PNNL

Abstract

The y-proteobacterium Shewanella oneidensis strain MR-1 is a respiratory versatile organism that can reduce a wide range of organics, metals, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the MR-1 genome were annotated as uncharacterized ‘hypothetical’ genes. We implemented an integrative approach using experimental and computational analyses to provide more detailed insight into their function. Global expression studies were conducted using RNA and protein expression profiling of cells cultivated under aerobic, suboxic, and fumaratereducing conditions, phosphate limitation and UV irradiation. transcriptomic and proteomic analyses confidently identified 538 ‘hypothetical’ genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all ‘hypothetical’ proteins). Publicly available analysis tools and databases and our own expression data were applied to improve the annotation of these genes. The annotation results were scored using a seven-category schema that ranked both confidence and precision of the functional assignment. We identified homologs for nearly all of these ‘hypothetical’ proteins (96%), thus allowing us to minimally classify them as ‘conserved proteins’. Computational and/or experimental evidence provided more precise functional assignments for 297 genes (categories 1-4; 55%). These improved functional annotations will significantly widen our understanding of vital cellular processes including signal transduction, ion transport, secondary metabolism, and transcription, as well as structural elements, such as cellular membranes. We propose that this integrative approach offers a viable means to undertake the enormous challenge of characterizing the rapidly growing number of ‘hypothetical’ proteins with each newly sequenced genome.