A novel mechanism of translational regulation in bacteria: de-repression of non-canonical start codons in response to oxidative stress

The harmless soil bacteria called streptomycetes are vital to human welfare because they are the source of the vast majority of antibiotics used by doctors to cure infectious diseases, as well as providing us with numerous other medicines used, for example, to treat cancer, and to help organ transplant patients. We have identified a master regulator (called a 'transcription factor') that switches the genetic machinery of these useful bacteria to allow them to survive in the presence of toxic molecules called Reactive Oxygen Species (a condition called "oxidative stress"). The aim of this work is to find out exactly how the activity of this master regulator is controlled at the molecular level. We also know that this same master regulator controls resistance to oxidative stress in the pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, the bacteria that cause the fatal diseases tuberculosis and diphtheria, respectively. This is important because white blood cells ingest pathogenic bacteria and expose them to Reactive Oxygen Species in order to kill them, and so the mechanisms by which disease-causing bacteria like M. tuberculosis and C. diphtheriae resist this oxidative killing are potentially important to human health.

The objectives will be addressed through a multidisciplinary programme incorporating genetics, functional genomics and biochemistry.

OBJECTIVE 1. DOES IF3 REPRESS SIGR TRANSLATION?
We will construct an IF3 controlled depletion strain either (i) by placing infC under the control of the tetracycline-inducible promoter or the theophylline riboswitch and then deleting the native infC gene, or (ii) by CRISPR interference. We will also introduce infC* alleles in a targeted manner and run genetic selections for enhanced translation of sigR from its GTC start codon. Once we have an infC depletion strain, or infC* mutants, we will use our SigR-Gus translational fusions (with GTC and ATG start codons) and Westerns to assess the role of IF3 in the regulation of SigR translation.

OBJECTIVE 2. HOW DOES IF3 SENSE OXIDATIVE STRESS?
We will use either anti-FLAG or polyclonal antibodies to pull down IF3 before subjecting the samples to tryptic digestion. MALDI-TOF MS will then be used to identify the redox state of IF3 (based on peptide mass shifts due to modification with dimedone or iodoacetamide), plus other modifications, and MS/MS will be used to pinpoint modified residues. If IF3 senses oxidative stress through oxidation of Cys57, we will be able to monitor the oxidative inactivation of IF3 by Mal-PEG-induced bandshifting in Western blots.

OBJECTIVE 3. GLOBAL TRANSLATIONAL CONTROL OF THE OXIDATIVE STRESS RESPONSE AND THE UNEXPLORED POTENTIAL OF IF3 AS A GLOBAL REGULATORY DEVICE.
To analyse translational regulation of the oxidative stress response at a global level, and to globally identify genes with non-canonical start codons, we will develop genome-wide ribosome profiling (Ribo-seq) for Streptomyces. In this method, mRNA fragments buried inside actively translating ribosomes are isolated and subjected to deep sequencing. This is the first method to allow genome-wide analysis of protein synthesis and it can track translation at single-nucleotide resolution.

WHO WILL BENEFIT FROM THIS RESEARCH? The outputs of this research will be of value to the pharmaceutical industry, and ultimately to the health sector and thus to patients.

HOW WILL THEY BENEFIT FROM THIS RESEARCH? Streptomycetes account for ~80% of commercially important antibiotics used in human medicine, 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 new antibiotics will depend on a much better understanding of the physiology of the organism as a whole. This improved understanding will also contribute to increased yield of existing antibiotics, making them cheaper. The SigR-controlled thioredoxin and mycothiol systems are of particular interest in beta-lactam-producing streptomycetes because they are responsible for maintaining supplies of the reduced substrate for isopenicillin-N-synthase, ACV, the key precursor of most beta-lactam antibiotics (the oxidised version of ACV is not a substrate for the enzyme). It also seems likely that the production of other streptomycete antibiotics will be influenced by SigR, for example through the reduction of the phosphospantetheine arms of PKSs and NRPSs (oxidation of the terminal thiol group renders the enzyme inactive). Thus the proposed fundamental study will be of direct interest to companies manufacturing streptomycete antibiotics because genetic manipulation of the SigR system might improve yield, making them cheaper. Further, the SigR regulatory paradigm was discovered in Streptomyces but was subsequently found to be an important element in the pathogenesis of medically important actinomycete relatives like M. tuberculosis and C. diphtheriae (sigR mutants show greatly reduced pathogenesis). New drugs targeted at SigR-RsrA could make M. tuberculosis and C. diphtheriae more sensitive to oxidative killing by white blood cells. Thus the proposed work has potential commercial and medical relevance through multiple paths.

WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH? Outputs with potential commercial impact will be identified during regular reviews of progress. Discoveries with potential commercial implications will be discussed (with a view to patenting) with Plant Biosciences Ltd (PBL), a technology transfer company jointly owned by BBSRC, JIC and The Sainsbury Laboratory. 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 2003), and Procarta Biosystems (founded 2008). Thus, there are established routes for delivery of IP arising from Streptomyces research at the JIC.

Mark Buttner and Morgan Feeney 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.

JIC has an excellent External Relations 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, in 2010 JIC Communications prepared a press release, interviews and other activities around the publication of research involving the Buttner lab into vancomycin resistance (published in Nature Chem. Biol.).