Abstract
The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology.
Highlights
Encoded reporters have revolutionized the study of bacterial protein-protein interactions
To explore the utility of split Renilla luciferase (Rluc) complementation for probing dynamic protein-protein interactions in V. cholerae, we applied the technology toward the goal of developing a novel chemotaxis assay that could in principle enable rapid, quantitative measurements of chemotaxis in response to chemotactic stimuli
Since previous BRET- and FRET-based analyses of chemotactic signaling in E. coli [14,23] demonstrated that the interaction of the CheY and CheZ proteins is directly proportional to chemotaxis pathway activity, we reasoned that fusion of Rluc fragments to homologous proteins from V. cholerae (CheY3 and CheZ, respectively) would enable a direct measure of chemotactic responses
Summary
Encoded reporters have revolutionized the study of bacterial protein-protein interactions. Protein fragment complementation assays (PCA), in which two inactive fragments of a monomeric reporter, such as bgalactosidase or green fluorescent protein, are fused to interacting proteins, have been used to measure binding interactions [2,3]. Both of these approaches typically suffer from limitations (e.g., lag time of transcriptional activation or fluorescent protein maturation, irreversible signal amplification over time) that preclude real-time analyses of dynamic interactions. A tractable assay that enables quantitative and sensitive measurements of dynamic interactions in real-time would be a powerful addition to the existing toolkit for studies of bacterial protein interactions
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