CrcA, a novel player in Pseudomonas aeruginosa carbon catabolite repression. Pseudomonas aeruginosa carbon catabolite repression

In the opportunistic human pathogen Pseudomonas aeruginosa (Pae), carbon catabolite repression (CCR) orchestrates the hierarchical utilization of N and C sources, and is known to impact virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the catabolite repression control protein Crc form repressive assemblies on target mRNAs that impede translation. Affected are different uptake functions and catabolic proteins for the assimilation of less preferred C sources. After exhaustion of the preferred C-source, the levels of the regulatory RNA CrcZ increase. CrcZ binds to and acts as a decoy for Hfq, which in turn relieves translational repression of target genes. Here, we asked whether the functionality of Crc can be affected, which would likewise be anticipated to contribute to a relief of CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se, we endeavoured to identify a protein interactor. In vivo co-purification studies, co-immunoprecipitation- and biophysical assays revealed that Crc binds to Pae O1 protein PA1677. Our structural studies support bioinformatics analyses that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA1677 resulted in de-repression of Hfq/Crc repressed target genes, strongly indicating that PA1677 acts as an antagonist of Crc. This opposing function of PA1677 can be reconciled with an extended lag phase in the absence of the protein during diauxic growth on a preferred and a non-preferred carbon source, i.e. with a delayed synthesis of functions required to metabolize the non-preferred carbon source. We present a working model, wherein PA1677, which we termed CrcA (catabolite repression control protein antagonist), diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc.

在机会性人类致病菌铜绿假单胞菌（Pseudomonas aeruginosa, Pae）中，碳分解代谢物阻遏（carbon catabolite repression, CCR）可调控氮源与碳源的层级化利用，且已知其可影响病原菌的毒力、抗生素抗性与生物被膜形成。在CCR过程中，RNA分子伴侣Hfq与分解代谢物阻遏调控蛋白Crc会在靶mRNA上形成阻遏性复合物，阻碍翻译进程。受该调控网络影响的包括多种负责摄取次优碳源的功能蛋白与分解代谢蛋白。当优先碳源被耗尽后，调控RNA CrcZ的表达水平会显著升高。CrcZ可结合并作为Hfq的诱饵分子，进而解除靶基因的翻译阻遏。本研究旨在探究Crc的功能是否可被调控——这一机制同样有望在优先碳源耗尽后促成CCR的解除。由于Crc本身无法直接结合RNA，我们尝试寻找其蛋白质互作因子。体内共纯化实验、免疫共沉淀实验与生物物理测定结果显示，Crc可结合铜绿假单胞菌O1蛋白PA1677。我们的结构研究验证了生物信息学分析结果：PA1677属于异分支酸酶样超家族。PA1677的异位表达可解除Hfq/Crc介导的靶基因阻遏，这强烈表明PA1677是Crc的拮抗剂。PA1677的这一拮抗功能可与以下表型相契合：在以优先碳源与非优先碳源进行二次生长时，缺失该蛋白会导致迟滞期延长，即代谢非优先碳源所需功能的合成出现延迟。我们提出了一个工作模型：我们将PA1677命名为CrcA（分解代谢物阻遏调控蛋白拮抗剂，catabolite repression control protein antagonist），其通过竞争性结合Crc，减少靶mRNA上功能性Hfq/Crc阻遏复合物的形成。