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

点击翻译按钮获取中文摘要

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.