title: Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.
abstract: Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track single LacI molecules in live cells by stochastically photoactivating and observing fluorescent proteins individually, there are no limitations on the copy number of the protein under study; as a result, we were able to study the behavior of LacI in bacterial strains containing the natural copy numbers (∼40 monomers), as well as in strains with much higher copy numbers due to LacI overexpression. Our results allowed us to determine the relative abundance of specific, near-specific, and non-specific DNA binding modes of LacI in vivo, showing that all these modes are operational inside living cells. Further, we examined the spatial distribution of LacI in live cells, confirming its specific binding to lac operator regions on the chromosome; we also showed that mobile LacI molecules explore the bacterial nucleoid in a way similar to exploration by other DNA-binding proteins. Our work also provides an example of applying tracking photoactivated localization microscopy to studies of low-copy-number proteins in living bacteria.
authors.affiliation: Gene Machines Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom.
authors.forename: Federico
authors.initials: F
authors.lastname: Garza de Leon
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: Sellars
authors.affiliation: Gene Machines Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom.
authors.forename: Mathew
authors.initials: M
authors.lastname: Stracy
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Stephen J W
authors.initials: SJW
authors.lastname: Busby
authors.affiliation: Gene Machines Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom. Electronic address: kapanidis@physics.ox.ac.uk.
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D058948:Cell Tracking; D002876:Chromosomes, Bacterial / Q000378:metabolism; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism*; D005453:Fluorescence; D018628:Gene Dosage; D056426:Genetic Loci; D056968:Lac Repressors / Q000378:metabolism*; D050296:Microbial Viability; D008853:Microscopy; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D056968:Lac Repressors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors