The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development [ChIP-seq]

Caulobacter species are dimorphic Gram-negative bacteria that inhabit diverse terrestrial and aquatic ecosystems. A suite of molecular sensory systems enables these microbes to control growth, development, and reproduction in response to levels of essential elements, including carbon, nitrogen, and phosphorus. Although the enhancer binding protein NtrC and its cognate sensor histidine kinase NtrB are well-established regulators of bacterial nitrogen assimilation, their precise functions in Caulobacter metabolism and cell development remained largely undefined. Deletion of C. crescentus ntrC slowed cell growth in complex medium, while ntrB and ntrC were essential for growth when ammonium was the sole nitrogen source due to their requirement for glutamine synthase (glnA) expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring glnBA transcription, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nitrogen limitation. We further defined the NtrC regulon through transcriptomic and ChIP-seq analyses, which demonstrated the role of NtrC as both a transcriptional activator and repressor. The chromosome of C. crescentus harbors dozens of direct binding sites for NtrC, with a significant proportion located near genes involved in polysaccharide biosynthesis. Numerous NtrC binding sites align with those of GapR, an essential protein involved in chromosome organization, and MucR1, a cell cycle regulator. This observation suggests that NtrC participates in the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. These developmental defects were restored by supplementing media with glutamine or by ectopic expression of the glnBA operon. This study establishes a regulatory connection between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. To investigate the NtrC direct regulon/DNA binding sites in Caulobacter by creating a 3xFLAG-tagged ntrC allele, which resulted in a 3xFLAG tag at the N-terminus of NtrC We performed NtrC DNA-binding analysis from ChIP-seq data of Caulobacter harboring 3xFLAG-ntrC grown in PYE to log phase

柄杆菌属（Caulobacter）物种是一类双形态革兰氏阴性菌，广泛栖息于各类陆地与水生生态系统中。一套完整的分子感知系统使这类微生物能够响应碳、氮、磷等必需元素的浓度变化，调控自身生长、发育与繁殖过程。尽管增强子结合蛋白NtrC及其同源组氨酸激酶传感器NtrB是公认的细菌氮同化调控因子，但它们在柄杆菌属代谢与细胞发育中的具体功能仍未得到明确阐释。敲除新月柄杆菌（C. crescentus）的ntrC基因会使复杂培养基中的细胞生长速率减慢；而当以铵盐作为唯一氮源时，ntrB与ntrC是细胞生长的必需基因，因为二者的正常表达是谷氨酰胺合酶（glutamine synthase, glnA）转录的前提条件。保守的IS3家族移动遗传元件的随机转座，通常可通过恢复glnBA的转录来挽救ntrC突变菌株的生长缺陷，这揭示了IS3转座可能在氮限制条件下参与柄杆菌属种群的进化塑造。本研究进一步通过转录组学与染色质免疫沉淀测序（ChIP-seq）分析，明确了NtrC的调控子，证实NtrC兼具转录激活与转录抑制双重功能。新月柄杆菌的染色体上存在数十个NtrC直接结合位点，其中相当一部分位于多糖生物合成相关基因的邻近区域。大量NtrC结合位点与GapR以及MucR1的结合位点重合：GapR是参与染色体组织的必需蛋白，MucR1则是细胞周期调控因子。这一现象提示NtrC参与细胞周期与细胞发育的调控过程。实验证实，NtrC功能缺失会导致细胞极柄伸长，并使细胞包膜多糖的合成量升高。通过在培养基中添加谷氨酰胺，或异位表达glnBA操纵子，可修复上述发育缺陷。本研究明确了柄杆菌属中NtrC、氮代谢、极形态发生与包膜多糖合成之间的调控关联。为探究柄杆菌属中NtrC的直接调控网络与DNA结合位点，本研究构建了在NtrC N端融合3xFLAG标签的ntrC等位基因；随后利用在PYE培养基中培养至对数生长期的携带该等位基因的柄杆菌的染色质免疫沉淀测序数据，开展了NtrC的DNA结合分析。