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

生物技术专业化学、生物工程、环境和运输系统分部注释微生物基因组中分泌组结合的小ORF的功能方法Robert KellyNorth卡罗莱纳州立大学CBET-0730091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胞质溶胶上。分泌蛋白组或胞外蛋白组在很大程度上被忽视，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，分泌体结合的小蛋白和肽通常不被注意。这些生物分子不仅难以识别，分离其成熟形式和表征，而且它们可能仅响应于特定的，有时是短暂的刺激而产生。在此，假设微生物基因组包含许多尚未注释的小开放阅读框（ORF），其编码在微生物生物学和生物加工中具有中心重要性的新型分泌体结合的生物分子，包括涉及种间和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，全基因组方法的发展，以战略性地探测微生物分泌组的动态，着眼于分泌组结合的小ORF（约100个氨基酸或更少）。虽然这里的重点将是在纯培养和混合培养中生长的模式超嗜热栖热袍菌海栖热袍菌，开发的方法也将适用于嗜热性较低的微生物。这种方法将通过追求通过初步努力获得的分泌体结合肽的线索来验证，除了探索这里确定的新的可能性。本项目的具体目标是：（1）证明转录反应信息可以用于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T. maritima和确定在较少嗜热细菌中的邻位肽是否在群体感应中起作用，以及（3）评估T.该项目的更广泛影响涉及在定量系统生物技术的新方面对生物分子工程研究生进行培训。通常情况下，每年有5-10名本科生参加这项研究，其中一些将被招募到该项目中，以使他们了解研究中的职业前景。实验室，包括生产和极端酶的纯化将开发和扩大在NCSU生物技术计划教授的课程中使用。通过网站提供的精心策划的数据库将提供T中带有小ORF注释的进展更新。海洋生物和其他微生物。

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.