The delta subunit of RNA polymerase, RpoE, is a global modulator of Streptococcus mutans environmental adaptation. Streptococcus mutans UA159

Transcriptional profiling of S. mutans rpoE knockout mutant comparing to the wild type. The mutation of rpoE cause dramatic changes in phenotypes. The goal is to determine the genes affected by rpoE. The delta subunit of RNA polymerase, RpoE, is wide spread in low G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak-promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth, and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth, and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were down-regulated in the ΔrpoE mutant in all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly up-regulation, of 330 non-coding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans, which is required for optimal growth and environmental adaptation. Overall design: 5 conditions, mutant vs wild type, biological replicates: 2, technical replicates: 2

变形链球菌（Streptococcus mutans, S. mutans）rpoE敲除突变体与野生型的转录组分析。rpoE基因的突变会引发表型的显著改变，本研究旨在明确受rpoE调控的基因。RNA聚合酶（RNA polymerase）的δ亚基（delta subunit）RpoE广泛分布于低G+C含量的革兰氏阳性菌中，被认为可通过阻断RNA聚合酶与弱启动子区域的结合、加速核心酶循环以促进RNA合成，进而增强转录特异性。尽管RpoE的生化特性已得到广泛研究，但该蛋白在体内（in vivo）的具体功能仍未得到深入阐释。本研究发现，在人类龋齿致病菌S. mutans中敲除rpoE会导致生长受损，并丧失包括生物膜形成、抗生素抗性以及环境胁迫耐受性在内的重要毒力性状。通过反式表达rpoE对突变体进行遗传互补，可使其表型恢复至野生型水平。荧光素酶（Luciferase）融合报告基因实验结果显示，rpoE在整个生长周期中均呈高转录水平，且酸性胁迫与过氧化氢胁迫均可抑制rpoE的表达。对指数生长期、早期稳定生长期的野生型与ΔrpoE突变体细胞，分别在酸性胁迫、过氧化氢胁迫以及两种胁迫共同处理下开展转录组分析，结果显示：在所有实验条件下，ΔrpoE突变体中参与组氨酸合成、苹果酸-乳酸发酵、生物膜形成以及抗生素抗性的基因均呈下调表达。此外，RpoE的缺失会导致碳水化合物与氨基酸的转运及代谢通路发生显著改变。值得注意的是，本研究还发现330个非编码区域（non-coding regions）存在表达差异，其中多数呈现上调表达。综上，本研究证实RpoE是S. mutans中重要的基因表达全局调控因子（global modulator），对于该菌的最优生长与环境适应不可或缺。实验整体设计：共设置5种实验条件，均为突变体与野生型对照；生物学重复（biological replicates）数：2次；技术重复（technical replicates）数：2次