pH Regulates Genes for Flagellar Motility, Catabolism, and Oxidative Stress in Escherichia coli K-12

Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown with aeration to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with a significance level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins. Keywords: Steady State Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Overnight cultures were diluted 1:1000 in potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 5.0, pH 7.0, and pH 8.7. Bacteria were cultured in baffled flasks (less than 10% volume filled) with rotation at 240 rpm, incubated at 37°C to an optical density at 600 nm of 0.3. For each of the three pH conditions, RNA was isolated from five independent cultures. Labeled cDNA was hybridized to Affymetrix antisense arrays according to standard procedures. To analyze the expression levels, Dchip software was used to generate model-based expression indices normalized to sample pH 7 replicate 1. ANOVA was used to identify genes with sitnificant expression differences among the three pH classes (p = 0.001). For genes showing significant differences, the Log2 expression ratios were determined for each pair of pH classes, and significance was determined by Tukey's test (p = 0.001).

本研究以稳态外界pH为影响变量，对比分析了大肠杆菌K-12 W3110（Escherichia coli K-12 W3110）的基因表达谱特征。将菌株在经钾离子修饰的Luria-Bertani培养基（LBK）中通气培养，该培养基采用50 mM HOMOPIPES缓冲至pH 5.0、7.0及8.7，于37℃、240 rpm转速下在装液量不超过10%的挡板三角摇瓶中培养至600 nm处光密度值为0.3。针对三种pH培养条件，分别从5个独立重复培养的样本中提取总RNA，反转录得到互补DNA（cDNA）后，按照标准流程与Affymetrix大肠杆菌反义基因芯片进行杂交。
采用显著性水平为0.001的方差分析（ANOVA），本分析方法对检测表达量差异达2倍的基因具有98%的检验效能。为每个基因及基因间区（intergenic region, IG）计算标准化表达指数。结果显示，三种pH条件下共存在763个基因及353个基因间区呈现差异表达。通过层级聚类分析得到6个特征明确的pH表达谱簇，分别命名为：酸高表达簇（Acid High，在pH 5.0时表达量最高）、酸低表达簇（Acid Low，在pH 5.0时表达量最低）、碱高表达簇（Base High，在pH 8.7时表达量最高）、碱低表达簇（Base Low，在pH 8.7时表达量最低）、中性高表达簇（Neutral High，在pH 7.0时表达量最高，在酸性或碱性条件下表达量降低）以及中性低表达簇（Neutral Low，在pH 7.0时表达量最低，在两种极端pH条件下表达量升高）。
鞭毛及趋化相关基因在pH 8.7（碱低表达簇）条件下被抑制，此时细胞通过维持反向pH梯度导致跨膜质子电位降低。高pH环境同时抑制质子泵细胞色素o（cytochrome o, cyo）以及NADH脱氢酶I和II的表达。与之相反，质子输入型ATP合酶F1Fo以及可减少质子外排的微需氧型细胞色素d（cytochrome d, cyd）在pH 8.7条件下被诱导表达。上述观测结果与下述模型相符：高pH环境会抑制消耗质子动力势的鞭毛合成，同时上调维持质子在细胞内留存的电子传递链及ATP合酶相关组分的表达。而酸性条件诱导的基因则与代谢速率升高相关的条件（如氧化应激）协同诱导表达。全部6个pH依赖表达簇均涵盖直接暴露于外界pH环境的包膜蛋白及周质蛋白。
综上，本研究揭示了以下三点结论：(1) 低pH环境会加速酸性物质消耗与质子外排，同时协同诱导氧化应激及热休克调控子的表达；(2) 高pH环境会加速质子输入，同时抑制耗能较高的鞭毛及趋化调控子的表达；(3) pH值可差异性调控大量周质蛋白与包膜蛋白的表达。
关键词：稳态，基因表达谱（gene expression profiles），大肠杆菌K-12 W3110（Escherichia coli K-12 W3110）。
补充实验细节：将过夜培养的菌液以1:1000的比例稀释至钾修饰型Luria-Bertani培养基（LBK）中，该培养基采用50 mM HOMOPIPES缓冲至pH 5.0、7.0及8.7，于37℃、240 rpm转速下在装液量不超过10%的挡板三角摇瓶中培养至600 nm处光密度值为0.3。针对三种pH培养条件，分别从5个独立重复培养的样本中提取总RNA。标记后的cDNA按照标准流程与Affymetrix反义基因芯片进行杂交。采用Dchip软件生成基于模型的表达指数，并以pH 7.0的重复样本1作为参照进行标准化。采用方差分析（ANOVA）筛选三种pH条件下存在显著表达差异的基因（p=0.001）。对于存在显著表达差异的基因，计算每两组pH条件间的log2表达比值，并通过Tukey检验（p=0.001）确定差异显著性。