MarR type regulator PA3458 is involved in osmoadaptation control in Pseudomonas aeruginosa (ChIP-seq)

Pseudomonas aeruginosa is a common bacterium, but also a facultative human pathogen, especially fatal for immunocompromised patients. It is dangerous due to its high adaptability and intrinsic antibiotic resistance mechanisms making treatment of P. aeruginosa infections particularly difficult. Sophisticated, complex, multilevel regulatory systems engaging huge repertoire of transcriptional regulators (TRs) are the main factors that direct cellular processes to keep cellular homeostasis and adapt to changeable conditions. The PA3458 gene encodes a putative MarR-type TR. Most representatives of MarR family are described as repressors involved in response to various environmental signals and control of different processes e.g. adhesion, virulence, antibiotic resistance. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in mRNA level of 133 genes, among them 100 was down-regulated, pointing out the role of PA3458 as a repressor. ChIP-seq analysis identified multiple PA3458 binding sites in P. aeruginosa genome suggesting its role as a global regulator. The preferred sequence motif recognised by PA3458 was identified. Many genes involved in stress response are under control of PA3458. The PA3459-PA3461 operon, divergent to PA3458 gene is regulated by PA3458. It encodes: asparagine synthase, GNAT-family acetyltransferase and glutamyl aminopeptidase, respectively engaged in the production of potent osmoprotectant an N-acetylglutaminylglutamine amide (NAGGN). Overall, the PA3458 is engaged in osmoadaptation control in P. aeruginosa. Overall design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1, was used in the experiment (Kawalek et al., 2020; BMC Genomics, 21:14). Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis was performed on P. aeruginosa PAO1161 ?PA3458 / pKKB2.12 (araC araBADp-PA3458-flag) (hereafter referred to as PA3458-F) as well as PAO1161 / pABB28.3 (araC araBADp-flag), empty vector control (hereafter called EV-F) strains. Cells were grown under selection in L broth with 0.02% arabinose until OD600 = 0.5). For each biological replicate, two independent cultures of each strain were pooled together. The immunoprecipitation was performed with commercially available polyclonal anti-FLAG antibodies (DYKDDDDK Tag polyclonal antibodies; PA1-985B; Invitrogen).

铜绿假单胞菌（Pseudomonas aeruginosa）是一种常见细菌，同时也是兼性人类致病菌，对免疫功能低下患者尤其具有致死性。该菌之所以具有危害性，源于其极强的适应性与固有抗生素耐药机制，这使得铜绿假单胞菌感染的治疗难度陡增。
一套精密复杂、层级多元的调控系统，借助庞大的转录调节因子（transcriptional regulators, TRs）库，主导细胞各项生命过程以维持细胞稳态并适应多变环境，是铜绿假单胞菌的核心特性来源。PA3458基因编码一个推定的MarR家族转录调节因子。MarR家族的绝大多数成员被报道为阻遏蛋白，参与响应各类环境信号，并调控黏附、毒力、抗生素耐药等多种生理过程。
对过表达PA3458的铜绿假单胞菌PAO1161开展转录组分析（transcriptional profiling），结果显示133个基因的mRNA水平发生显著变化，其中100个基因表达下调，这表明PA3458发挥阻遏蛋白的功能。染色质免疫共沉淀测序（ChIP-seq）分析在铜绿假单胞菌基因组中鉴定到多个PA3458结合位点，提示PA3458作为全局调控因子发挥作用。研究还鉴定出PA3458识别的偏好性序列基序。诸多参与应激响应的基因均受PA3458的调控。与PA3458基因反向转录的PA3459-PA3461操纵子，同样受PA3458的调控。该操纵子分别编码天冬酰胺合成酶、GNAT家族乙酰转移酶以及谷氨酰氨基肽酶，这些酶参与强效渗透压保护剂——N-乙酰谷氨酰谷氨酰胺酰胺（N-acetylglutaminylglutamine amide, NAGGN）的合成。综上，PA3458参与调控铜绿假单胞菌的渗透压适应过程。

实验整体设计：本研究使用PAO1的衍生菌株铜绿假单胞菌PAO1161（基因型为leu⁻、r⁻、利福平耐药（Rif^R）），相关实验信息引自Kawalek等人2020年发表于《BMC Genomics》的研究（21:14）。本研究对两种菌株开展染色质免疫共沉淀测序（ChIP-seq）分析：其一为PAO1161 ΔPA3458 / pKKB2.12（携带araC araBADp-PA3458-flag，后续简称为PA3458-F菌株）；其二为PAO1161 / pABB28.3（携带空载体pABB28.3，其表达araC araBADp-flag，后续简称为EV-F对照菌株）。菌株在含0.02%阿拉伯糖的LB肉汤培养基中进行抗性筛选培养，直至光密度600nm（OD600）值达到0.5。每个生物学重复均将同一菌株的两份独立培养物混合后进行后续实验。免疫沉淀实验采用商业化多克隆抗FLAG抗体（DYKDDDDK Tag多克隆抗体，货号PA1-985B，购自Invitrogen）。