Transcriptional profiling of an evolved polymorphism in E. coli. Escherichia coli K-12

Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to understand how stable polymorphism arose in an Escherichia coli population that evolved for 765 generations under continuous glucose limitation. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguish evolved clones from their common ancestor. However, when co-evolved clones are cultured separately their transcriptional profiles differ markedly from that ancestor, and do so in ways that are consistent with our understanding of how E. coli adapts to glucose limitation. All adaptive clones exhibit reduced activity of the stationary-phase sigma factor σS and increased expression of glucose transport genes, including the glycoporin LamB and the galactose transporter MglABC. Other expression differences, such as up-regulation of acetyl-CoA synthetase, are clone-specific and confirm previous reports of acetate cross-feeding in this system. When co-evolved clones are cultured together, transcription profiling reveals another class of genes whose expression in the dominant clone differs from that observed when the clone is cultured by itself. Many of these genes are part of the CpxR-mediated stress response. CpxR activation in monoculture likely results from extracellular accumulation of acetate that is removed by acetate-scavenging strains in co-culture. Targeted gene sequencing reveals that global regulatory mutations in σS as well as small-scale regulatory mutations in the maltose and acetyl CoA synthetase operons contribute to the evolution of cross-feeding. Finally, we identified two mutations in the founder that likely pre-disposed the experimental population to develop specialists that thrive on overflow metabolites. Subsequent mutations that lead to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Observations that polymorphism readily evolves in an asexual population, that adaptive mutants arise without large-scale change in genome architecture, and that morphs have both common and unique patterns of gene expression influenced by whether they are cultured separately or together, underscore the importance of regulatory change, founder genotype, and the biotic environment in the adaptive evolution of microbes. Overall design: Four isolates of E. coli evolved under long-term glucose limitation were grown in glucose-limited chemostat culture to steady state. Each isolate was grown separately (i.e in monoculture) in triplicate and three of the four (CV101, CV103, CV115 and CV116) were grown as a consortium in duplicate. Each experiment, or biological replicate, was sampled twice for monoculture experiments and once for consortium experiments. Total RNA from each sample was competitvely hybridized against total RNA from the common ancestor of the isolates grown at the same time in a separate chemostat using the same feed medium. For monoculture experiments, three hybridizations were done for each experiment: two hybridizations comparison for the first sample and a single hybridization for the second (with the exception of sample Jv116-1A which was only hybridized twice due to lack of sufficient RNA). For consortium experiments, two hybridizations were done for each biological replicate.

由单一克隆株构建并在资源限制条件下传代培养的微生物种群可发生多态性。本研究旨在探究在持续葡萄糖限制条件下进化765代的大肠杆菌（Escherichia coli）种群中，稳定多态性的形成机制。除优势克隆中存在的29 kb缺失外，进化克隆与其共同祖先之间未出现大规模基因组变异。然而，共进化克隆单独培养时，其转录组谱与共同祖先存在显著差异，且差异模式符合我们对大肠杆菌适应葡萄糖限制机制的认知。所有适应性克隆均表现出稳定期σ因子σS活性降低，以及葡萄糖转运基因表达上调，包括孔蛋白LamB与半乳糖转运蛋白MglABC。其余表达差异（如乙酰辅酶A合成酶的上调）则为克隆特异性，验证了该系统中乙酸交叉营养的既往研究结果。当共进化克隆共同培养时，转录组分析揭示了另一类基因：其在优势克隆中的表达模式与单独培养时存在显著差异。这类基因中有许多属于CpxR介导的应激反应通路。单独培养时的CpxR激活，可能源于胞外乙酸积累；而在共培养体系中，乙酸清除菌株可消除该积累。靶向基因测序结果显示，σS的全局调控突变，以及麦芽糖与乙酰辅酶A合成酶操纵子的小规模调控突变，均参与了交叉营养性状的进化。最后，我们在起始菌株中发现了两个突变，它们可能使实验种群易于进化出依赖溢出代谢物存活的营养特化株。后续导致营养特化的突变则表明，该系统中补偿性突变较功能获得性突变更为关键。本研究观察到：无性繁殖种群中可快速形成多态性；适应性突变的产生不伴随基因组结构的大规模改变；不同形态型的基因表达模式存在共性与特异性，且该模式受培养方式（单独/共同培养）的影响。这些结果凸显了调控变化、奠基者基因型以及生物环境在微生物适应性进化中的重要性。

总体实验设计：将在长期葡萄糖限制条件下进化得到的4株大肠杆菌分离株，置于葡萄糖限制的恒化培养体系中培养至稳态。分别将每株分离株单独培养（即单培养），设置3个生物学重复；同时将4株分离株中的3株（CV101、CV103、CV115及CV116）以菌群形式共培养，设置2个生物学重复。单培养实验的每个生物学重复采样2次，共培养实验的每个生物学重复采样1次。将每个样本的总RNA与同期在相同进料培养基的恒化器中培养的共同祖先菌株的总RNA进行竞争性杂交。单培养实验中，每个生物学重复设置3次杂交：2次针对首次采样样本的杂交，以及1次针对第二次采样样本的杂交（样本Jv116-1A因RNA量不足仅完成2次杂交）。共培养实验中，每个生物学重复设置2次杂交。