Understanding the role of small regulatory RNAs in the Gram-positive model organism Bacillus subtilis

Bacillus subtilis is a bacterium that lives in the soil and is important in some industrial processes. It has been widely adopted by scientists as a "model organism", delivering general insights into how bacterial cells function. Working on B. subtilis brings many benefits: it is not dangerous; it can be easily manipulated to allow in-depth studies; many aspects of its biology are very well understood allowing observations to be put into context. Insights obtained from work on B. subtilis are of relevance to a wide range of related bacteria, including pathogens that cause infections in humans and animals, such as Staphylococcus aureus (including MRSA), Clostridium difficile and the anthrax bacillus.

B. subtilis, like many other bacteria, faces an uncertain environment and therefore has to regulate the expression of genes and synthesis of proteins to meet changing circumstances. For many years the central dogma of molecular biology has been that "DNA makes RNA makes Protein", with the assumption that interactions between proteins and DNA control the production of RNA, which then makes protein. However, we now recognise, from studies on a range of organisms from humans to bacteria, the existence a wide variety of "non-coding RNAs" that are not directly responsible for protein production, but instead regulate gene expression in a number of ways. Small regulatory RNAs are a group of non-coding RNAs that bind to other RNA molecules and regulate their function.

Through a pioneering study of gene expression in B. subtilis, which was published in the prestigious Science magazine, the applicant and her collaborators have identified over 150 small regulatory RNAs in this organism. This provides us with a timely and exciting opportunity to investigate the function of these newly discovered RNAs, exploiting a range of innovative high-throughput molecular-biology approaches, twinned with access to cutting-edge high-throughput sequencing technologies, to shed light on this new regulatory landscape. This research will fill a significant gap in our current knowledge for an important bacterial species, with the potential to enhance our ability to manipulate the organism during industrial processes and to improve our understanding of related pathogenic organisms.

Non-coding RNAs represent an overlooked level of gene regulation, over and above that played by proteins, with an elegant complexity that impacts on many poorly understood aspects of bacterial cell biology. Determining the function of ncRNAs during growth and development represents one of the primary challenges of 21st Century biology.

Building on a landmark study published in Science magazine, which revealed the transcriptional landscape of Bacillus subtilis in >100 different conditions and identified >1500 non-coding segments, including 153 potential small regulatory RNAs (srRNAs)--the applicant is uniquely well placed to exploit a timely opportunity to advance our understanding of a key model organism and shed light on how these newly discovered srRNAs contribute to transcriptional and post-transcriptional regulation.

We have developed an analytical pipeline that allows us to predict targets of srRNAs. In this project, we will create a bank of deletion mutants in srRNAs, along with complemented strains, that will provide a useful resource for the research community. We will perform high-throughput phenotyping of srRNA mutants (including some already in our possession), iteratively refining our predictions and models. We will develop a system for transient expression of srRNAs suitable for use in Gram+ bacteria. We will perform RNASeq to determine effects of srRNA overexpression on the transcriptome. We will exploit translational fusions and other approaches to investigate the mechanistic basis and functional significance of srRNA-target interactions.

B. subtilis is an ideal organism for this study, given the vast corpus of knowledge and the extensive set of tools already available. The strains, reagents and protocols generated during this project will contribute to the tool kit for this model organism, while the novel insights we are certain to gain will have implications for bacteriology, systems biology, synthetic biology and for biology as a whole.

Regulation is the fundamental basis for all cell functions. This proposal aims to understand how bacteria use their specific regulatory tool of non-coding RNA to control responses to cellular and environmental perturbation. Non-coding RNAs have been identified in the past decade in all species where they have been looked for and to gain a holistic understanding of all regulatory events it is important that their function is fully explored.

The immediate audience for the knowledge that will be generated is the scientific community. A large active research community based both in the UK, Europe and the rest of the world, use Bacillus subtilis to explore basic research questions and also for applied research. In industry this organism acts as an important biological workhorse. The knowledge that will be generated in understanding how ncRNAs impact on Bacillus biology will be of interest to both groups, as understanding regulation is not only key to producing target molecules in an industrial setting, but also crucial for academic scientists interested in understanding cell function.

The Bacillus Industrial Platform (BACIP) is a collection of European industrial companies who use Bacillus as a key production host for a range of biotechnological applications. They share a keen interest in understanding how Bacillus functions and in further exploiting it for production. Although no direct product is expected from this research, this group are likely to be interested in the data as they look for new manners by which to manage their production hosts. Using specific srRNAs it may be possible to control RNA decay rates and in doing so increase numbers of mRNA molecules in the cell, therefore resulting in an increase in production. BACIP support the annual meeting of BACELL that is held within Europe and brings scientists and industry together. The work will be showcased by the PDRA and the PI at this meeting, the biannual Gram-positive genomics meeting and other meetings where it is seen to be relevant. Attending the meetings will also be an ideal opportunity to develop new collaborations.

The knowledge that will be generated by the research proposed here will be published in leading international journals, to ensure that research is seen by the widest audience. The PI will give invited seminars, participate in outreach within schools and participate in university open days. The project will train a PDRA in a wide range of areas including manipulating and handling B. subtilis, analysis of large integrated data sets and in skills in project management. The research will offer a number of spin off projects that will act as research work for bachelors or masters students.