H-076. Global Mapping of Transcription Start Sites of Geobacter sulfurreducens

K. Juarez1, A. Mendoza1, L. Olvera1, L. Vega-Alvarado2, B. Taboada2, V. Jiménez3, M. Olvera1, E. Morett1, D. R. Lovley4;
1Inst. de Biotecnologia, Univ. Nacional Autonoma de Mexico, Cuernavaca, Morelos, MEXICO, 2Centro de Ciencias Aplicadas y Desarrollo Tecnologico, Univ. Nacional Autonoma de Mexico, Mexico, MEXICO, 3Centro de Ciencias Genomica, Univ. Nacional Autonoma de Mexico, Cuernavaca, Morelos, MEXICO, 4University of Massachusetts, Amherst, MA.

Geobacteraceae is the predominant family of bacteria during in situ bioremediation of uranium-contaminated groundwater and on the surface of electrodes harvesting energy from a variety of organic sources. The completion of the genome-sequences from multiple members of this group provides the opportunity to apply experimental and computational analyses to obtain new insights into their regulatory processes. Although the availability of the genome sequence of Geobacter sulfurreducens has made it possible to predict operon structure, promoters and transcriptional factors binding sites, only a few have been experimentally determined. While such predictions are based mainly on what is known for other bacteria, the experimental determination of an important set of transcription start sites (TSS) would contribute not only to improving the knowledge about the promoter and operon structure in this bacterium, but also increase the predictive capacity for those cases not experimentally determined. Based on a large scale TSS mapping methodology that we have previously developed for E. coli, which has allowed us to map several hundred TSS in this organism, we implemented two approaches for G. sulfurreducens: directed mapping using a modification of the 5’RACE protocol; and global mapping of TSS using pyrosequencing technology. The modified 5'RACE protocol allowed us to experimentally map new TSS in G. sulfurreducens and confirm some TSS previously reported, demonstrating that this methodology also efficiently works in this organism. The second methodology used consisted of a variation of the standard pyrosequencing methodology to sequence cDNA fragments, this approach allows the clonal amplification of individual cDNA fragments and sequencing till the 5'end, to identify the TSS. This information is being integrated in a reliable database of promoters and transcription start sites of G. sulfurreducens that will ultimately be linked to analyses of transcriptional regulatory systems providing a database that can be easily accessed.