Molecular mechanism of environmental stress sensing by bacterial Zinc-containing Anti-Sigma factors

A critical evolved property of all cells is their ability to sense and respond to environmental change. This is especially true of bacteria that often have to live in inhospitable and fluctuating environments. An active yet still poorly understood area of research is how bacteria sense environmental change and the mechanisms they deploy to respond to such changes. This proposal focuses on these questions in the soil-living organism Streptomyces coelicolor, which is an ideal model for two reasons. First, its genome sequence shows the organism is well armed with genes that encode proteins likely to be involved in responding to environmental stresses but the mode of action of these have yet to be described. Second, the genus Streptomyces is the source of a multitude of commercially important antibiotics, anticancer agents and immunosuppressants, the production of which are likely linked to the organism's ability to respond to environmental changes and so by understanding the underlying mechanisms we may be able to affect the production of such therapeutic molecules. Our proposal focuses on one specific group of stress sensors which are in fact complexes of two proteins: One is a sigma factor that directs the main cellular enzyme responsible for the production of specific RNA molecules (RNA polymerase) to produce proteins which allow the cell to respond to environmental change; the other is an anti-sigma factor that binds to the sigma factor and blocks its ability to bind RNA polymerase. It is the anti-sigma factor's job to sense the environmental stress. It is known that this sensing mechanism involves the disabling of the anti-sigma factor, which releases the sigma factor to coordinate the cellular response to the stress. Although it has been over 15 years since we first described the presence of what is now recognised as a widespread group of ExtraCytoplasmic Function (ECF) sigma factors (Streptomyces alone has over 50 of them encoded in its genome), we still know surprisingly little about how anti-sigma factors bind ECF sigma factors or how environmental stresses disable them. We have recently uncovered the mechanism by which Streptomyces responds to a particular form of oxidative stress (the main causative agent of ageing) known as disulfide stress. Disulfides are covalent bonds formed between the sulfur atoms of two cysteine amino acids, which are ordinarily found in proteins that get secreted from cells (e.g. hormones such as insulin) to help stabilise them in the harsh extracellular environment. Such bonds however are toxic for proteins inside the crowded environment of the cell cytoplasm where they can cause the inactivation of enzymes and the aggregation of proteins. Streptomyces responds to the appearance of intracellular disulfide bonds by inactivating a specific anti-sigma factor (RsrA), which releases its sigma factor, sigma R, to mount an anti-oxidative response. We have determined the three dimensional structure of RsrA in its resting state, i.e. before it binds sigma factor. Comparison to related protein complexes reveals that RsrA engages in a new form of molecular recognition in which the protein essentially turns itself inside-out to bind sigma R. We have also determined the structure of the deactivated form of RsrA in which an internal disulfide blocks the ability of the protein to turn inside-out. This proposal aims to capitalise on these novel observations. We will investigate how RsrA turns inside-out to bind its sigma factor. We rationalise that this mechanism could be the basis for other forms of environmental stress sensing by this large group of cellular regulators. We will therefore uncover the activation signals for a select few anti-sigma factor/sigma factor pairings, which have yet to be studied, and compare them to the RsrA/sigma R complex. Our goal is to determine if the mechanism we have discovered represents a new paradigm in environmental stress sensing in microbes.

The objectives of our proposal will be addressed through a multidisciplinary programme incorporating molecular genetics, functional genomics, biochemistry, biophysics and structural biology. Objective i (JIC/York)- Uncovering the activation mechanisms for novel ZAS protein/sigma factor pairings in S. coelicolor. We will focus on four cytoplasmic ZAS protein/sigma factor complexes that have yet to be characterised and for which activation signals are unknown. By analogy with our past work on RsrA, we will identify these signals by first identifying the regulons for the complexes (through the creation of null mutants, ChIp-on-chip analysis & microarray transcriptional profiling, qRT-PCR). This will provide clues of the activating signals (e.g. oxidative stress responses), which will be tested on reporter constructs in vivo and on isolated (overexpressed, purified) proteins in vitro, the latter provided to York for biochemical and biophysical analysis to test whether IOPPR is the basis for signal perception. JIC will also characterise in vivo responses of RsrA mutants supplied by York that are potentially compromised in signal sensing. Objective ii (York/JIC)- The structure of RsrARed-Zn bound to sigma R from S. coelicolor or a closely related organism determined either by X-ray crystallography or NMR. Crystallization screens of newly produced ZAS/sigma factor complexes from JIC Objective iii (York/JIC)- The mechanistic basis for IOPPR in the RsrARed-Zn/sigma R complex. This will be accomplished through kinetic and thermodynamic investigations of the complex where we will investigate the role of RsrARed-Zn conformational dynamics by NMR and single molecule methods and probe the relationship between fold and function of RsrA residues, especially hydrophobic residues that contribute both to the protein hydrophobic core and the interface with sigma R. These studies will yield mutants that will be supplied to JIC for in vivo testing of disulfide stress responses

WHO WILL BENEFIT FROM THIS RESEARCH? The outputs of this research will be of value to fundamental scientists and to applied scientists working in the pharmaceutical industry. HOW WILL THEY BENEFIT FROM THIS RESEARCH? Streptomyces account for ~80% of commercially important antibiotics, and are also a rich source of other types of bioactive molecules such as anticancer agents and immunosuppressants, currently accounting for ~$40 billion of revenue annually in the pharmaceutical industry worldwide. There is no doubt that the full exploitation of genetic engineering in Streptomyces for the production of both existing and novel compounds will depend on a much better understanding of the physiology of the organism as a whole. The SigR-controlled thioredoxin system is of particular interest in beta-lactam-producing streptomycetes because it plays an important role in maintaining supplies of the reduced substrate for isopenicillin-N-synthase, ACV (the key precursor of most beta-lactam antibiotics). It also seems likely that the production of other streptomycete antibiotics may be influenced by expression of the thioredoxin system, for example through the reduction of the phosphospantetheine arm of polyketide ACPs. The proposed fundamental study will therefore be of direct interest to companies manufacturing streptomycete secondary metabolites. Further, the SigR-RsrA regulatory paradigm was discovered in Streptomyces but was subsequently found to be an important element in the pathogenesis of medically important (but genetically less tractable) actinomycete relatives like Mycobacterium tuberculosis and Corynebacterium diphtheriae, showing this work has commercial and medical relevance through multiple paths. WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH? We will disseminate our results to the academic community through publications and presentations at relevant scientific conferences. We will publish in open access journals wherever practical. Outputs with potential commercial impact will be identified during regular reviews of progress between the collaborating laboratories. Discoveries with potential commercial implications would be discussed (with a view to patenting) with Plant Biosciences Ltd (PBL), a technology transfer company based at JIC that is jointly and equally owned by the Gatsby Charitable Foundation and the JIC. The purpose of PBL is to bring the results of research in plant and microbial sciences at the Centre into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC. Streptomyces research is prominent in PBL's portfolio. As an illustration, two spin-out companies have been established based on JIC Streptomyces group patents: Novacta Biosystems Ltd, founded at JIC in 2003 and now based at Welwyn Garden City Biopark, where it employs about 30 people; and Procarta Biosystems, founded at JIC in 2008. Thus, there are well established routes for delivery of IP arising from Streptomyces research at the JIC. Mark Buttner and the staff working on the grant will participate in the JIC Teacher-Scientist Network (TSN), give public lectures on e.g. antibiotic resistance and the need for new antibiotics, make presentations to the Friends of the JIC, and to the general public through open days, as was held most recently on 13.09.09. JIC has an excellent Communications Department (http://www.jic.ac.uk/corporate/media-and-public/index.htm) and, where appropriate, we will work proactively with them to approach and interact with the press and broadcast media to publicise this scientific area in general and the outputs of the grant. As an illustration, JIC Communications are currently preparing a press release, interviews and other activities around the publication of research involving the Buttner lab into vancomycin resistance, to be published in Nature Chem Biol on 11.04.10.