Functional Approaches for Annotating Secretome-bound Small ORFs in Microbial Genomes

生物技术项目化学、生物工程、环境和运输系统部门用于在微生物基因组中注释分泌组结合的小orf的功能方法迄今为止，几乎所有的微生物功能基因组学和蛋白质组学研究都集中在细胞质上。“分泌组”或细胞外蛋白质组在很大程度上被忽视了，尽管它在理解自然和工业生物加工环境的微生物生理学和生态学方面至关重要。特别是，分泌组结合的小蛋白质和肽通常不被注意。这些生物分子不仅难以识别、分离成熟形态和表征，而且可能仅在特定的、有时是短暂的刺激下产生。在这里，我们假设微生物基因组包含许多尚未被注释的小开放阅读框架（orf），这些框架编码在微生物生物学和生物加工中至关重要的新型分泌组结合生物分子，包括涉及物种间和物种内信号传导和抗微生物功能的肽/蛋白质。该项目的科学和智力价值涉及到定量的、全基因组的方法的发展，以战略性地探测微生物分泌组的动力学，着眼于分泌组结合的小orf（约100个氨基酸或更少）。虽然这里的重点是在纯培养和混合培养中培养的超嗜热海洋热菌模型，但所开发的方法也将适用于不太嗜热的微生物。除了探索这里确定的新可能性外，还将通过对通过初步努力获得的分泌体结合肽的研究来验证这种方法。该项目的具体目标是：(1)证明转录反应信息可用于战略性地关注微生物分泌组的蛋白质组学分析，以鉴定和分离成熟形式的新型分泌组结合小蛋白和肽；(2)研究与海洋T.分泌组结合肽相关的群体依赖信号机制，并确定不太嗜热的细菌中的同源肽是否在群体感应中发挥作用。(3)评估海洋藻分泌组结合的小orf的新线索，重点是热稳定的抗微生物肽。该项目更广泛的影响涉及培养定量系统生物技术新方面的生物分子工程研究生。通常每年有5-10名本科生参与这项研究，其中几名将被招募到该项目中，以使他们了解研究领域的职业前景。将开发和扩大生产和纯化极端酶的实验室，用于NCSU生物技术项目的课程。通过网站提供的一个精心策划的数据库将提供在海苔菌和其他微生物中进行小ORF注释的最新进展。

Biotechnology ProgramDivision of Chemical, Bioengineering, Environmental, and Transport SystemsFunctional Approaches for Annotating Secretome-Bound Small ORFs in Microbial GenomesRobert KellyNorth Carolina State UniversityCBET-0730091To date, almost all microbial functional genomics and proteomics studies have focused on the cytosol. The "secretome", or extracellular proteome, has been largely ignored, despite its critical importance in understanding the microbial physiology and ecology of both natural and industrial bioprocessing environments. In particular, secretome-bound small proteins and peptides typically go unnoticed. These biomolecules are not only difficult to identify, isolate in their mature forms, and characterize, but they may only be produced in response to specific, and sometimes transient, stimuli. Here, it is hypothesized that microbial genomes contain many yet to be annotated small open reading frames (ORFs) that encode novel secretome-bound biomolecules of central importance in microbial biology and bioprocessing including peptides/proteins implicated in inter- and intra-species signaling and anti-microbial functions. The scientific and intellectual merit of this project relates to the development of quantitative, genome-wide approaches to strategically probe the dynamics of microbial secretomes with an eye towards secretome-bound small ORFs (about 100 amino acids or less). While the focus here will be on the model hyperthermophile Thermotoga maritima grown in pure and mixed cultures, the methodology developed will be applicable to less thermophilic microorganisms as well. This approach will be validated by pursuing leads on secretome-bound peptides obtained through preliminary efforts, in addition to exploring new possibilities that are identified here. The specific objectives of this project are to: (1) demonstrate that transcriptional response information can be used to strategically focus proteomics analysis of microbial secretomes to identify and isolate mature forms of novel secretome-bound small proteins and peptides, (2) investigate population-dependent signaling mechanisms related to a putative secretome-bound peptide in T. maritima and determine if orthologous peptides in less thermophilic bacteria play roles in quorum sensing, and (3) evaluate new leads on secretome-bound small ORFs in T. maritima, with a focus on thermally stable anti-microbial peptides.The broader impacts of this project relate to the training of biomolecular engineering graduate students in a new aspect of quantitative systems biotechnology. Typically 5-10 undergraduates participate in this research each year and several will be recruited to the project to expose them to career prospects in research. Labs that incorporate production and purification of extremophilic enzymes will be developed and expanded for use in courses taught in the NCSU Biotechnology Program. A curated database provided through the website will provide updates on progress with small ORF annotation in T. maritima and other microorganisms.