Insights Into Microbial Metal Reduction Through Implementation Of Proteomic-Based Techniques: A Focus On The Gram-Positive Bacterium Desulfotomaculum Reducens Mi-1

Abstract

Certain microorganisms are capable of extracellular metal reduction. Fe(III) is an abundant element in the Earth's crust and serves as a common terminal electron acceptor in anaerobic environments. Decades of research have focused on pathways of extracellular electron transfer to metals since the discovery of enzymatic microbial metal reduction in the late 1980's. Most of these studies have analyzed two model genera of Gram-negative proteobacteria, specifically Geobacter and Shewanella. It is now understood that a variety of phylogenetically diverse microorganisms are capable of metal reduction, including species of Gram-positive bacteria, but studies on microbial metal reduction are scarce outside of the Gram-negative model organisms. In this dissertation, parallel proteomic approaches were employed in order to study microbial metal reduction. Several studies focused on Desulfotomaculum reducens MI-1, a Gram-positive sulfate-reducing bacterium capable of Fe(III), Mn(IV), Cr(VI), and U(VI) reduction. This included a 'top-down' proteomic approach (activity screens of fractionated proteins) to identify proteins capable of Fe(III) reduction. Fe(III) reductases identified from D. reducens, which were also shown to have in vitro Cr(VI) and U(VI) reductase activity, were Dred_2421 and Dred_1685-6 (a protein complex). A 'bottom-up' proteomic approach was also utilized in order to perform comparative proteomic analysis. The proteomes of D. reducens were compared during sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. This was the first global comparative proteomic analysis of a Grampositive organism cultivated on either sulfate or Fe(III)-reducing conditions. Based on differential abundance patterns, certain proteins were predicted to be involved in either Fe(III) or sulfate reduction in D. reducens. These included proteins within several heterodisulfide reductase-containing loci with previously unknown function. Evidence for flavin-based electron bifurcation was also revealed in this comparative proteomic study. Another proteomic technique employed was targeted biomarker peptide quantification. Various peptide biomarkers of metal reductases from diverse metal-reducing bacteria were created. Using multiple reaction monitoring (MRM) mass spectrometry, these peptides were quantified in laboratory cultures including Fe(III)-reducing co-cultures established between D. reducens and Geobacter sulfurreducens PCA. These co-cultures exhibited enhanced rates of both soluble and insoluble Fe(III) reduction as well as increased rates of pyruvate oxidation. Furthermore, D. reducens and G. sulfurreducens cells grew faster in co-culture than in pure cultures. Altogether, these observations suggest formation of a mutually beneficial association. Along with the targeted MRM technique, global comparative proteomic analysis was performed on D. reducens-G. sulfurreducens co-cultures in order to provide further biological insight. Interestingly, multiple proteins previously associated with Fe(III) reduction in G. sulfurreducens (including multiheme c-type cytochromes and type IV pili-related proteins) were significantly increased in abundance during growth with D. reducens. In summary, through employment of varied proteomic techniques, this work strives to progress the study of microbial metal reduction, with a particular focus on the Gram-positive bacterium D. reducens.