Bacterial chromosome structure and transcription

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

Bacteria are microscopic free living organisms that are found nearly everywhere on earth, including in the human body. Their actions have big impacts on the environment at all levels and they also affect human health and happiness. Bacterial cells are organised in a different way to animal cells, notably with respect to how they handle their DNA. In animal cells, the DNA is packaged into individual chromosomes that are kept in a separate membrane-bound compartment of the cell called the nucleus. For most bacteria, their DNA consists of millions of base pairs in a single chromosome that is free in the main cell compartment. This creates a logistic problem since bacterial cells are small and, in order to fit the DNA into the cell, it has to be highly compacted by folding. Microscopy studies have shown that, in many bacteria, the chromosome is restricted to a part of the cell called the nucleoid. We are interested in how proteins interact with bacterial chromosome DNA in order to compact it into the nucleoid, and over a dozen different proteins that contribute to the compaction have now been identified. Whilst we understand the actions of many of these proteins when bound at individual DNA targets, we have little idea how these proteins act together on a bigger scale to organise DNA in the bacterial nucleoid.

This proposal is prompted by the recent discovery of specific locations on the chromosome of a common bacterium, Escherichia coli, where the amount of bound protein is especially high. It has been suggested that these highly occupied targets act as the organising centres of the nucleoid by clustering together segments from different parts of the chromosome. It is thought that this clustering is essential to the compaction of the Escherichia coli chromosome and that similar mechanisms operate in most bacteria. Hence our aim is to identify the proteins that bind at these targets and start to build up a detailed protein occupancy map of the Escherichia coli chromosome. To achieve this, we will exploit a newly developed method called DNA sampling. Having identified the proteins that bind at different targets, we next want to build up a DNA proximity map by identifying chromosome segments that are far apart in the DNA sequence but clustered together in the 3-dimensional space of the nucleoid. One of the problems with doing this is that bacterial nucleoids are not fixed structures and each locus on the DNA may well make short-lived interactions with many other loci. Hence, to capture transient interactions, we will use a method called chromatin conformation capture, and, by combining it with high throughput sequencing, we will be able to record the different interactions. Taken together, this information will allow us to build up a picture of the different interactions that hold the Escherichia coli nucleoid together.

Finally, we will investigate the possibility that the folding of gene DNA into a bacterial nucleoid affects its ability to be expressed. This is most likely because the folding restricts the accessibility of certain DNA elements that must be recognised by the proteins that initiate gene expression. We already have some preliminary data to show that this is the case for some of the regions of high protein binding. Hence, we are planning to use state-of-the-art fluorescence microscopy to find out where these transcriptionally silent loci are positioned in the nucleoid. These experiments will provide important information for modellers who want to predict patterns of expression from the DNA base sequence of any bacterium.

Technical Summary

Recent work has identified specific regions of the Escherichia coli chromosome that are transcriptionally silent but have high protein binding, and it has been suggested that these act as organising centres for the folding and compaction of DNA into the nucleoid. These will be used as a starting point for a study that aims to build a detailed protein occupancy map of the E. coli chromosome, a DNA proximity map of the nucleoid, together with transcriptional and accessibility maps.

We propose to exploit our newly developed 'DNA sampling' methodology to identfy the proteins present in at least a dozen high protein binding regions. Chromatin immunoprecipitation in combination with high throughput sequencing will then be used to identify DNA segments that cluster at these locations. In parallel experiments, we will develop a combination of chromatin conformation capture (3C) and high throughput sequencing to locate DNA segments that are far apart on the 1-dimensional chromosome base sequence but close in the 3-dimensional space of the nucleoid. This will establish a DNA proximity map for the whole E. coli chromosome.

In preliminary work, using our 'gene doctoring' chromosome engineering technique, we have exploited a novel lactose operon promoter::gfp fusion to show that certain locations in the E. coli chromosome are unfavorable for gene expression. We will extend these studies and exploit fluorescence microscopy directly to visualise locations within the nucleoid where expression is disfavoured or favoured.

Overall, the research will provide insights into bacterial nucleoid organisation, identify new targets for anti-bacterial therapies and provide a framework for predictions of gene expression patterns from whole genome base sequences.

Planned Impact

This proposal will have high impact as it will open up completely new aspects of nucleoid organisation. This impact will be due to the fact that the research will elucidate novel complexes that play a key role in bacterial well being and their consequences on gene expression. Thus, the completion of the proposed research should benefit those wishing to understand and manipulate the processes of gene regulation. The study of high protein occupancy segments of a bacterial chromosome will also reveal potential targets for new antimicrobials and hence there will be benefits to public health and wellbeing in the longer term. As detailed in the previous section, the work will benefit a number of individuals worldwide that study bacterial transcription. It should also have wider academic benefit in areas such as nucleic acid-protein interactions, computational modelling of cell-based systems and developmental biology.

E. coli is used extensively as a 'factory' for the production of heterologous proteins, including those with commercial and clinical value. Thus, understanding its nucleoid has potential economic impact on the nation's wealth in the medium term. Beneficiaries include the commercial private sector that produces proteins using recombinant DNA technology and the wider public through improved health and wellbeing.

IP stemming from this proposal will be managed by the applicants with the assistance of University of Birmingham Research and Commercial Services (RCS: http://www.rcs.bham.ac.uk/) that not only offers expertise in the identification of novel intellectual property with commercial potential, but seed capital to finance spin out companies and ongoing strategic and financial support to maximise the chances of success.

The applicants will also disseminate their findings, when appropriate, through publication in scientific journals and presentations at national and international meetings. The findings will also be disseminated to the general public through newspaper articles, university open days and engagement with local schools or youth organisations. The applicants have all participated in these activities.

Publications

10 25 50
 
Description We have discovered that gene expression in bacteria is dependent on the location of the gene in the bacterium as well as on its nature. In our most recent work, we have discovered that this is because the supply of RNA polymerase differs at different locations in the E. coli cell. This becomes critical in rapidly growing cells. Right at the end of the project, we were able to show that certain genetic loci in E coli move physically when they are transcribed and this was published as Sellars et al in 2017.
Most recently, we have found that our findings affect the expression of antibiotic resistance determinants
Exploitation Route Our results are important for those who seek to exploit bacteria as factories, and also have important consequences for computational biologists who try to model bacterial gene expression.

Most recently, Peter Freddolino's group has managed to apply a high throughput approach to understanding the effects of location on bacterial transcription.His publication is a stunning confirmation of our published work:
Scholz SA, Diao R, Wolfe MB, Fivenson EM, Lin XN, Freddolino PL.
High-Resolution Mapping of the Escherichia coli Chromosome Reveals Positions of
High and Low Transcription. Cell Syst. 2019 Mar 27;8(3):212-225.e9. doi:
10.1016/j.cels.2019.02.004. Epub 2019 Mar 20. PubMed PMID: 30904377; PubMed
Central PMCID: PMC6508686.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.ncbi.nlm.nih.gov/pubmed/?term=busby+sj
 
Description They have altered the way we think about bacteria They have contributed to the development of a SynBio project funded by the EEC FP7 prtogramme and a Catalyst grant
First Year Of Impact 2015
Sector Environment,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Economic

 
Title Providing strains, methods 
Description We provided contructs to facilitate others who wanted to engineer their bacteria 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact Lots of groups use materials derived from our lab and this has continued after the award finished 
 
Title Collaboration with RegulonDB team in Cuernavaca, Mexico 
Description Together, we have been developing methods for factoring new-generation data into Regulon DB 
Type Of Material Data handling & control 
Year Produced 2018 
Provided To Others? Yes  
Impact Together with a consortium of users, we have submitted a position paper to Nature Reviews genetics: it is currently in revision 
URL http://regulondb.ccg.unam.mx/
 
Description Collaboration with Indian Institute of Science, Bangalore 
Organisation Indian Institute of Science Bangalore
Department Department of Microbiology & Cell Biology
Country India 
Sector Academic/University 
PI Contribution Our researches concerning nucleoid structure and function in E. coli complement those of our partners in Bangalore who are working on Mycobacteria
Collaborator Contribution Our partners in Bangalore are working on Mycobacterial nucleoid proteins
Impact Joint publication
Start Year 2012
 
Description Public Understanding of Science activities 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact During the period of the grant, I did 6 Schools visits, mainly talking to 6th forms. Different Schools had different formats. I also gave a presentation to local Biology Teachers (organised by the Royal Society of Biology)
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016,2017