Defining adaptive processes in a model bacterial species by integrating proteomic and metabolomic analytical strategies

与BBSRC相关：该项目将KCL和ASTA联系起来，与BBSRC战略政策领域“生物科学的系统方法”和“生物科学的技术开发”（见http://www.bbsrc.ac.uk/funding/grants/priorities/）直接相关。如下文影响部分所述，预计还将在与抗菌素耐药性和农业有关的BBSRC战略政策领域取得效益。目的：利用尖端技术研究促进铜绿假单胞菌对其种内多样性传播的复杂调控因子。背景：新的DNA测序技术的到来导致了人类和许多环境微生物组数据的爆炸式增长。虽然这些信息提供了一个物种的全面目录，其中许多目前还不能培养，但这些数据并没有深入了解微生物的功能，也没有提出如何传播到新栖息地的尖锐问题。微生物的栖息地是多种多样的。然而，一些关键物种可以超越它们原来的“边界”，在新的生态位定居。尽管几个研究中心试图绘制通常具有高度抗生素耐药性的物种从环境到人类的传播路线，但使微生物离开其正常栖息地并在新地点定居的驱动力目前尚不清楚。这项研究将研究铜绿假单胞菌适应潜力的关键调节因子，因为它具有极端的代谢多功能性和“无处不在”的新位点定植。此外，该物种及其近缘分类群广泛应用于许多工业过程，包括生物修复。此外，由于铜绿假单胞菌在染色体上具有显著的耐药基因能力，它是目前我们最大的治疗挑战之一，并对环境构成巨大威胁。假设：该项目背后的潜在假设是，适应性过程的表型可塑性，选择性地使微生物离开其正常栖息地并在新地点定居，是由过多的关键调节因子和信号分子驱动的，在铜绿假单胞菌的情况下，这占其基因组的近10%。然而，目前的证据表明，适应性过程的表型可塑性存在于蛋白质组和代谢组水平。一旦转录，许多相关的蛋白质/代谢物可能被修饰（翻译后修饰- PTM）以促进它们的转化。因此，绘制这些过程不仅涉及这些复杂生物分子的检测和定量，还涉及其修饰的蛋白质形态。直到最近，蛋白质和ptm的高通量检测还在生物科学的范围之外。今天，有了新型的质谱（MS）和核磁共振光谱，这种分析是可能的，但技术和技能一直局限于相对较少的专业实验室。计划：这个工业案例奖学金将建立在监督小组在学术界（KDB， AJM）和工业界（OB， HNS）对铜绿假单胞菌的工作经验的基础上，研究与其种群结构相关的代谢组和蛋白质组。将从对水、污水、土壤等生态位高度适应的精选谱系中选择具有代表性的菌株进行比较深入的分析，以阐明调控标记。这些将通过对每个鉴定标记的RNA表达分析来验证，以证实它们的分布和表达水平。该项目将与学生一起开发，将训练和实验工作结合起来，在四年的重叠期间进行，如下所述。

BBSRC RELEVANCE: This project, linking KCL and ASTA, is of direct relevance to BBSRC Strategic Policy areas "Systems approaches to the biosciences" and "Technology development for the biosciences" see http://www.bbsrc.ac.uk/funding/grants/priorities/). As in the Impact section below, benefits in BBSRC Strategic Policy areas relating to antimicrobial resistance and agriculture are also expected outcomes.AIM: To utilise cutting-edge technologies to investigate the complex regulators that facilitate transmission of Pseudomonas aeruginosa against its intra-species diversity.BACKGROUND: The arrival of novel DNA sequencing technologies have led to an explosion of data on the microbiome of man and numerous environments. While such information provides a comprehensive catalogue of species, many of which cannot be cultured at present, the data give no insight into the function of the microbe nor the incisive question of how transmission to a new habitat occurs. The habitats that microbes colonise are diverse. A few key species can however transcend their original "boundaries" to colonise new niches. Despite attempts by several research centres to map routes of transmission of often highly antibiotic resistant species from the environment to man, the driving forces that enable a microbe to leave its normal habitat and colonise a new site are not currently known. This study will investigate key regulators of the adaptive potential of Pseudomonas aeruginosa as a model due to the extreme metabolic versatility and "ubiquitous" colonisation of new sites that it displays. This species and closely related taxa are moreover used widely in many industrial processes including bioremediation. In addition, due to its remarkable capacity to harbour antibiotic resistance genes chromosomally, P. aeruginosa is currently one of our greatest therapeutic challenges and poses an immense environmental threat.HYPOTHESIS: The underlying hypothesis behind this project is that the phenotypic plasticity for adaptive processes that selectively enables a microbe to leave its normal habitat and colonise a new site is driven by a plethora of key regulators and signaling molecules, which in the case of P. aeruginosa accounts for nearly 10% of its genome. Current evidence indicates however that the phenotypic plasticity for adaptive processes lies at the level of the proteome and metabolome. Once transcribed, many of the proteins/metabolites involved may be modified (post-translational modification - PTM) to facilitate their transition. Mapping these processes therefore involves not only detection and quantitation of these complex biomolecules but their modified proteoforms. Until recently, high throughput detection of proteins and PTMs were outside the reach of bioscience. Today, with novel forms of Mass spectrometry (MS) and NMR spectroscopy, such analyses are possible but the technologies and skills have been limited to relatively few specialist laboratories.PLAN: This Industrial CASE Studentship will build upon the experience of the supervisory team's work on P. aeruginosa from both academia (KDB, AJM) and industry (OB, HNS) to investigate the metabolome and proteome in relation to its population structure. Representative strains from selected lineages where hyper-adaptation to several ecological niches such as water, sewage, soil etc, will be selected for comparative in-depth analysis to elucidate regulatory markers. These will be validated using RNA expression analysis of each identified marker to corroborate their distribution and expression level. The project will be developed with the student combining training and experimental work through overlapping periods during the four years as outlined below.