Integrative “Omics”-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses

Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level (“expressome”). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.细菌始终面临各类胁迫环境，因此会启动特异性响应以保障自身的适应与存活。此前学界普遍认为，胁迫响应的调控主要发生在转录水平。在光合细菌球形红杆菌（Rhodobacter sphaeroides）中，其对单线态氧的响应由替代σ因子（sigma factors）启动。此外，其他适应性调控机制还包括转录后与翻译后事件，若要深入解析复杂调控网络的运作逻辑，必须将这些事件纳入考量范围。为解决这一研究缺口，本研究整合了三层调控组学的数据：(1) 不同时间节点下的总mRNA水平，可揭示转录组（transcriptome）的动态变化；(2) 多聚核糖体组分中的mRNA，可反映翻译调控的相关特征，即翻译组（translatome）；(3) 采用基于SILAC（Stable Isotope Labeling by Amino acids in Cell Culture）的质谱分析法对蛋白质丰度进行定量，即蛋白质组（proteome）分析。单线态氧胁迫响应展现出极强的动态特征，涵盖短期效应与后期适应两个阶段，其中部分动态变化可归因于σ因子RpoE与RpoH2，二者可调控对应的调控子（regulons）形成差异化的表达动力学特征。胁迫诱导型操纵子（operons）内基因呈现的极性表达模式，提示了胁迫响应中动态精细调控的另一潜在途径。除转录激活之外，本研究还在转录后层面（即翻译组）观测到基因的显著诱导表达现象，借此鉴定出了新的潜在调控因子，并将群体感应（quorum sensing）相关基因关联至单线态氧胁迫响应通路。值得注意的是，基于SILAC的分析不仅揭示了光合蛋白质随胁迫发生的丰度下降现象，还鉴定出19个全新的开放阅读框（open reading frames, ORF），其中部分已通过RNA测序（RNA-seq）得到验证。本研究提出，如本文所述的比较组学策略，将有助于构建系统层面的多层级表达图谱，即表达组（"expressome"）。最终，结合RNA-seq的深度质谱分析有望成为未来各类生物基因组重新注释的首选工具，并助力解析生物如何适应多变的环境条件。