Bacterial chromosome structure and transcription

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.

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.

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.