Supplementary_TABLE_S9_Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis.xlsx

Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, we aimed to progress in the characterization of this regulation by dealing with three convergent aspects: the effector enzymes involved, the master regulator CRP, and the dependence on glucose availability.
Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 relevant scenarios. These conditions arise when the wild type and four isogenic mutants (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and R-type mutants of protein CRP) are studied under three levels of glucose availability (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). The Q-type emulates a permanent stage of CRPacetylated, whereas the R-type emulates a permanent stage of CRPdeacetylated. The data were analyzed by an optimized factorial microarray method (Q-GDEMAR).
Results: (a) By analyzing one mutant against the other, we were able to unravel the true genes that participate in the interaction between ΔcobB/ΔpatZ mutations and glucose availability; (b) Increasing stages of glucose limitation appear to be associated with the up-regulation of specific sets of target genes rather than with the loss of genes present when glucose is in excess; (c) Both CRPdeacetylated and CRPacetylated produce extensive changes in specific subsets of genes, but their number and identity depend on the glucose availability; (d) In other sub-sets of genes, the transcriptional effect of CRP seems to be independent of its acetylation or deacetylation; (e) Some specific ontology functions can be associated with each of the different sets of genes detected herein.
Conclusions: CRP cannot be thought of only as an effector of catabolite repression, because it acts along all the glucose conditions tested (excess, limited, and exhausted), exerting both positive and negative effects through different sets of genes. Acetylation of CRP does not seem to be a binary form of regulation, as there is not a univocal relationship between its activation/inhibitory effect and its acetylation/deacetylation stage. All the combinatorial possibilities are observed. During the exponential growth phase, CRP also exerts a very significant transcriptional effect, mainly on flagellar assembly and chemotaxis (FDR = 7.2 × 10−44).

研究背景：尽管在真核生物中，乙酰化/去乙酰化调控发挥多种多效性效应，但在大肠杆菌（Escherichia coli）中，该调控过程的研究较为有限且相关机制尚不明晰。因此，本研究旨在通过三个交汇方向推进该调控系统的表征工作：涉及的效应酶、核心调控因子环腺苷酸受体蛋白（CRP），以及葡萄糖可利用性的依赖效应。
研究方法：本研究分析了大肠杆菌BW25113（Escherichia coli BW25113）在14种相关实验条件下的转录响应。这些条件设置为：分别在三种葡萄糖可利用水平下（分批培养中的葡萄糖限制恒化培养、葡萄糖过量培养及葡萄糖耗尽培养），对野生型菌株与4株同基因敲除突变株（去乙酰化酶CobB缺陷株、N(ε)-赖氨酸乙酰转移酶PatZ缺陷株、CRP蛋白的Q型与R型突变株）进行研究。其中Q型突变模拟CRP永久乙酰化状态，R型突变则模拟CRP永久去乙酰化状态。本研究采用优化后的析因微阵列分析方法（Q-GDEMAR）对数据进行分析。
研究结果：(a) 通过对不同突变株进行两两比较分析，本研究明确了参与ΔcobB/ΔpatZ突变与葡萄糖可利用性之间相互作用的真实靶基因；(b) 葡萄糖限制程度的逐步升高，与特定靶基因集的上调表达显著相关，而非与葡萄糖过量培养下存在的基因丢失相关；(c) 去乙酰化CRP与乙酰化CRP均可对特定基因子集造成广泛的转录变化，但其调控的基因数量与种类均依赖于葡萄糖可利用水平；(d) 在其他基因子集中，CRP的转录调控效应似乎与其乙酰化/去乙酰化状态无关；(e) 本研究检测到的各类基因子集，均可与特定的基因本体（Gene Ontology）功能相关联。
研究结论：CRP不能仅被视为分解代谢物阻遏的效应因子，因为它在所有受试葡萄糖条件（过量、限制及耗尽）下均发挥调控作用，并通过不同的基因集同时产生正向与负向调控效应。CRP的乙酰化调控并非二元化机制，因为其激活/抑制效应与乙酰化/去乙酰化状态之间并不存在单一明确的对应关系，所有组合可能性均已被观测到。在指数生长期，CRP同样可发挥显著的转录调控作用，主要涉及鞭毛组装与趋化性通路（错误发现率（False Discovery Rate）= 7.2 × 10⁻⁴⁴）。