163. Exploring Whole Genome Sequence Information for Defining the Functions of Unknown Genes and Regulatory Networks in Dissimilatory Metal Reduction Pathways 

Jizhong Zhou¹, Douglas Lies², Gary Li¹, Rebecca Clayton³, Kenneth H. Nealson², Claire Fraser³, James M. Tiedje^4 
1Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; ²Department of Geology and Planetary Sciences, Jet Propulsion Laboratory and California Institute of Technology, Pasadena, CA 91109; ³The Institute of Genomic Research, Rockville, MD 20850; and ⁴Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824-1325 
zhouj@ornl.gov 

The goal of this project is to explore whole genome sequence information to understand the genetic structure, functions, regulatory networks and mechanisms of dissimilatory metal reduction pathways. The following objectives will be pursued: (1) To identify the genes involved in dissimilatory metal reduction pathways in MR-1; (2) To generate and characterize deletion mutants for defining the functions of the unknown genes expressed under metal reducing conditions; (3) To understand the metabolic and genetic control of gene expression at the genome level under iron reducing conditions; and (4) To explore genetic diversity of the dissimilatory iron reduction pathways in selected thermophilic and psychrophilic iron-reducing bacteria. To achieve these objectives, we will construct microarrays consisting of all ORFs from MR-1, and use them to monitor gene expression patterns under different growth conditions for identifying the genes involved in dissimilatory metal reduction and for defining the putative functions of unknown ORFs. We will also use them to compare the gene expression patterns when MR-1 is shifted from aerobic to anaerobic iron-reducing conditions, and to compare the gene expression patterns between wild type and specific regulatory mutants for understanding the metabolic control and regulatory networks of iron reduction pathways. In addition, we will generate and characterize specific deletion mutants for defining the functions of unknown ORFs. Finally, we will use the microarrays to assay the genetic diversity of iron reduction pathways at the genomic level among representative thermophilic and psychrophilic iron-reducing bacteria. 

To optimize the conditions for microarray hybridization, we are constructing prototype microarrays containing genes involved in anaerobic metabolisms to understand how these genes are regulated under anaerobic conditions. As a part of this project, nine psychrophilic iron-reducing bacteria have also been isolated from Siberia and Alaska permafrost soils, deep marine sediments and Hawaii deep sea water. These bacteria are also able to reduce cobalt, chromium at low temperature. Phylogenetical analysis showed that they are closely related to Shewanella and Vibrio species. In addition, we are using sequences of genes known from mutational studies to be involved in metal reduction (mirAB) as hybridization probes to search for homologues in additional Shewanella species and other metal-reducing bacteria.