Visualising a bacterial stress response: gene product localisations movements and gene regulation at single molecule level in live bacterial cells

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute

Abstract

Bacteria comprise one of the most abundant life forms on the planet, and contribute hugely to, for example, the major carbon, nitrogen, sulphur and phosporus cycles and transformations that characterise a functional and healthy global living environment. Despite huge advances in understanding how the components of the bacterial cell work, little is known about how they are organised within the living cell, nor how such organisations impact upon the functionality of the cell. Using a method that enables us to visualise single molecules within living cells, we will look in a quantitative way at the dynamics of the cellular components that let the cell survive a stress that damages its membrane. In effect we will be studying the cell in action, and will be able to describe the cell in its pre-stressed state as well as in its adapted state. Gaining an appreciation and understanding of how the cells components are organised for function is important in establishing principles of signaling and the networks of interactions that allow cells to grow and adapt to new environments.

Technical Summary

To date, much of our in depth understanding of cell functionality has been at the level of the detailed biochemical/biophysical characterisations of key cellular components. Advances in the field of single molecule, single cell imaging now make it possible to obtain quantitative information on the cellular localisations, interactions, dynamics and gene expressions of key effector and control proteins in important microbial cells. We plan to examine at the single molecule and single cell level how the E.coli cell adapts to stress that causes its inner membrane to lose integrity. This stress is important in a range of significant contexts, including protein export scenarios that support virulence in bacteria. One protein we will study, PspA, has a homologue in VIPP1, needed for thylakoid biogenesis. We will study in vivo (i) the states of self association of PspA and its localisations, to understand how it switches from negative regulator to effector, (ii) the localisations and dynamics of the effector protein PspG, the localisations and self associations of the transcriptional control protein PspF, and finally (iii) the expression dynamics of the pspG promoter. The latter will for the first time allow us to determine if expression of an activated and complexly controlled promoter does or does not show the geometric distribution of expression bursts seen with simple repression control as in lac. At present it is even unknown whether expression bursts are generally seen or not. The gene expression studies are important in understanding how variations in expression levels between cells might be caused, and for deducing whether different types of gene regulation strategies give rise to rather different types of outputs. Different types of outputs may well be better suited to some purposes than others, dependepending upon the type of gene product being produced.

Publications

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Gilburt JAH (2017) Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy. in Angewandte Chemie (International ed. in English)

 
Description This grant has enabled us to develop an approach based on the imaging of the photobleaching steps of a fluorescent protein tagged protein complex to determine its stoichiometry in living cells. We have found that a stable repressive PspF-PspA complex is located in the nucleoid of an E. coli cell, transiently
communicating with the inner membrane via PspA. We have also established that the PspF as a hexamer stably binds only one of the two psp promoters at a time, suggesting that psp promoters will fire asynchronously.
Exploitation Route The code for protein complex stoichiometry analysis based on single molecule photobleaching has been used by other labs in the US and UK.
Sectors Education

Environment

Pharmaceuticals and Medical Biotechnology

 
Description The code developed for the measurement of the stoichiometry of protein complexes in living cells has been adopted by several other labs in the US and UK.
First Year Of Impact 2011
Sector Education,Environment,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Economic

 
Description Institute of Chemical Biology DTC PhD Studentship 2 (Imperial College London)
Amount £85,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 08/2013 
End 09/2017
 
Description Project grant/Leverhulme Trust (co-PI)
Amount £241,000 (GBP)
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2013 
End 01/2016
 
Title Single molecule data for unphosphorylated Aurora-A (Gilburt et al, Chemical Science 2019) 
Description Raw and partially processed single molecule intensity histogram and dwell time histogram data for the following publication: James A H Gilburt, Paul Girvan, Julian Blagg, Liming Ying, Charlotte A Dodson (2019) Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation. Chemical Science. DOI: 10.1039/c8sc03669a Please cite our publication in any use of this data. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
 
Description Application of CRISPR-Cas9 Technology in Fast Detection of Transgenic Products 
Organisation Chinese Academy of Sciences
Department Institute for Nutritional Sciences
Country China 
Sector Learned Society 
PI Contribution Investigate the molecular mechanisms and corresponding thermodynamic parameters of the detection system using single-molecule and other biophysical techniques.
Collaborator Contribution 1. Prepare main components of the detection system, including Spy-dCas9 protein, specific sgRNA, double-stranded target DNA, DNA-RNA hybrid antibody, and so on. 2. Confirm the effectiveness of the detection system by in vitro approaches, and modify the reaction conditions. 3. In cooperation with the partner, further improve the specificity and sensitivity of the detection system based on its molecular mechanisms. 4. Quantitatively detect the heterologous gene fragments in samples from transgenic products by applying the system.
Impact multi-disciplinary, biosensor/biophysics/spectroscopy
Start Year 2016
 
Description Collaboration with Guangdong University of Technology, China 
Organisation Guangdong University of Technology
Department Institute of Biomedical and Pharmaceutical Sciences
Country China 
Sector Academic/University 
PI Contribution Provide research facilities to express, purify and label protein. Host a postdoctoral researcher form the partner organisation to carry out single molecule fluorescence measurement.
Collaborator Contribution Salary of the postdoctoral researcher
Impact Multi-disciplinary: biochemistry, biophysics, drug discovery
Start Year 2017
 
Description Structural and functional characterisation of a G-quadruplex in the promoter of ß1-adrenergic receptor 
Organisation Chinese Academy of Sciences
Department Shenzhen Institute of Advanced Integration Technology
Country China 
Sector Academic/University 
PI Contribution Provide guidance for the project and carry out single molecule FRET measurements.
Collaborator Contribution Cell biology and other biophysical measurements
Impact Multi-disciplinary, biophysics/biochemistry/cell biology/cardiovascular science
Start Year 2016
 
Title LABELLED KINASES FOR DRUG DISCOVERY 
Description A kinase or kinase fragment comprising a first label and a second label, wherein: the kinase or kinase fragment has a first conformation and second conformation; one or both of the first and second label are fluorophores; the first label and second label comprise an interacting pair capable of interacting with each other by static quenching or Dexter quenching or PET (photoinduced electron transfer) or exciplex formation to produce a quenched pair; and wherein the first and second labels are positioned on the kinase or kinase fragment such that when the kinase or kinase fragment is in the first conformation the labels are distal to each other such that static quenching or PET or Dexter quenching or exciplex formation does not occur and when the kinase or kinase fragment is in the second conformation the labels are brought into close proximity such that static quenching or PET or Dexter quenching or exciplex formation of one or both labels occurs. 
IP Reference WO2018167502 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact The new tool is expected to be widely applicable to the kinase drug development program.
 
Description Guest speaker of Netherhall House 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A public lecture titled from DNA double helix to personalised medicine

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Invited seminar/Peking University 2009 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Seeing is believing: single molecule fluorescence approaches to biology and medicine, Department of Chemical Biology, Peking University, Beijing, July 2009

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description Invited seminar/University of Cambridge 2009 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Seeing is believing: single molecule fluorescence approaches to biology and medicine, Department of Chemistry, University of Cambridge, July 2009.

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description Invited seminar/University of Warwick 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Participants in your research or patient groups
Results and Impact Seeing Is Believing: Single Molecule Fluorescence Approaches to Biology, Warwick Mathematics Institute, University of Warwick, May 2010.

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Visit to Guangzhou Zengcheng Economic and technological Development Zone and short presentation to the business leaders and managing team of the Development Zone 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Visiting R&D facilities, give short presentation about translational research activities of the group
Year(s) Of Engagement Activity 2016
 
Description Visit to Hainan Medical School, Haikou, China 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact short presentation of research and dialogue with leaders, doctors and graduate students of the medical school
Year(s) Of Engagement Activity 2016
 
Description Visit to Sanya People's Hospital 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Visit research labs and patient wards, science exchange via short presentations. This activity marks the beginning of the Academic Platform of the Hospital.
Year(s) Of Engagement Activity 2016