Genomic and Proteomic Analysis of the Alkaline-Tolerance Response (AlTR) in Listeria monocytogenes 10403S. Listeria monocytogenes 10403S

Information regarding the Alkaline Tolerance Response (AlTR) in Listeria monocytogenes is very limited. In this study, L. monocytogenes was shown to exhibit a significant adaptive alkaline tolerance response (AlTR) following a 1-h exposure to mild alkaline (pH 9.5), which is capable of protecting cells from subsequent lethal alkali stress (pH 12.0). Treatment of adapted cells with protein synthesis inhibitor chloramphenicol has revealed that AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of AlTR, we compared differential gene expression and protein content at pH 9.5 using microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively. By interfacing the results of both approaches this study showed strong evidence that alkali tolerance response in L. monocytogenes functions as to minimize excess alkalisation and energy expenditures while mobilizing available carbon sources. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. The variability observed between results of cDNA arrays and 2D may account for posttranslational modifications. Interestingly, several alkali induced genes/proteins can provide a cross protective overlap to other types of stresses. Alkaline pH provides therefore L. monocytogenes with nonspecific multiple-stress resistance that may be vital for survival in the human gastrointestinal tract as well as within food processing systems where similar alkaline conditions as the ones used in this study prevail. Keywords: Transcriptomic and Proteomic Comparison Study Overall design: Overnight grown Listeria monocytogenes 10403S culture (100 µl) was inoculated into a 250 ml Erlenmeyer flask with 100 ml of BHI, and cultures were incubated at 30° C with shaking at 200 rpm. Cells were harvested by centrifugation when the optical density at 600 nm reached ~0.4, and then rapidly transferred to prewarmed (30 ° C) alkali adjusted BHI pH 9.5 (Adapted cultures) or not adjusted BHI (Control cultures) for 60 min. The pH of BHI medium was adjusted to 9.5 with a glycine-NaOH-NaCl buffer. Control and alkali adapted cells were used for transcriptomic and proteomic studies. In array analysis several controls were employed to minimise technical and biological variations, and ensure the quality of the data, i.e. i) each ORF was present in duplicate in each array, ii) each RNA preparation was used to make probes for at least two-separate arrays with reversal of incorporated dye and iii) three independent RNA batches from each condition were analysed. Only differences of ≥ 2 fold change in the levels of gene expression, which also had SAM 'q' values of 0.05, were recorded as significant.

目前关于单核细胞增生李斯特菌（Listeria monocytogenes）的耐碱反应（Alkaline Tolerance Response, AlTR）的相关研究信息十分有限。本研究证实，单核细胞增生李斯特菌经1小时温和碱胁迫（pH 9.5）处理后，可产生显著的适应性耐碱反应，该反应能够保护菌体免受后续致死性碱胁迫（pH 12.0）的伤害。
使用蛋白质合成抑制剂氯霉素处理适应性胁迫后的菌体，结果表明AlTR至少部分依赖于蛋白质合成。
为更全面地解析AlTR的生理学机制与调控通路，本研究分别采用微阵列（microarray）技术与二维凝胶电泳（two-dimensional gel electrophoresis, 2D）结合质谱（mass spectrometry）的方法，对比分析了pH 9.5条件下的差异基因表达与蛋白质含量变化。
通过整合两种技术的实验结果，本研究证实单核细胞增生李斯特菌的耐碱反应可通过减少过量碱化作用与能量消耗，同时调动可利用碳源来发挥功能。
适应性胞内基因表达涉及毒力相关基因、通用应激反应基因、细胞分裂相关基因以及细胞壁结构改变相关基因，同时包含大量功能未知的基因。
cDNA微阵列与二维凝胶电泳结果之间的差异，可能与翻译后修饰有关。值得注意的是，多个碱诱导基因/蛋白可与其他类型的胁迫产生交叉保护重叠效应。
因此，碱性环境可赋予单核细胞增生李斯特菌非特异性多重胁迫抗性，这对于其在人类胃肠道以及与本研究使用的碱性条件相似的食品加工环境中的存活至关重要。
关键词：转录组与蛋白质组比较研究 整体实验设计：将过夜培养的单核细胞增生李斯特菌10403S菌株菌液（100 μl）接种至装有100 ml脑心浸液培养基（BHI）的250 ml三角烧瓶中，于30 ℃、200 rpm振荡培养。当600 nm处光密度值达到约0.4时，通过离心收集菌体，随后快速转移至预热至30 ℃、经甘氨酸-氢氧化钠-氯化钠缓冲液调节pH至9.5的BHI培养基（适应性胁迫组）或未调节pH的BHI培养基（对照组）中培养60分钟。
对照组与碱胁迫适应性菌体分别用于转录组与蛋白质组学研究。在微阵列分析中，为尽可能减少技术与生物学误差、保证数据质量，采用了多项质控措施：① 每个开放阅读框（ORF）在每张微阵列中均设置重复位点；② 每份RNA样品均用于制备至少两组探针，且标记染料互为反向；③ 对每种条件下的3个独立RNA批次进行分析。仅当基因表达水平变化倍数≥2倍，且显著性微阵列分析（Significance Analysis of Microarrays, SAM）的q值≤0.05时，才被记为具有显著性差异。