Fig 2B uncropped images.

Burkholderia thailandensis strain E264 (BtE264) 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 BtE264 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 pathway pilus (csuFABCDE) and a two-component regulatory system (bfmSR) are necessary and sufficient for generating efficient biofilms and for conferring a selective advantage during biofilm growth. BfmSR functionality is required, as deletion of either bfmS or bfmR, or a mutation predicted to abrogate phosphorylation of BfmR, abrogates biofilm formation. However, duplicate copies of the csuFABCDE genes are not required. Instead, we found that BfmSR controls expression of csuFABCDE and bfmSR by activating a promoter upstream of csuF during biofilm growth or when the 208.6 kb region, or just bfmSR, are duplicated. Single cell analyses showed that duplication of the 208.6 kb region is sufficient to activate BfmSR in 75% of bacteria during planktonic (BfmSR ‘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 BtE264 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.泰国伯克霍尔德菌E264菌株（Burkholderia thailandensis strain E264，BtE264）及其近缘菌株可通过两个近乎完全一致的插入序列元件（insertion sequence element）间的RecA依赖重组，随机复制染色体I上一段长208.6 kb的区域。由于同源重组以恒定的低水平发生，BtE264的种群始终存在异质性：携带该区域2个或更多拷贝的细胞（Dup+）在生物膜生长过程中具有生长优势并成为优势种群，而仅携带单拷贝的细胞（Dup–）则在浮游生长条件下更具适应性。此外，仅Dup+菌株可在24小时内于液体培养基中形成"高效"生物膜。我们明确了一段包含编码古伴胞-引导通路菌毛（archaic chaperone-usher pathway pilus，csuFABCDE）基因以及双组分调控系统（two-component regulatory system，bfmSR）的亚区域，其重复拷贝是形成高效生物膜，以及在生物膜生长过程中获得选择优势的必要且充分条件。BfmSR的功能不可或缺：删除bfmS或bfmR基因，或引入可破坏BfmR磷酸化的突变，均会完全阻断生物膜形成。但csuFABCDE基因的重复拷贝并非必需。进一步研究发现，BfmSR可通过在生物膜生长阶段，或当208.6 kb区域（或仅bfmSR）发生复制时，激活csuF上游的启动子，从而调控csuFABCDE与bfmSR的表达。单细胞分析显示，在浮游生长（BfmSR"关闭"状态）条件下，208.6 kb区域的复制足以使75%的细菌激活BfmSR通路。综上，我们的研究数据表明，确定性双组分信号转导与随机复制介导的该双组分调控系统（TCS）激活相结合，形成了一种风险对冲策略，可使BtE264在环境快速变化时存活——从利于浮游生长的条件转变为需要形成生物膜的条件，例如东南亚与澳大利亚北部土壤中可能遭遇的环境。本研究同时凸显了转座元件（transposable elements）对细菌种群进化的积极影响。