircles. And both formation and loss of tandem copies is conducted by stochastic RecA-mediated homologous recombination. Because DNA recombination is constantly occurring at a low rate, populations are always genotypically heterogenous, composed of cells with different copy numbers of the 208.6 kb region. As observed before, multiple copies of the 208.6 kb region alter phenotypes, which can influence the fitness of cells with (Dup+) or without (Dup-) a duplication in different conditions. For example, when in conditions requiring planktonic growth and growth in colonies, Dup- cells are advantaged, while growth in biofilms or the center of colony biofilms advantages Dup+ bacteria. I identified that duplication of two operons within the 208.6 kb region is required and sufficient for rapid biofilm formation and the selective advantage during biofilm growth in Dup+ cells. These operons, aplFABCDE and bubSR, encode an archaic chaperone-usher pilus and a two-component regulatory system respectively. Surprisingly, BubSR is inactive in cells with a single copy of the region during growth in M63, yet duplication of bubSR leads to BubSR activation. When BubSR is active, BubR positively regulates transcription of the aplFABCDE genes, leading to pilus-mediated surface attachment and biofilm formation. We posit that the combination of stochastic DNA duplication and deterministic two-component regulatory signaling work as a bet-hedging strategy, creating a Dup+ population that contains active BubSR regardless of environmental conditions, therefore allowing B. thailandensis to survive when conditions rapidly change to require biofilm growth.