Improved Understanding of Microbial Iron and Sulfate Reduction Through a Combination of Bottom-up and Top-down Functional Proteomics Assays

Richardson, Ruth¹ 

[1]Cornell Univ., Ithaca, NY (United States)

关键词: iron reduction;    Geobacter;    Anaeromyxobacter;    Desulfotomaculum;    Shewanella;    proteomics;    targetted proteomics;    heterologous expression;    bioremediation biomarkers;   

DOI  :  10.2172/1239632
RP-ID  :  None
PID  :  OSTI ID: 1239632

学科分类：生物科学（综合）

美国|英语

来源: SciTech Connect

【 摘 要 】

Our overall goal was to improve the understanding of microbial iron and sulfate reduction by evaluating a diverse iron and sulfate reducing organisms utilizing a multi-omics approach combining ???top-down??? and ???bottom-up??? omics methodologies. We initiated one of the first combined comparative genomics, shotgun proteomics, RTqPCR, and heterologous expression studies in pursuit of our project objectives. Within the first year of this project, we created a new bioinformatics tool for ortholog identification (???SPOCS???). SPOCS is described in our publication, Curtis et al., 2013. Using this tool we were able to identify conserved orthologous groups across diverse iron and sulfate reducing microorganisms from Firmicutes, gamma-proteobacteria and delta-proteobacteria. For six iron and sulfate reducers we also performed shotgun proteomics (???bottom-up??? proteomics including accurate mass and time (AMT) tag and iTRAQ approaches). Cultures include Gram (-) and Gram (+) microbes. Gram (-) were: Geobacter sulfureducens (grown on iron citrate and fumarate), Geobacter bemidjiensis (grown on iron citrate and fumarate), Shewanella oneidiensis (grown on iron citrate and fumarate) and Anaeromyxobacter dehalogenans (grown on iron citrate and fumarate). Although all cultures grew on insoluble iron, the iron precipitates interfered with protein extraction and analysis; which remains a major challenge for researchers in disparate study systems. Among the Gram (-) organisms studied, Anaeromyxobacter dehalogenans remains the most poorly characterized. Yet, it is arguably the most versatile organisms we studied. In this work we have used comparative proteomics to hypothesize which two of the dozens of predicted c-type cytochromes within Anaeromyxobacter dehalogenans may be directly involved in soluble iron reduction. Unfortunately, heterologous expression of these Anaeromyxobacter dehalogenans ctype cytochromes led to poor protein production and/or formation of inclusion bodies, even when we co-expressed several genes known to be important for assembly of cytochrome holoenzymes. We confirmed the proteomics trends at the RNA level by designing specific primer sets for hypothesized iron reductases in Anaeromyxobacter dehalogenans, and performed Reverse Transcription-qPCR. AD_0127 was 20 fold upregulated only on iron citrate conditions. AD_0127 is described as a hypothetical protein, but Pfam predicts it to be C554 type cytochrome having a possible role in nitrification (Wang et al., in preparation).