Controlling chromosome structure in starved bacteria

DNA can be envisaged as a long piece of string and, just like string, if not carefully wound, DNA becomes a tangled mess. This presents a problem for living organisms because they have to package their string of DNA within the confines of a small compartment (the cell). Cells have evolved numerous mechanisms to package DNA and, for more complicated organisms (for example humans and plants) we have a reasonably good understanding of how these mechanisms work. However, our understanding of chromosome folding in bacteria (such as the familiar organisms E. coli and Salmonella) is less well developed. In our work we propose to identify mechanisms used by bacteria to organise their DNA. In particular we will pay attention to DNA organisation in starved bacteria. This is important because, in starved bacteria, the DNA string is packaged much more tightly than normal. It is believed that this is a protection mechanism to ensure that DNA is not damaged in the harsh environments bacteria may encounter when not growing in optimal conditions. For example, E. coli transferred to a chopping board from contaminated meat would employ such protection mechanisms until it encountered a more favourable environment (i.e. the gut of you or I). Our study will focus on a protein called Curved DNA Binding Protein A (CbpA) that binds to the DNA and is produced by E. coli cells only when they are starved. Our goals are to determine, exactly how CbpA structures DNA, which parts of the DNA are associated with CbpA, and how CbpA activity is regulated. Because CbpA is also produced by bacteria related to E. coli, such as Salmonella, our findings should also be applicable to these organisms.

All living organisms must organise their DNA content within the confines in the cell. The way in which DNA is organised influences gene expression, chromosome segregation, DNA replication and thus all aspects of cell biology. Staggeringly, despite having determined how DNA is organised in many higher organisms, we still have little understanding of the DNA folding processes that drive chromosome condensation in bacteria. The emergence of bionanotechnology and large scale genomic techniques, as tools for dissecting the characteristics of bacterial chromosomes, will be exploited in this application. Recent work has shown that Escherichia coli, and other bacteria, radically re-model their chromosomes in response to environmental stress. This is most notable during periods of starvation, when the chromosome is super compacted. The E. coli Curved DNA Binding Protein A (CbpA) is present at 16,000 copies per cell during periods of starvation, is associated with the folded bacterial chromosome, and is conserved in many proteobacteria. Despite this we know little about the function of the CbpA protein. Thus, the overriding aim of this work is to better understand how the chromosome of starved E. coli cells is structured by CbpA. Our two primary goals are to determine how CbpA packages the DNA and which areas of the chromosome are bound with CbpA. We will use a combination of nano-scale imaging and high throughput genomic technologies to answer these questions. Atomic Force Microscopy will be used to visualise purified CbpA-DNA complexes in vitro and Chromatin Immunoprecipitation, coupled with DNA microarray analysis, will be exploited to measure chromosome-wide DNA binding by CbpA in vivo. These avenues of research will be supplemented by studies to determine how CbpA activity is regulated, both at the transcriptional and post-translational level. We expect to identify new DNA-folding mechanisms employed by bacteria to survive in harsh environments.

We anticipate that, by applying a unique combination of exciting new techniques to a fundamental biological question, we will maximise the impact of our research. Three main groups will benefit from the work described here (detailed below). Academic: The most obvious group to benefit from our work will, of course, be the world-wide academic research community. Firstly, because bacterial chromosome structure is so poorly understood, we expect to identify new DNA folding and stress response mechanisms applicable to many bacterial species. Secondly, the techniques that we will use are beginning to have a great impact on the way in which bacterial DNA binding proteins are studied. Hence we expect that the technical advancements made during our study will be of great interest. Finally, the project will specifically benefit the post-doctoral researcher employed to undertake the laboratory work. There is an increasing need for multidisciplinary researchers at the biophysical-life science interface and the training provided by this project will provide the successful candidate with a fantastic set of cutting edge skills. The primary mechanisms of engagement with the academic community will be the attendance of research conferences and the publication of our data in high-profile journals. The impact of our work on this particular group is therefore likely to be rapid. Industrial: My group has a longstanding relationship with Oxford Gene technology (OGT) a commercial DNA microarray fabrication company. We have worked closely with OGT for 5 years to design and promote the use of high-density DNA microarrays to study protein-DNA interactions in bacteria. We expect to continue our relationship with OGT to further develop this technology and promote its application. Because our relationship with OGT is well-developed the impact of this collaboration will be rapid and will likely involve the production of new commercial tools for studying bacteria. In addition to existing collaborations, we fully expect to engage with new industrial partners through our nano-scale imaging of protein-DNA complexes. The nanotechnology industry is growing rapidly and it is clear that biological molecules can be utilised to design nano-machinery. We will exploit our expertise in this area to capture the interest of nanotech companies. Links will be forged by attending appropriate international meetings and by exploiting the publicity machinery of the University. We expect that the impact of our work on the bionanotechnology industry is likely to come to fruition in the medium to long term as nanotechnology becomes a more frequently encountered tool. Public Sector: Understanding the mechanisms employed by bacteria to survive harsh conditions is of key importance when designing strategies to control these organisms. Therefore, we also expect that our work will be of interest to government funded organisations responsible for the management of disease. In addition, we will contribute to the development of technology to better characterise the response of bacteria to anti-microbial agents. Through our work with OGT (described above), genomic techniques for studying DNA binding in bacteria are beginning to find application in the study of medically important micro-organisms such as Mycobacterium tuberculosis, where gene regulatory proteins involved in the transcriptional response to beta-lactam antibiotics have been studied. Communication of our work will be facilitated by publication in appropriate high-impact journals and presentation to a wider audience via the media outlets of the University.