Malasarn, Davin (2007) Molecular and environmental studies of bacterial arsenate respiration. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-02232007-132917
Arsenate [As(V)]-respiring bacteria that reduce As(V) to arsenite, As(III), for energy production have been implicated as possible catalysts for arsenic mobilization into drinking water supplies. To understand how this metabolism contributes to arsenic geochemistry, this thesis explores the dynamics of As(V)-respiratory gene expression, the impact of As(V) respiration on microbial ferric [Fe(III)] reduction, and biochemical properties of the arsenate respiratory reductase, ARR.
Using sequences for arrA, a gene encoding the terminal reductase involved in As(V) respiration, degenerate PCR primers were designed to amplify a diagnostic region of the gene in multiple As(V)-respiring isolates. These primers were used to track arrA transcription in microcosm studies involving synthetic sediments. arrA was required for As(V) reduction in this context, and the gene was expressed in contaminated sediments at Haiwee Reservoir in Olancha, CA.
To understand the impact of As(V) respiration on Fe(III) reduction, native microbial consortia from Haiwee Reservoir and pure cultures of the genetically tractable Shewanella sp. strain ANA-3 were incubated with As-sorbed hydrous ferric oxide (HFO), and rates of As(V) and Fe(III) reduction were determined. As(V) reduction occurred simultaneously with or prior to Fe(III) reduction, consistent with the idea that electron acceptor utilization is determined by thermodynamic favorability. Furthermore, the presence of sorbed As(III) increased rates of Fe(III) reduction, potentially by increasing HFO surface area.
Lastly, the expression, assembly, and kinetic properties of ARR from ANA-3 were characterized. ARR is a soluble periplasmic heterodimer that is expressed during early exponential growth and persists into late stationary phase. The enzyme contains molybdenum, Fe, and sulfur cofactors. It has a Km of 5 µM, a Vmax of 11,111 µmol As(V) reduced . min-1 . mg protein-1, and reduces only As(V). Mutational analysis of the residues corresponding to the diagnostic region of arrA mentioned above resulted in loss of enzyme activity.
This work brings us closer to being able to quantify and predict the contribution of As(V) respiration to the solubilization of arsenic from sediments. Structural studies, the development of probes to detect ARR, and comparisons of ARR from different bacterial species are now possible.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||ARR; arsenate respiration; arsenate respiratory reductase; iron reduction; Shewanella sp. strain ANA-3|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||8 February 2007|
|Author Email:||dmalasarn (AT) gmail.com|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||27 Feb 2007|
|Last Modified:||26 Dec 2012 02:31|
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