Supplementary_TABLE_S10_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).

背景：在真核生物中，乙酰化/去乙酰化调控发挥多种多效性作用，而在大肠杆菌（Escherichia coli）中，此类调控过程的功能更为局限，相关研究也相对不足。为此，本研究从三个相互关联的维度推进该调控机制的解析：涉及的效应酶、核心调控蛋白CRP，以及葡萄糖可利用性的依赖效应。
方法：本研究针对大肠杆菌BW25113的转录响应，在14种相关培养条件下展开分析。这些条件设置为：野生型菌株与4株同基因缺失突变株（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）分别在三种葡萄糖可利用水平下培养，即葡萄糖限制恒化培养、葡萄糖过量分批培养及葡萄糖耗尽分批培养。其中，Q型突变体模拟CRP持续乙酰化的状态，R型突变体则模拟CRP持续去乙酰化的状态。实验数据采用优化的析因微阵列分析方法（Q-GDEMAR）进行处理。
结果：(a) 通过对比两株突变株的转录谱差异，我们明确了ΔcobB/ΔpatZ突变与葡萄糖可利用性之间互作所涉及的真实功能基因；(b) 葡萄糖限制程度的升高，与特定靶基因集的上调表达呈现显著相关性，而非仅对应葡萄糖过量培养下的基因丢失现象；(c) 去乙酰化CRP与乙酰化CRP均可对特定基因子集产生广泛的转录调控变化，但其调控的基因数量与种类取决于葡萄糖可利用水平；(d) 在另一部分基因子集的调控中，CRP的转录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, GO）功能类别相对应。
结论：CRP不应仅被视为分解代谢物阻遏效应的效应因子，因其在所有测试的葡萄糖培养条件（过量、限制及耗尽）下均发挥作用，并通过不同的基因集同时产生正向与负向调控效应。CRP的乙酰化并非二元调控模式，其激活/抑制效应与乙酰化/去乙酰化状态之间不存在单一明确的对应关系，所有组合可能性均有观测到。在指数生长阶段，CRP同样可产生显著的转录调控效应，主要涉及鞭毛组装与趋化功能（错误发现率（FDR）= 7.2 × 10⁻⁴⁴）。