The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics (ChIP-seq dataset)

The regulatory roles of proteins associating with DNA-dependent RNA polymerase (RNAP) during transcription elongation are poorly characterized in bacteria. A forward genetic selection for Caulobacter crescentus cell cycle mutants revealed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR associates with promoters and coding sequences (CDSs) and tracks with active RNAP. Loss of TrcR causes a cell division cycle and growth defect and an insufficiency in the essential cell cycle regulator CtrA. TrcR also protects cells from the quinolone antibiotic nalidixic acid that induces a multi-drug efflux pump and from the RNAP inhibitor rifampicin (Rif) that prevents transcription elongation. TrcR interacts biochemically and genetically with RNAP and no longer associates with chromatin when RNAP is inhibited with Rif. We show that these TrcR functions and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella sp., indicating that TrcR represents a new antibiotic target. Overall design: Examination of TrcR, RpoD and RNAP holoenzyme whole genome binding/occupancy (ChIP-Seq) in WT Caulobacter crescentus and mutants in PYE and following Rifampicin and Nalidixic Acid treatments and TrcR whole genome occupancy/binding in WT Sinorhizobium meliloti before and after Rifampicin treatment.

细菌中转录延伸阶段与DNA依赖RNA聚合酶（DNA-dependent RNA polymerase, RNAP）结合的蛋白质的调控功能，目前尚缺乏充分研究。本研究通过对新月柄杆菌（Caulobacter crescentus）细胞周期突变体开展正向遗传筛选，发现了功能未被表征的DUF1013家族蛋白（TrcR，转录细胞周期调控因子）。TrcR可结合启动子与编码序列（coding sequences, CDSs），并随活性RNAP一同移动。TrcR缺失会引发细胞分裂周期与生长缺陷，同时导致关键细胞周期调控因子CtrA的表达量不足。此外，TrcR可使细胞抵御诱导多药外排泵表达的喹诺酮类抗生素萘啶酸（nalidixic acid），以及阻断转录延伸的RNAP抑制剂利福平（rifampicin, Rif）。TrcR可与RNAP发生生化与遗传互作；当RNAP被利福平抑制时，TrcR将不再结合染色质。本研究证实，TrcR的上述功能及其依赖RNAP的染色质招募机制，在共生性中华根瘤菌属（Sinorhizobium sp.）与致病性布鲁氏菌属（Brucella sp.）中均具有保守性，这表明TrcR可作为新型抗生素靶点。实验整体设计：分别在PYE培养基中，以及经利福平与萘啶酸处理后，检测野生型（wild type, WT）新月柄杆菌及其突变株中TrcR、RpoD与RNAP全酶的全基因组结合/占据情况（染色质免疫共沉淀测序，Chromatin Immunoprecipitation Sequencing, ChIP-Seq）；同时检测野生型苜蓿中华根瘤菌（Sinorhizobium meliloti）在利福平处理前后的TrcR全基因组占据/结合情况。