Prophage host interactions: pulling back the curtains on Pseudomonas puppet masters

前噬菌体是已经整合到细菌基因组中的病毒(噬菌体)，但它们对宿主成功的贡献被严重低估。我们的目标是提供一个更详细的前噬菌体对细菌宿主的影响的视图，这将极大地扩大我们对已知它们是重要的系统的理解，但不是它们是如何重要的。铜绿假单胞菌，一种植物、动物和人类的病原体，将是我们的模式原噬菌体/细菌宿主系统。我们将特别关注利物浦流行毒株(LES)和致病性较低的模式毒株PAO1。众所周知，LES毒株携带多个已被证明可增强其竞争力的前驱噬菌体。LES噬菌体不编码任何已知的毒素或毒力因子，但与铜绿假单胞菌代谢途径和其他生物学特征的改变有关。前噬菌体序列在几乎所有的细菌菌株中都能找到。然而，人们实际上对这些先知携带的遗传信息知之甚少。事实上，75%或更多的前噬菌体基因被注释为假想的(通常被称为噬菌体暗物质)。这些假设的序列可能在许多不同的噬菌体中共享，但除非它们被鉴定为毒力相关因子，否则这些序列与宿主生物学的相关性通常被忽略。最近，一些暗物质的重要性已经被阐明并被证明是调节细菌宿主基因或促进细菌生存的。此时此刻，当我们在控制耐多药细菌感染传播的战斗中失利时，迫切需要更好地了解控制细菌生物学的所有遗传因素。这些数据将有助于制定更好的控制细菌感染的知情策略，也有助于理解目前用于发展噬菌体疗法的裂解噬菌体。我们的目标将揭示前噬菌体“傀儡主人”影响铜绿假单胞菌生物学和适应性的隐藏机制：我们已经从铜绿假单胞菌的LES菌株中提纯了多个可诱导的前驱噬菌体。我们已经构建了一套精确的工具和菌株来研究每个前驱体(单独和联合)对一个特征良好的模式宿主菌株的直接影响，而不是一个没有前驱体的菌株。我们将使用尖端技术，结合基因组结构、基因表达变化和可能的调节因子的知识，揭示前驱体影响细菌宿主的不同方式。克隆已识别的调节子和突变体构建将使原噬菌体基因与功能途径相关联。我们将监测识别的前噬菌体编码元件在许多不同的环境参数下的影响，这些参数反映了铜绿假单胞菌的生态位。这一结合的方法将阐明三个同居的LES前驱体与它们的宿主之间的相互作用，以更好地了解它们对细菌行为的控制。应用和好处：这些研究不仅将使人们更好地了解铜绿假单胞菌的生物学，而且这些技术也可以应用于其他噬菌体-宿主系统。近年来，用于识别细菌基因组中的前驱噬菌体的生物信息学工具有了很大的改进。但没有像我们这样的功能研究，噬菌体基因的相关性仍然是暗物质的一部分。识别未知基因的功能(在噬菌体数据库中保守)将改变噬菌体生物学领域和我们对微生物学的基本理解。

Prophages are viruses (bacteriophages) that have integrated into bacterial genomes, but their contribution to the success of their hosts has been grossly under-estimated. We aim to provide a more detailed view of prophage influence on the bacterial host, which will greatly expand our understanding of a system where they are known to be important, but not how they are important.Pseudomonas aeruginosa, a pathogen of plants, animals and humans will be our model prophage/bacterial host system. We will specifically focus on the Liverpool Epidemic Strain (LES) and, a less pathogenic model strain, PAO1. The LES strain is known to carry multiple prophages that have been proven to enhance its competitiveness. The LES prophages do not encode any known toxins or virulence factors but have been associated with altered metabolic pathways and other biological traits of P. aeruginosa. Prophage sequences are found in almost all bacterial strains. However, very little is actually known about the genetic information that these prophages carry. In fact 75% or more of most prophage genes are annotated as hypothetical (often referred to as phage dark matter). These hypothetical sequences may be shared across many different phages but, unless they have been identified as virulence-related factors, the relevance of these sequences to the biology of the host is generally ignored. Recently, the importance of some of this dark matter has been elucidated and shown to regulate bacterial host genes or to promote bacterial survival. At this point in time when we are losing the battle in controlling the spread of multidrug resistant bacterial infections, there is an urgent need to better understand all of the genetic elements that are controlling bacterial biology. These data will help to produce better informed strategies for the control of bacterial infection and also aid the understanding of lytic bacteriophages that are currently being used in the development of phage therapy. Our objectives will uncover the hidden mechanisms by which prophage "puppet masters" affect the biology and fitness of P. aeruginosa: We have purified multiple inducible prophages from the LES strain of P. aeruginosa. We have constructed a precise set of tools and strains to investigate the direct effect of each prophage (separately and in combination) on a well-characterised model host strain, compared to a strain where the prophages are absent. We will use cutting edge techniques, combining knowledge of genome architecture, changes in gene expression and putative regulators to reveal the different ways that prophages impact their bacterial hosts. Cloning of identified regulators and mutant construction will enable association of prophage genes to functional pathways. We will monitor the impact of identified prophage-encoded elements under many varied environmental parameters that reflect the niches of P. aeruginosa. This combined approach will elucidate the interactions between three cohabiting LES prophages and their host to understand better their control of the bacterial behaviour.Applications and benefits: Not only will these studies inform a better understanding of P. aeruginosa biology, but the techniques can be applied to other phage-host systems. Bioinformatic tools have been much improved in recent years, for identifying prophages in bacterial genomes. But without functional studies such as ours, the relevance of the phage genes remains part of the dark matter. Identifying function of unknown genes (that are conserved across phage databases) will transform the field of phage biology and our fundamental understanding of microbiology.