Interplay of bacterial transcription and chromosome organisation in vivo

Our study uses ultra-sensitive microscopes to observe important processes in the gene expression, which is the path that leads from the genetic information (stored in DNA, the molecule that forms the chromosomes of living organisms) to the manufacturing of proteins (the molecules that make up most of the machines and structures of living cells). Specifically, the work focuses on the process of gene transcription, which is performed by protein machines called RNA polymerases. These tiny biological machines read DNA and copy the information into a messenger molecule (messenger RNA), and ensure that the right genes are expressed at the right time, the right place, and at the required level.

Much of what we know about how RNA polymerase works to transcribe DNA to RNA comes from studies with purified proteins and DNA in the test tube; these involve simple mixtures of RNA polymerase with DNA sequences and accessory transcription proteins that can make transcription faster or slower. However, the mechanisms of transcription in actual living organisms and cells can be very different, due to the myriad of other biological components that are present in cells, and due to the way that the genes are packaged in the "bacterial nucleoid", which is a tightly packed structure made of the bacterial DNA and some of its proteins. An example of the complexity that characterises gene transcription in living cells is the fact that genes that are being transcribed appear to be on the surface of the nucleoid, and not buried deeply into it. Another example of complexity is that RNA polymerases seem to operate in large teams ("clusters"), with the number of team members and the location of the team depending on how many nutrients the cells have in their environment, and how fast they are growing.

To study the process of gene transcription in its natural environment of living cells, and understand how this process is organised and controlled, we will use advanced fluorescence microscopy to look the position of labelled RNA polymerases and specific genes in living bacterial cells. We will use the bacterium Escherichia coli, which is a simple model organism for understanding biological mechanisms. A special feature of our work is that it is performed using a special microscope (a "single-molecule fluorescence microscope"). This microscope is carefully designed to allow detection and monitoring of individual (single) fluorescent molecules inside living cells (as opposed to conventional microscopes that require thousands or millions of fluorescent molecules).

Using our powerful microscope to record movies of the position of individual genes and RNA polymerase molecules, we will see how genes change their position in the cell as they are being transcribed, and analyse the influence of other proteins that are known to control the amount of transcription in cells. We will also study how the individual RNA polymerase teams are organized, how many members the teams have, and how the teams come together or disband. Finally, we will study whether RNAP teams co-operate to work more efficiently, and if this is the case, we will examine how the teams assemble to achieve this high transcription efficiency. Our studies will improve our understanding of how gene expression works in living cells, and help other scientists to build more efficient artificial cells, as well as to develop new pharmaceuticals that will improve health by disabling the RNAP teams of dangerous microbes.

We propose to study the molecular mechanisms controlling the spatial organisation of transcription and RNA polymerase (RNAP) in living bacterial cells. In bacteria, despite the lack of a nuclear envelope and many eukaryotic DNA-packaging proteins, the chromosome is still highly condensed into a structured object, the nucleoid. However, the spatial organization of transcription within the nucleoid, and the interplay between transcription and DNA organization, remain poorly understood. Cell-imaging studies on the subcellular organization of transcription in fixed cells identified large RNAP clusters within fixed E. coli cells, but the precise chromosomal sites, exact RNAP stoichiometry, and spatial organization within these clusters remain elusive. Further live-cell work from our group reported molecular information on the RNAP subcellular localization, and revealed a strong interplay between transcriptional activity and chromosomal organisation, with movement of gene loci out of the nucleoid interior as transcription and translation increase.

We propose to test the hypothesis of gene relocation to the nucleoid surface during the expression of single genes, and study the importance of transcription-translation coupling for gene relocation. We will also engineer E.coli strains with a single ribosomal RNA operon to characterise single unitary transcription clusters in vivo and test the hypothesis that they form on maximally transcribed ribosomal RNA operons. Further, we will use E.coli strains with two ribosomal RNA operons to characterise potential interactions between unitary clusters. Finally, we will examine the presence of inter-cluster interactions and spatial organisation within RNAP "super-clusters", and study their kinetics of assembly. Our tools will be advanced single-molecule tracking and high-resolution imaging in living E.coli cells, with many assays involving multi-colour monitoring of the location of genes and proteins.

Apart from the academic beneficiaries, our proposed interdisciplinary work, which encompasses aspects of engineering, physical, biomedical and life sciences, will also benefit many end-users in the government, hospitals, and industry.

The technology development linked with our work will have many short-to-mid term benefits. The development of cutting-edge single-molecule imaging methods will benefit the scientific instrumentation and microscopy industry, since industrial teams can adapt our bacterial-imaging methods, software, and assays (cell segmentation, particle tracking, diffusion analysis, clustering, protein counting) to make them available to users interested in studying or detecting bacteria at the single-cell level and at single-molecule sensitivity, with long-term effects on our ability to diagnose, control, and eliminate devastating diseases caused by deadly bacteria.

Our quest for higher temporal and spatial resolution while operating at the maximum possible sensitivity of a single molecule will also inspire novel microscopes and optics, which can lead to commercialization of these technologies, leading to jobs and wealth creation. The wide availability of such technologies (along with biosensors and diagnostic assays) will in turn benefit researchers, health services and the general public through improved healthcare and well-being.

Our single-molecule and single-cell assays should benefit scientists and engineers in clinical labs and in the biotechnology industry (microscopists, microbiologists, and biochemists) who develop rapid antimicrobial susceptibility tests; such assays are central for evaluating antibiotic resistance and selecting the best treatment for patients suffering from bacterial infections. As a result, our work will should benefit the general public by improving public health in the mid-term.

Our work will help the development of biosensors for bacterial detection, benefiting many clinical, biotechnological, environmental, and biosecurity laboratories. These biosensors can be introduced into cells, or may constitute engineered bacterial cell lines produced through a synthetic-biology path.

Our novel fluorescent labelling of DNA, coupled with microscopy and image-analysis tools for single-cell biology, will benefit many applied biology sectors, including systems biology, and synthetic biology. Synthetic biologists and biotechnologists will benefit by our understanding of how RNA polymerase assemblies maximize the production of RNAs and proteins in vivo, both from experimental and theoretical perspective. Our sophisticated analysis of large data sets, especially images, should inspire industrial computational scientists and software engineers, who develop efficient algorithms to process "big data".

Our study is also an example of basic bioscience underpinning health, since it will lead to long-term health benefits due to improved rational therapeutic strategies and new antibiotics which target RNA polymerase and its assemblies in bacteria; such therapies are sorely needed due to the alarming levels of antibiotic resistance, and will benefit the pharmaceutical industry and their stakeholders. Understanding the basic mechanisms of bacteria and providing new ways to study their organization and regulation will lead to long-term advances in agriculture, since many bacteria are involved in a wide range of agricultural practices.

The PDRA involved in this work will interact with researchers from physical and biological backgrounds and acquire important skills desirable in academic, industrial or clinical environments including skills in molecular biology, assay development, statistical analysis, image analysis, and data mining. The proposed work will also strengthen the connection between an academic lab and an SME in the area of fluorescence microscopy.