Genetic investigations of the iron and arsenic reduction pathways of the bacterium Shewanella sp. str. ANA-3

In arsenic contaminated sediments arsenate is often associated with iron(III) hydroxides and can be released through microbial iron(III) dissimilatory reduction and arsenate respiration. Whether arsenic reducing, iron reducing or arsenic/iron reducing microorganisms contribute most to arsenic release in groundwater is a matter of ongoing debate. Shewanella sp. str. ANA-3 is a bacterium that utilizes an arsenate respiratory reductase (encoded by arrA/B) to reduce arsenate to arsenite coupled to oxidation of an electron donor. Additionally, ANA-3 has a cluster of metal reducing genes (mtrCABDEF/omcA) that encode c-type decaheme cytochromes and non-cytochromes some of which are involved in reduction of soluble and insoluble metal oxides such as iron and manganese hydroxides.;Chapter 2 of this dissertation is a reprint from a published book that gives an overview of microbial iron and arsenic metabolisms. Chapter 3 of this dissertation addresses the hypothesis that the iron reduction pathway of ANA-3 contributes most to arsenic release from arsenic-ferrihydrite minerals. This chapter describes the effect mutating mtrCAB/omcA and arrA have on ANA-3's ability to reduce iron and arsenic from arsenate-ferrihydrite minerals. Various strains of ANA-3 were characterized using arsenate and ferrihydrite batch incubations: an iron(III) metal reducing mutant (FERM), an arsenate metal reducing mutant (ARM-1) and a biological control (FARM) unable to reduce iron(III) or arsenate. Mutant strains were incubated in the presence of ferrihydrite with or without pre-adsorbed arsenate. Results showed that the FERM mutant was capable of reducing arsenate at wildtype levels but lacked ferrihdyrite reducing capability. Moreover, the ARM-1 mutant was able to reduce ferrihydrite at wildtype levels but showed a significant decrease when arsenate was present. Thus, under batch conditions, it appears that the arsenate reduction pathway alone can release arsenic from arsenate-ferrihydrite minerals. However, when the iron reduction pathway is absent, ANA-3 cannot reduce ferrihydrite as efficiently from arsenate-ferrihydrite minerals.;Many microorganisms can dissolve minerals in the subsurface via extracellular electron transfer. In Shewanella sp. str. ANA-3, certain c-type cytochrome proteins consisting of multiple heme groups are involved in the extracellular reduction of solid phase electron acceptors such as iron(III) and manganese oxides. Multi-heme cytochromes are unique to Bacteria and Archaea and we are just starting to understand how they facilitate electron transport to extracellular electron acceptors. Chapter 4 addresses the hypothesis that all ten hemes of the decaheme cytochrome MtrA from ANA-3 are necessary for iron(III) reduction. Because no crystal structure is available for MtrA, we used a site directed mutagenesis method to investigate the role of the decahemes of MtrA in the reduction of soluble and low soluble iron(III). Our approach involved disrupting the function of each individual heme by mutating proximal and distal histidine residues coordinated to each heme, and then tested their ability to reduce ferrihydrite or iron(III)-citrate. Mutating the proximal histidine of all hemes (with the exception of one), led to loss of ferrihydrite reduction ability. In contrast, mutating the distal histidine of each heme led to various levels of ferrihydrite reduction. All heme mutants were capable of iron(III)-citrate reduction, although to varying degrees. Differences in MtrA protein expression were also observed depending on which residue was mutated. These findings suggest all ten hemes of MtrA may not be necessary for iron(III) reduction and that more than one heme could serve as an entry or exit point for electron transfer. (Abstract shortened by UMI.)...