Tracking Low-Copy Transcription Factors in Living Bacteria:The Case of the lac Repressor

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

Competitive binding of MatP and topoisomerase IV to the MukB hinge domain.

Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the Escherichia coli SMC complex, MukBEF, and matS-bound MatP in the chromosome replication termination region, ter, results in depletion of MukBEF from ter, a process essential for efficient daughter chromosome individualization and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParC2E2), so that their chromosome distribution mirrors that of MukBEF. We demonstrate that MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding, which occurs with the same dimer to dimer stoichiometry. Furthermore, we show that matS DNA competes with the MukB hinge for MatP binding. Cells expressing MukBEF complexes that are mutated at the ParC/MatP binding interface are impaired in ParC binding and have a mild defect in MukBEF function. These data highlight competitive binding as a means of globally regulating MukBEF-topoisomerase IV activity in space and time

Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase

Horizontal gene transfer permits rapid dissemination of genetic elements between individuals in bacterial populations. Transmitted DNA sequences may encode favourable traits. However, if the acquired DNA has an atypical base composition, it can reduce host fitness. Consequently, bacteria have evolved strategies to minimize the harmful effects of foreign genes. Most notably, xenogeneic silencing proteins bind incoming DNA that has a higher AT content than the host genome. An enduring question has been why such sequences are deleterious. Here, we showed that the toxicity of AT-rich DNA in Escherichia coli frequently results from constitutive transcription initiation within the coding regions of genes. Left unchecked, this causes titration of RNA polymerase and a global downshift in host gene expression. Accordingly, a mutation in RNA polymerase that diminished the impact of AT-rich DNA on host fitness reduced transcription from constitutive, but not activator-dependent, promoters

Single-molecule imaging of DNA gyrase activity in living Escherichia coli

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ~12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ~2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ~8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies

Single-molecule studies of DNA-binding proteins in live bacteria

Protein-DNA interactions are critical to many important biological functions, from transcription to DNA replication. To better understand these processes we need to look at molecular details, such as the stoichiometries and binding kinetics of these proteins. However, focusing on the molecular level can miss the bigger picture; we also need to understand how protein-DNA interactions shape the organisation of chromosomes and cause phenotypical changes over the whole cell. In this thesis I describe the construction of a super-resolution fluorescence microscope to image single molecules in live bacteria, and show how analysis with tools like single-particle tracking allow individual proteins specifically bound to DNA to be distinguished from mobile molecules, offering a new perspective on protein-DNA interactions, from the molecular level to the length scale of whole bacterial cells. I detail how I have applied these techniques to answer key questions in transcription, chromosome organisation, and DNA segregation in Escherichia coli. Firstly, I looked at RNA polymerase (RNAP) to study how transcription affects the organisation of the nucleoid. Discriminating specifically bound RNAPs showed that low levels of transcription can occur throughout the nucleoid, but clustering analysis and 3D Structured Illumination Microscopy (SIM) showed that dense clusters of transcribing RNAPs format the nucleoid periphery, indicating a movement of gene loci out of the bulk of DNA as levels of transcription increase. Furthermore, I developed an assay to characterise the search process and non-specific DNA interactions of RNAP, which I also apply to a diverse selection of other DNA-binding proteins. I also characterized the in vivo behaviour of the type II topoisomerase, TopoIV. Imaging both subunits of TopoIV, combined with over-expression of unlabelled subunits, allowed the fraction of functional enzymes to be determined. Measuring the duration of catalytic events indicated that the majority of active TopoIV molecules catalyse decatenation. Finally, I studied MukBEF, an SMC (Structural Maintenance of Chromosomes) complex that acts in chromosome segregation, to show that TopoIV and MukB interact directly in vivo and determine the dissociation constant and turnover of this TopoIV-MukB complex.</p

Single-molecule studies of DNA-binding proteins in live bacteria

Protein-DNA interactions are critical to many important biological functions, from transcription to DNA replication. To better understand these processes we need to look at molecular details, such as the stoichiometries and binding kinetics of these proteins. However, focusing on the molecular level can miss the bigger picture; we also need to understand how protein-DNA interactions shape the organisation of chromosomes and cause phenotypical changes over the whole cell. In this thesis I describe the construction of a super-resolution fluorescence microscope to image single molecules in live bacteria, and show how analysis with tools like single-particle tracking allow individual proteins specifically bound to DNA to be distinguished from mobile molecules, offering a new perspective on protein-DNA interactions, from the molecular level to the length scale of whole bacterial cells. I detail how I have applied these techniques to answer key questions in transcription, chromosome organisation, and DNA segregation in Escherichia coli. Firstly, I looked at RNA polymerase (RNAP) to study how transcription affects the organisation of the nucleoid. Discriminating specifically bound RNAPs showed that low levels of transcription can occur throughout the nucleoid, but clustering analysis and 3D Structured Illumination Microscopy (SIM) showed that dense clusters of transcribing RNAPs format the nucleoid periphery, indicating a movement of gene loci out of the bulk of DNA as levels of transcription increase. Furthermore, I developed an assay to characterise the search process and non-specific DNA interactions of RNAP, which I also apply to a diverse selection of other DNA-binding proteins. I also characterized the in vivo behaviour of the type II topoisomerase, TopoIV. Imaging both subunits of TopoIV, combined with over-expression of unlabelled subunits, allowed the fraction of functional enzymes to be determined. Measuring the duration of catalytic events indicated that the majority of active TopoIV molecules catalyse decatenation. Finally, I studied MukBEF, an SMC (Structural Maintenance of Chromosomes) complex that acts in chromosome segregation, to show that TopoIV and MukB interact directly in vivo and determine the dissociation constant and turnover of this TopoIV-MukB complex.</p

ALICE’s new meeting room

Building 3294 at Point 2, ALICE’s new meeting roo