Post-translation regulation of antibiotic production in Streptomyces: the loaded gun hypothesis.

Around three quarters of the antibiotics we use in human medicine are derived from the natural products of soil bacteria and fungi. We call these natural products specialised metabolites because they have a specialised function, which is usually to kill other bacteria and fungi in the highly competitive soil environment. These molecules can also be toxic to worms, insects and even plants and many have been used in medicine to treat parasite infections and as herbicides or pesticides in agriculture.

The biggest producers of specialised metabolites are Streptomyces bacteria which make around 50% of all known antibiotics. These bacteria are incredibly important to humans but we have relatively little understanding of how they control the production of their specialised metabolites. This is important because they only make around 10% of their specialised metabolites when we grow them in the laboratory. We know this from sequencing all the DNA in their cells which shows they have the instructions and capacity to make many more. If we can understand how they control their production we should be able to engineer strains to switch on production of all the specialised metabolites in all of the >600 known Streptomyces species and discover many new and potentially useful natural products, including antibiotics.

As part of our efforts to identify the master regulators of antibiotic production, we characterised a DNA binding protein called MtrA which is found in all Streptomyces species. MtrA controls antibiotic production in all the Streptomyces strains that have been tested so far and is part of a signal transduction pathway called a two-component system. These signalling systems are common in bacteria.

MtrA is a response regulator, and these proteins are typically transcription factors which control gene expression by binding to promoter DNA. The DNA binding activity of MtrA is controlled by MtrB, a sensor kinase which spans the cell membrane and senses a signal outside the cell and then phosphorylates and activates MtrA inside the cell. MtrA binds to around 80% of the predicted biosynthetic gene clusters for specialised metabolites in Streptomyces coelicolor and S. venezuelae. MtrA also binds to other transcription factors which is unusual in bacteria and to enzymes involved in making specialised metabolites which, to our knowledge, has never been described before for any other bacterial transcription factor.

In this proposal we will use S. venezuelae as a model to characterise the regulation of antibiotic production by MtrA since it controls chloramphenicol production by binding to the transporter genes and to the enzyme CmlS, which catalyses the final step in the biosynthetic pathway. It appears that antibiotic biosynthesis does not occur from scratch. Instead we hypothesise that it is like a loaded gun, the precursor is made but the final step is blocked by MtrA. When MtrA is switched off it is like pulling the trigger - the final step occurs, and the active antibiotic is made, the transport genes are expressed and the active compound is exported from the cell. In this project we will test this "loaded gun" hypothesis and the results will likely change the way we think about bacterial transcription factors and the regulation of antibiotic biosynthesis.

MtrA also binds to a closely related response regulator called Vnz13500 which is conserved in Streptomyces species and probably also activated by MtrB suggesting its functions are complex in S. venezuelae. To test whether these functions are conserved in other streptomycetes we will use the distantly related S. coelicolor which is also experimentally tractable. Published data from our and other groups suggests S. coelicolor MtrAB and SCO3008 (its Vnz13500 homologue) are involved in controlling production of its antibiotic undecylprodigiosin and we will test if it does this at both the transcriptional and post translational levels.

MtrAB is a two-component system that is highly conserved in the Actinobacteria, a phylum which contains around a third of all known bacteria. Crucially we have found that MtrAB is conserved in all Streptomyces species where it is a master regulator of antibiotic production that coordinates sporulation with specialised metabolism, most likely in response to desiccation and osmotic stress. Streptomyces bacteria make numerous specialised metabolites including 55% of clinically used antibiotics but they only produce around 10% of the specialised metabolites they encode under lab conditions. Clearly there is a need to understand how biosynthesis is regulated and initiated so we can unlock their cryptic gene clusters and find new antibiotics.

This proposal builds on our recent discovery that S. venezuelae MtrA acts as a transcriptional regulator, as expected, but also acts post translationally to control antibiotic biosynthesis, by interacting directly with biosynthetic enzymes. We have evidence that it controls chloramphenicol biosynthesis and export at both the transcriptional level, by repressing expression of the transporter genes cmlF and cmlN, and post translationally by binding to the halogenase enzyme, CmlS

In this project we will gain better mechanistic understanding of this phenomenon since to our knowledge nothing like this has ever been reported before. This adds a new and unforeseen level of complexity to the regulation of antibiotic production.

S. venezuelae MtrA also interacts with a closely related (and conserved) response regulator called Vnz13500 and we predict they form homo- and heterodimers, all of which are controlled through phosphorylation/dephosphorylation by the sensor kinase MtrB. We will use S. venezuelae and S. coelicolor as models to understand the roles of MtrAB and the orphan response regulator Vnz13500/Sco3008 in controlling antibiotic production in these bacteria at the transcriptional and post translational levels.