Q-290. Identification and Analysis of Genes Involved in Anaerobic Nitrate-Dependent Fe(II) Oxidation

S. R. Taft1, K. A. Weber2, J. D. Coates3, L. A. Achenbach1;
1Southern Illinois Univ., Carbondale, IL, 2Univ. of California, Berkeley, CA, 3Univ. of California Sch. of Med., Berkeley, CA.

Bioremediation of soluble radionuclides and heavy metals potentially represents a cost-effective alternative to current removal methods. Although some naturally-occurring microbial communities can act directly upon these contaminants, others may use a different metabolism that indirectly transforms the soluble compounds into insoluble aggregates. Anaerobic iron oxidation at circumneutral pH is a process of interest since oxides that are formed may bind to soluble radionuclides, rendering them insoluble. Because the genetics of this metabolism is not known, we have taken molecular-based approaches to analyze differential expression among cultures of Dechloromonas aromatica RCB grown in Fe(II)-oxidizing conditions using nitrate as the electron acceptor. Microarray analysis was used to identify genes that were upregulated in comparison to acetate controls and northern blotting was used to confirm mRNA expression. RNA arbitrarily primed (RAP)-PCR, a method that randomly reverse-transcribes RNA into cDNA, was also used to identify differential expression that might not have been observed in the microarray, and the resulting RAP-PCR bands were sequenced and submitted to northern analysis. Upregulation in the microarray and RAP-PCR approaches were observed mainly in genes that had been annotated as hypothetical proteins, transmembrane proteins, and enzymes possibly involved in signal transduction and protein transport. More specifically, genes of interest include a signal transduction histidine kinase, several proteins of unknown function, several transmembrane proteins, and a protein phosphatase. These targeted genes will be subjected to deletion analysis and complementation experiments to verify their involvement in nitrate-dependent iron oxidation. Employing these approaches will help us further understand this metabolism, which will prove valuable when designing and assessing bioremediative strategies.