Manipulating two-component systems to activate cryptic antibiotic pathways in filamentous actinomycete bacteria
Lead Research Organisation:
John Innes Centre
Department Name: Molecular Microbiology
Abstract
Most 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 specialised functions that help the producing organisms survive in their highly competitive soil environment, but they are not required for the organism to grow in the laboratory. These molecules are often toxic to other bacteria and fungi, but some of them can also kill worms, insects and even plants and many have been used as medicines and as herbicides and pesticides in agriculture. They are incredibly useful and valuable to humans.
The biggest producers of specialised metabolites are a genus of bacteria called Streptomyces, which make 55% of the antibiotics we use as medicines. These bacteria are important to human survival, but we have relatively little understanding of how they control the production of their specialised metabolites. This is important because they only make 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, we just don't know how to switch on their production. If we can understand how to do this we can discover new antibiotics that will help us treat drug resistant infections and address the antimicrobial resistance crisis.
The biosynthetic pathways for each specialised metabolite are encoded by genes which are clustered together on the chromosome and we call them biosynthetic gene clusters (BGCs). If we can activate all of these BGCs we will be able to discover new antibiotics. Up to 25% of the BGCs in any Streptomyces genome also encode two-component systems which sense environmental signals and respond by altering target gene expression. These systems also interact with biosynthetic enzymes to exert multilevel (transcriptional and post translational) control of antibiotic biosynthesis. We have recently shown that by simply over-producing the two component systems encoded by individual BGCs we can switch on antibiotic production in strains that don't usually have antibiotic activity. This is exciting as it suggests we are switching on some of the 90% of biosynthetic pathways that are usually silent under laboratory conditions and this simple technique could enable us and other researchers around the world to discover new antibiotics and other useful molecules.
In this proposal we will characterise all seven of the BGC-encoded two component systems in our model organism Streptomyces formicae to identify the genes they control, the proteins they interact with and the specialised metabolites that are produced when we over-express these two component systems. This will identify new antibiotics and will also give us detailed understanding of the roles of BGC-encoded two-component systems in a single organism. We will then extend this to five other strains we have cultured from diverse environments to see if over-expressing the two component systems encoded in their BGCs activates antibiotic production in these organisms. We will share the data, results and techniques widely through public databases, preprints, publications and on http://actinobase.org, a wiki we set up to share protocols and information for working with actinomycetes. We hope that the outputs from this project will help the global research community to activate antibiotic production in their strains of interest and so accelerate the rate of antibiotic discovery.
The biggest producers of specialised metabolites are a genus of bacteria called Streptomyces, which make 55% of the antibiotics we use as medicines. These bacteria are important to human survival, but we have relatively little understanding of how they control the production of their specialised metabolites. This is important because they only make 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, we just don't know how to switch on their production. If we can understand how to do this we can discover new antibiotics that will help us treat drug resistant infections and address the antimicrobial resistance crisis.
The biosynthetic pathways for each specialised metabolite are encoded by genes which are clustered together on the chromosome and we call them biosynthetic gene clusters (BGCs). If we can activate all of these BGCs we will be able to discover new antibiotics. Up to 25% of the BGCs in any Streptomyces genome also encode two-component systems which sense environmental signals and respond by altering target gene expression. These systems also interact with biosynthetic enzymes to exert multilevel (transcriptional and post translational) control of antibiotic biosynthesis. We have recently shown that by simply over-producing the two component systems encoded by individual BGCs we can switch on antibiotic production in strains that don't usually have antibiotic activity. This is exciting as it suggests we are switching on some of the 90% of biosynthetic pathways that are usually silent under laboratory conditions and this simple technique could enable us and other researchers around the world to discover new antibiotics and other useful molecules.
In this proposal we will characterise all seven of the BGC-encoded two component systems in our model organism Streptomyces formicae to identify the genes they control, the proteins they interact with and the specialised metabolites that are produced when we over-express these two component systems. This will identify new antibiotics and will also give us detailed understanding of the roles of BGC-encoded two-component systems in a single organism. We will then extend this to five other strains we have cultured from diverse environments to see if over-expressing the two component systems encoded in their BGCs activates antibiotic production in these organisms. We will share the data, results and techniques widely through public databases, preprints, publications and on http://actinobase.org, a wiki we set up to share protocols and information for working with actinomycetes. We hope that the outputs from this project will help the global research community to activate antibiotic production in their strains of interest and so accelerate the rate of antibiotic discovery.
Technical Summary
Streptomyces spp. make specialised metabolites (SMs) that account for 55% of clinically used antibiotics. However, they encode biosynthetic pathways for ten times more SMs than they make in the laboratory. Artificially expressing the genes for these cryptic pathways does not usually switch on production of the SMs suggesting there are additional levels of control. We recently discovered that response regulators (RRs), which are part of two component systems, regulate gene expression but also interact with biosynthetic enzymes to activate or repress biosynthetic pathways. This post-translational control likely explains why gene expression alone is not enough to activate specialised metabolite biosynthesis.
Our model organism, Streptomyces formicae, encodes 41 biosynthetic pathways and seven of the gene clusters encoding these pathways also include two component systems. We hypothesised these two component systems activate their own pathways and when we individually over-expressed five of these it switched on antibiotic production in all five mutant strains. This suggests we have switched on pathways that are usually cryptic and is exciting because it potentially provides an easy way to switch on cryptic pathways. Up to 25% of biosynthetic gene clusters in public databases encode two component systems.
In this project we will test all seven two component systems in S. formicae to see if over-expressing any one of them can activate their respective pathways. We will identify the genes they control, determine which of them interact with biosynthetic enzymes and identify the antibiotics produced by the over expression strains. Finally we will test two component system over expression in five other actinomycete strains (4 Streptomyces and 1 Amycolatopsis) which we have isolated from diverse environments to see if we can use this as a general tool to activate the production of antibiotics that are cryptic in the wild type parent strains.
Our model organism, Streptomyces formicae, encodes 41 biosynthetic pathways and seven of the gene clusters encoding these pathways also include two component systems. We hypothesised these two component systems activate their own pathways and when we individually over-expressed five of these it switched on antibiotic production in all five mutant strains. This suggests we have switched on pathways that are usually cryptic and is exciting because it potentially provides an easy way to switch on cryptic pathways. Up to 25% of biosynthetic gene clusters in public databases encode two component systems.
In this project we will test all seven two component systems in S. formicae to see if over-expressing any one of them can activate their respective pathways. We will identify the genes they control, determine which of them interact with biosynthetic enzymes and identify the antibiotics produced by the over expression strains. Finally we will test two component system over expression in five other actinomycete strains (4 Streptomyces and 1 Amycolatopsis) which we have isolated from diverse environments to see if we can use this as a general tool to activate the production of antibiotics that are cryptic in the wild type parent strains.
