Identification And Analysis Of Genes Involved In Anaerobic Nitrate-Dependent Iron Oxidation

Author

Stacey Rae Taft, Southern Illinois University CarbondaleFollow

Date of Award

1-1-2009

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Achenbach,Laurie

Abstract

The mobility of trace metals and radionuclides released into aquatic and terrestrial environments by mining, industrial processes, and municipal waste disposal practices is an area that deserves significant scientific, public health, and regulatory attention. Indirect microbial interaction offers one potential mechanism for immobilizing these contaminants. For example, many metals, such as uranium and chromium, are less soluble once bound as iron oxide precipitates, thus inhibiting the spread of soluble heavy metals and radionuclides within groundwater and halting them from contaminating additional areas. Microbial iron oxidation is known to contribute to the immobilization of heavy metals and radionuclides in contaminated sites. A safe, cost-effective decontamination method for these materials is the association of radionuclides with iron oxides formed via microbial pathways, thus precipitating them out of solution and providing a promising technique for in situ bioremediation. Nitrate-dependent iron-oxidizing bacteria have been shown to play an important role in the retention of soluble uranium by forming iron oxides that absorb onto soluble U(VI) particles, rendering them immobile as U(VI)-iron oxides. Dechloromonas aromatica strain RCB is a β-proteobacterium that has been cultured and extensively studied in our laboratory and is capable of reducing perchlorate and anaerobically oxidizing benzene, humic acids, and ferrous iron. In addition, a newly-isolated β-proteobacterium, Diaphorobacter sp. strain TPSY, is of interest due to its ability to anaerobically oxidize humic acids, uranium, and ferrous iron. Thus, these two strains have enormous bioremediative potential and are prime candidates for in situ bioremediation. Microarray analysis was used to identify genes induced under iron-oxidizing conditions and RNA dot blotting was used to confirm mRNA expression in D. aromatica. As a follow-up, RNA arbitrarily primed (RAP)-PCR, a method used to randomly reverse-transcribe RNA into cDNA, was also used to identify expression that might not have been observed in the microarray. Genes that were identified from both microarray and RAP-PCR experiments include several hypothetical proteins, lipoproteins, and transmembrane proteins located in major operons, as well as genes annotated as signal transduction enzymes, c-type cytochromes, and proteins involved in chemotaxis, flagellar, and pilus development. Suicide vectors were used to create deletion mutations targeting the genes of interest. Additionally, transposon mutagenesis was used in Diaphorobacter sp. TPSY to identify any potential iron-oxidizing mutants. Out of seven TPSY mutants deficient in iron oxidation, four were identified as genes that encode an inner membrane protein, a signal transduction protein, a putative lipoprotein downstream of a cytochrome c, and a regulatory DNA-binding protein. Mutants were confirmed for their inability to oxidize iron by measuring Fe(II) concentrations over time with a ferrozine assay. The identification of genes involved in microbial anaerobic nitrate-dependent iron oxidation will prove to be a valuable asset when designing and assessing bioremediative strategies.