Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling. 1. Introduction The pursuit of a systems-level understanding of bacterial physiology requires not only knowledge about the identity, function, and relative abundance of proteins, but also insight into the subcellular localization of these proteins. Subcellular protein localization is linked to protein function, potential protein-protein interactions, and to interactions between a cell and its exterior environment. The observation of proteins in unexpected cellular compartments gives clues about the presence of possible alternate functions. Hence, there is a growing appreciation for the presence of bacterial “moonlighting proteins,” that is, those proteins that have a secondary function depending on subcellular location [1–3]. Experimentally verified localization also provides a foundation for describing proteins that are “hypothetical,” uncharacterized, or that contain domains of unknown function. Furthermore, with the increasing use of systems biology approaches, including genome-scale models of metabolism [4] and regulation to study microbial functions, experimentally founded protein localization on a global scale is necessary to produce more accurate model constraints. Subcellular proteomics has emerged as
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