Environmentally relevant responses in different Escherichia coli pathotypes: a functional genomics study of motility and associated regulons

在自然界中，细菌可能是朋友，就像我们消化系统中的“好”细菌一样，也可能是敌人，有时是危及生命的病原体。大肠杆菌既可以是朋友，也可以是敌人，它也是一种“模式”细菌，人们对它进行了大量研究，以了解细菌的结构和功能。这种认识主要基于一种名为K-12的无害菌株，它于1922年被分离出来。今天，K-12已经很好地适应了实验室，它甚至不能在人类的消化道中生存。但也有其他独特的大肠杆菌致病类型，包括大肠杆菌O157。完整的基因组DNA序列已确定为K-12和其他几种病型。在每个大肠杆菌中，大约有20%的基因是独特的，这是一个很大的惊喜，因为人们曾预计大肠杆菌作为一个物种在基因上是相当同质的。那么，K-12是一个很好的代表整个物种的模型吗？为了找到答案，我们将分析和比较K-12和其他病理类型对诱导它们改变行为的条件的遗传反应方式，重点关注它们在液体中“游泳”的能力（运动性）以及使它们能够在环境中生存和坚持的相关特性。新的“功能基因组学”技术，基于基因组序列的知识，能够同时监测基因组中每个基因的表达。这显示了每个基因在特定条件下如何参与细菌的行为。使用这些技术只是为了填补我们对K-12如何运作的知识空白，这是很诱人的。但是，这将忽视分析已知基因组序列的不同病理类型与它们在自然界中的行为之间的关系的潜力。我们建议充分利用这一潜力。大肠杆菌中的一些转录网络，包括由称为转录因子或tf的蛋白质控制的基因亚群（“规则”），由于它们参与细胞的基本生物化学和生理学而受到广泛关注。这在功能基因组学方法分析的网络选择中得到了回应。例如，已经发表了3项关于FNR调节参与缺氧适应的独立研究。相比之下，对由TF复合物FlhDC控制的运动调节的实验研究要少得多。我们认为这种TF在全球细胞转录网络中形成了一个重要的节点或“交叉点”，与其他介导金属和氧化应激反应的细胞相互作用。FlhDC调节子还对细胞内产生的一种重要的、很少被探索的信号分子——环二gmp做出反应。众所周知，这种分子与大肠杆菌和相关物种以一种被称为“生物膜”的生长模式在固体表面上定植的能力有关，这种模式的运动受到强烈抑制。我们将使用功能基因组学方法来分析FlhDC和相关tf（称为Fur和SoxS）控制的全基因组转录活性。我们将施加TFs通常被激活的条件，并监测代表性病型中基因表达的全局变化。在平行实验中，我们将研究tf被删除的突变体中的基因表达。我们还建议使用新的方法来研究TF与DNA不同区域的结合，并调节这些区域基因的表达。这将提供一幅明确的图片，说明这些规则是如何对刺激做出反应的，以及它们随后调节哪些细胞功能以使细胞适应环境挑战。因此，我们将获得令人兴奋的见解，了解由FlhDC和相关tf介导的反应多样性在这种独特的重要细菌的不同病理类型中，并首次产生基因表达模式的全球比较及其在同一细菌物种的不同代表之间的控制。

In nature bacteria may act as friends, like 'good' bacteria in our digestive systems, or foes, sometimes as life-threatening pathogens. Escherichia coli can be either friend or foe, and is also a 'model' bacterium, much studied to understand bacterial structure and function. This knowledge is based mainly on a harmless strain named K-12, isolated in 1922. Today, K-12 is so well adapted to the laboratory that it cannot even survive in the human digestive tract. But there are other distinctive 'pathotypes' of E. coli that can cause disease, including E. coli O157. Complete genomic DNA sequences have been determined for K-12 and several other pathotypes. In each, about 20% of its genes are unique, a big surprise since it had been expected that E. coli as a species would be genetically quite homogeneous. So, is K-12 a good model representing the species as a whole? To find out, we will analyse and compare the ways K-12 and other pathotypes respond genetically to conditions that induce them to change their behaviour, focusing on their ability to 'swim' in liquids (motility) and related properties that enable them to survive and persist in the environment. New 'functional genomics' techniques, based on knowledge of genome sequences, enable the expression of every gene in the genome to be monitored simultaneously. This shows how each gene is involved in the behaviour of the bacteria under given conditions. It would be tempting to use these techniques just to fill the gaps in our knowledge of how K-12 functions. But this would neglect the potential to analyse different pathotypes, of known genome sequence, in relation to their behaviour in nature. We propose to exploit that potential to the full. Some transcription networks in E. coli, comprising subsets of genes ('regulons') controlled by proteins called transcription factors or TFs, have received much attention due to their involvement in the basic biochemistry and physiology of the cell. This has been echoed in the choice of networks that have been analysed by functional genomics methods. For example, 3 independent such studies of the FNR regulon involved in adaptation to the absence of oxygen have already been published. In contrast, there has been far less experimental investigation of the regulon that controls motility, governed by the TF complex FlhDC. We believe this TF forms a significant node or 'crossing point' in the global cellular transcription network, interacting with, among others, those that mediate responses to the presence of metals and to oxidative stress. The FlhDC regulon also responds to an important and little-explored signal molecule made within the cell, called cyclic di-GMP. This molecule is known to be involved in the ability of E. coli and related species to colonise solid surfaces in a growth mode known as a 'biofilm' in which motility is strongly repressed. We will use functional genomics methods to analyse the genome-wide transcription activity controlled by FlhDC and associated TFs named Fur and SoxS. We will impose conditions in which the TFs would normally be activated, and monitor global changes in gene expression in representative pathotypes. In parallel experiments we will study gene expression in mutants in which the TFs have been deleted. We also propose to use new methods to study TF binding to different regions of DNA, modulating expression of the genes in those regions. This will provide a definitive picture of how the regulons respond to stimuli and what cellular functions they then modulate to allow the cell to adapt to environmental challenges. We will thus gain exciting insights into the diversity of responses mediated by FlhDC and related TFs in different pathotypes of this uniquely important bacterium, and generate for the first time a global comparison of patterns of gene expression and their control among different representatives of the same bacterial species.