By 2050 10 million lives could be claimed a year by drug resistant infections. We must develop new strategies for antimicrobial drugs. Often in infections bacteria form biofilms, requiring concentrations of antibiotics up to 1000 fold higher to be treated. Cyclic dipeptides are molecules produced by organisms in all domains of life, and their function is unknown. They can inhibit bacterial growth and/or biofilm formation, albeit by undetermined mechanisms. The majority of the biological effects caused by cyclic dipeptides are inter-species and in some instances inter-kingdom, mediating host pathogen interactions. I will study enzymes from gram-positive and gram-negative bacteria involved in the production of different cyclic dipeptides. I will characterise each enzyme biochemically and structurally and determine their substrate scope. I will produce novel molecules, which will be used to disrupt growth and biofilm formation in Pseudomonas aeruginosa and Staphylococcus aureus growing alone and in bacterial co-cultures. I will combine genetic and chemoproteomic approaches to determine the molecular targets of cyclic dipeptides in P. aeruginosa and S. aureus. I will validate targets using bacterial mutants and biochemical assays. The identification of molecular targets of cyclic dipeptides will unveil crucial pathways for inter-species interactions and identify novel antimicrobial targets and molecules. By 2050 one person will die every three seconds from antibiotic resistant bacterial infections. We need to develop new drugs to combat antibiotic resistance. Many bacteria grow by sticking to a surface in structures called biofilms. Some bacteria are completely resistant to antibiotics when in biofilms. Cyclic dipeptides are molecules that can disrupt bacterial growth and biofilm formation. They are common in nature but their biological function is unknown. I will study the enzymes that make these molecules, how they are affecting bacteria and how this information can be used to our favour. Then, I will manipulate these enzymes into making new molecules of my choosing, to be tested for antimicrobial activity. I will focus on disrupting growth and biofilm formation in two organisms that cause infections in humans. This project will increase our knowledge of bacterial infections and teach us new ways to inhibit bacterial growth and/or biofilm formation.