K-117. In Vivo Footprinting Analysis of Carbon Metabolism in Shewanella oneidensis MR-1

Y. Liang1, J. Chen1, H. Gao1, K. H. Nealson2, J. M. Tiedje3, J. K. Fredrickson4, J. Zhou1;
1Univ. of Oklahoma, Norman, OK, 2Univ. of Southern California, Los Angeles, CA, 3Michigan State Univ., East Lansing, MI, 4Pacific Northwest Natl. Lab., Richland, WA.

Most of studies on Shewanella oneidensis MR-1 have focused on anaerobic respiration, especially metal reduction and related biological pathways because it is distinguished by its ability to use an array of compounds as electron acceptors. Little has been done to decipher the bacterial aerobic metabolism, especially carbon utilization. Given that transposon mutagenesis has been well established in S. oneidensis, genetic footprinting appears to a good choice for assaying gene function on a genomewide scale. In this report, we describe the development of genetic footprinting in S. oneidensis based on in vivo transposon mutagenesis and in vitro T7 transcription. With the approach, we carry out a genome-wide, comprehensive experimental assessment of the S. oneidensis MR-1 genes important for aerobic growth on lactate or glycyl-glutamate. Meanwhile, conventional microarray hybridization was employed to provide a control for evaluating the efficacy of this approach at the whole-genome scale. Potential target pathways were deduced from the footprinting data. Compared to the data from conventional transcriptional analysis, conserved and indispensable functions revealed by the footprinting analysis were reaffirmed for further analysis. Overall, signal intensity of over 400 hundreds of genes was found to be significantly altered by either method, suggesting a global impact by different carbon sources. The major categories of these genes included energy metabolism, protein fate, and transportation. The most promising targets are validated by direct knockouts and functional analysis of the resulting mutants.