The role of a porin-cytochrome fusion in neutrophilic Fe oxidation: insights from functional charactarization and metatranscriptomics
Date
2018
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Publisher
University of Delaware
Abstract
Fe-oxidizing bacteria (FeOB) are an ecologically-important group of organisms found in a wide range of environments. However, mechanisms for microbial Fe oxidation, particularly within neutrophilic microaerophilic FeOB, such as the Zetaproteobacteria and Gallionellaceae, are not well understood and lag behind our knowledge of other metabolisms. Acidophilic FeOB are thought to carry out Fe oxidation through an outer membrane cytochrome, Cyc2, functionally-verified as an Fe oxidase in acidophiles. Recently, a distant homolog to Cyc2 has been identified in the proteome of a neutrophilic Zetaproteobacterium Mariprofundus ferrooxydans PV-1, but has not yet been functionally-verified. We identified homologs of cyc2 in the genomes of a wide variety of organisms, including most known neutrophilic FeOB. The protein is also encoded by many organisms not previously associated with Fe oxidation. The overall aim of this thesis is to provide functional evidence for Cyc2’s role as an Fe oxidase in neutrophilic FeOB and demonstrate its relevance to ecosystems dominated by neutrophilic FeOB.
Using an Escherichia coli expression system, we heterologously expressed Cyc2 from the neutrophilic FeOB PV-1 (Cyc2PV-1), and showed that it binds a heme c cofactor. Expression of Cyc2 conferred Fe oxidation to the host E. coli strain as evidenced by accelerated Fe oxidation in cell suspensions expressing Cyc2PV-1. Controls lacking 1) the cyc2 gene, 2) the cyc2 cytochrome, or 3) only just the heme cofactor all failed to confer Fe oxidation to E. coli. We found that acceleration of Fe oxidation by Cyc2PV-1 was dependent on micromolar amounts of oxygen; no Fe oxidation was observed under anaerobic conditions, suggesting that oxygen is the final electron acceptor in our whole-cell assays. Moreover, sodium azide partially inhibited Fe oxidation. These results provide support for Cyc2’s role as an Fe oxidase in neutrophilic FeOB.
In addition, I analyzed previously-published metatranscriptome data for expression of cyc2 homologs in environments associated with active microbial Fe oxidation. Using a custom Cyc2 HMM, we re-examined the dataset from the Rifle alluvial aquifer, previously published by Jewell et al. [ISME 10, 2106-2117, (2016)] and found that cyc2 homologs are highly-expressed by organisms closely related to known FeOB within the Gallionellaceae family. We also detected cyc2 transcripts from Gallionellaceae within the iron seep metatranscriptome published by Quaiser et al. [PLOS ONE, 9, 7 (2014)], even though Gallionellaceae form only a small fraction of the microbial community there. Taken together, functional characterization of Cyc2PV-1 and detection of cyc2 in environmental metatranscriptomes improve our understanding of mechanisms for microbial Fe oxidation, and highlight the potential for cyc2 to be used as a genetic marker for microbial Fe oxidation in diverse ecosystems.