Understanding and manipulating a conserved and essential transcription factor to activate antibiotic production in Streptomyces species

Almost all the antibiotics used in human medicine were discovered >50 years ago and most disease-causing bacteria are now resistant to one or more of these drugs. This means humans are facing a return to the pre-antibiotic era, an alarming situation that has been described as 'antibiotic Armageddon'. The government commissioned O'Neill review on AntiMicrobial Resistance (AMR) estimates that if we do not kick-start antibiotic discovery efforts now then by 2050 drug resistant infections will kill more people than cancer, an estimated 10 million a year.

Most of the antibiotics we currently use are natural products derived from the secondary metabolites of soil bacteria and the most important group are called Streptomyces, which make 50% of all known antibiotics. Streptomyces are incredibly important to humans and although scientists have already discovered lots of antibiotics from these bacteria we now know that they only found the really easy to find compounds, the low hanging fruit. Genome sequencing over the last 15 years has revealed that Streptomyces bacteria only make about 25% of their secondary metabolites under laboratory conditions which means that from 1940-60, the so-called golden age of antibiotic discovery, scientists were barely sampling their capability. The rest are called 'silent' secondary metabolites because they do not make them in the lab. The good news is this means we have a big advantage over scientists working in the 20th century - if we can find ways to switch on production of all the silent secondary metabolites in the >600 known species we will find lots of new antibiotics that can enter the clinical trials pipeline. This is the earliest stage in antibiotic discovery and it is vital that we increase our efforts now because it takes 10-15 years to get drugs through clinical trials and approved for use in humans. Probably <1% of antibiotics will be suitable for treating disease so the more natural products we can discover from Streptomyces in the next few decades the better.

One way to activate the production of silent secondary metabolites is to understand the natural signals and signalling pathways that control their production in the soil and this is the focus of our research. If we can manipulate those signalling pathways we can force the bacteria to make all of their antibiotics in the laboratory. Ideally we want to identify signalling pathways which effect antibiotic production in all 600+ known Streptomyces species and this is the subject of our proposal. We have identified a signalling pathway consisting of two proteins called MtrA and MtrB and found this is the only conserved and essential pathway in the genus Streptomyces. This means this MtrAB two-component system is found in every single sequenced Streptomyces strain!

MtrA is a DNA binding protein and its activity is controlled by the signal sensing protein MtrB. If we disrupt the pathway by deleting the mtrA gene it is lethal. If we delete the mtrB gene it removes the need for an environmental signal to activate the pathway and results in over-production of active MtrA protein which switches on production of antibiotics that are usually silent in the wild-type strains. However, simply over-producing MtrA does not work, we HAVE to remove MtrB as well. In this project we will analyse MtrAB in two model species called S. coelicolor and S. venezuelae. We will determine how MtrB controls MtrA activity, why MtrA is active in the absence of MtrB and why and how MtrA activates the production of silent secondary metabolites. We will also try to make gain of function MtrA proteins that are always active and see if we can use them to switch on antibiotic production in our model strains and in two new talented Streptomyces species that we have isolated and genome sequenced. We call them talented because they appear to encode many novel secondary metabolites and MtrA may allow us to discover new antibiotics from these strains.

MtrAB is a two-component system that is highly conserved in the phylum Actinobacteria, which contains around a third of all known bacteria. Crucially we have found that MtrA is the only conserved and essential response regulator in antibiotic-producing Streptomyces species. We have shown that deleting the mtrB (sensor kinase) gene results in over-production of active MtrA which in turn activates the production of secondary metabolites, including antibiotics, under conditions where they are silent (not produced) in wild-type strains of the distantly related model species S. coelicolor or S. venezuelae. This suggests manipulating MtrA activity could be used to activate silent antibiotic clusters in some or all of the 600+ known Streptomyces species.

ChIP-seq analysis to identify MtrA targets revealed that MtrA binds to two sites at the origin of DNA replication oriC and also to the promoters of dnaA and adpA, which control DNA replication in Streptomyces, and to the promoter of ftsZ, which controls cell division. This suggests a key role for MtrA in the regulation of DNA replication and cell division. Deletion of mtrB up-regulates antibiotic production, which is usually linked to cell division, and down-regulates siderophores and other secondary metabolites that are only required in actively growing (non dividing) cells. Our data suggest therefore that MtrA is essential because it coordinates DNA replication, cell division and secondary metabolism in Streptomyces. This makes it fundamentally important to study and potentially very useful.

We will measure MtrA activity and function in wild-type and mtrB strains throughout the growth cycles of the model organisms S. coelicolor and S. venezuelae to understand its basic cellular function. We will exploit this knowledge to make MtrA expression constructs to specifically induce antibiotic production in model strains and two new talented species we identified with multiple novel antibiotic biosynthetic gene clusters.

The proposed work will generate significant impacts and we describe below who will benefit and the mechanisms in place to show how that impact will be achieved.

1. TRAINING. This project will offer excellent training and career opportunities to the appointed microbiology PDRA. Training will be provided in chemical techniques including analysis of culture extracts and quantification of antibiotics using LC-MS. The project is likely to lead to high impact publications and s/he will also be encouraged to present at academic conferences and take part in public engagement events, including at least one big public exhibition during this project and SAW Trust workshops in schools. The grade 5 technician we employ 0.5 FTE will also be offered broad training in molecular microbiology as will visitors to the lab, including A level students, undergraduates and masters students.

2. DISSEMINATION TO SCIENTISTS AND THE GENERAL PUBLIC. Two-component systems are the major way by which bacteria sense and respond to their environment and many research groups around the world are investigating their functions and trying to rewire their activities for synthetic biology applications. Our work will be of general interest to those groups and to scientists interested in the regulation of bacterial DNA replication and cell division but particularly those working on filamentous actinomycetes like streptomycetes, a group that make two thirds of all known antibiotics. Many academic and industrial scientists around the world are developing tools to switch on secondary metabolite biosynthetic pathways in a quest to identify novel and useful bioactive natural products. We know that only a quarter of the secondary metabolites encoded by any actinomycete strain are actually made under laboratory conditions so the easiest way to discover new natural products is to unlock the silent pathways in the thousands of strains already cultured from soil and other environments. Our work will be of interest and benefit to those research groups who might try over-expressing MtrA and / or deleting mtrB to switch on silent gene clusters. We will share our results freely with scientists through publication in high quality open access journals, dissemination at academic conferences and more widely through Twitter and press releases and on our group websites. Expression constructs will be deposited with AddGene. Hutchings has a strong track record in public engagement in schools, through public lectures and at major public science events including the Royal Society Summer Science Exhibition 2014, the BBSRC Great British Bioscience Festival 2014 and Big Bang Science Fair 2015. Hutchings won a UEA award in 2015 for his outstanding contribution to public and community engagement and will continue to engage widely with the public through all available avenues to talk about his group's work on Streptomyces natural product antibiotics.

3. ECONOMIC - NEW TOOLS. Materials developed during this project that activate silent gene clusters and production of novel natural products will be made freely available to academics and industrial scientists (subject to MTA) to stimulate and assist antibiotic discovery efforts aimed at tackling the AMR crisis. We will also exploit these tools under separate on-going research projects with our own collection of actinomycetes, including many novel strains, which were isolated from the cuticles of leafcutter ants and the nests of other fungus-growing ants collected in South America and Africa. Hutchings is building a natural products discovery pipeline with Wilkinson and other partners on the Norwich Research Park and these new tools will contribute to their efforts. Intellectual property arising from this work will be protected and licensed by UEA's Research, Enterprise and Engagement Office.