Metabolomics of Antifungal Bacteria

In soil Pseudomonas fluorescens is an extremely important organism, as it grows alongside plants and crucially, protects them from other more harmful bacteria and fungi.

The remarkable antimicrobial properties of P. fluorescens come courtesy of a set of proteins called siderophores, which it secretes into the surrounding soil. Siderophores bind iron molecules extremely efficiently, which are essential to support life. Once bound by the siderophore, the iron is taken up into P. fluorescens, which both promotes its own growth and deprives any other microorganism of their benefit.

Knowledge of the metabolic pathways controlling siderophore production is consequently extremely useful. In a bid to find out more about these pathways, the NMR centre undertook a detailed analysis of the myriad of small molecules, or metabolites produced by P. fluoresecens.

Investigating such a complex mixture of small molecules is far from straightforward. To start with, the NMR team developed a method for growing P. fluorescens that allowed them to vary the nutrients supplied to it and subsequently monitor the effects on its metabolites. They then collected metabolites from P. fluorescens grown with different food sources under extremely closely controlled conditions and identified them from their characteristic NMR spectra. Finally, differences in the metabolite populations from P. fluorescens grown on different substrates were highlighted using principal component analysis.

This investigation identified clusters of metabolites associated uniquely with different food sources. One metabolite in particular, putrescene, known to be involved in siderophore production, was elevated when P. fluorescens was fed on glycerol. Identifying the chemicals produced when P. fluorescens uses different food sources provides a valuable insight into the interactions between its many complex metabolic pathways. 

Above: Principal Component Analysis of the metabolites associated with P. fluorescens grown on four food sources.

Compelling though it is, understanding the metabolic pathways in P. fluorescens is only the start of the story. The method developed for analysing bacterial metabolites has since been adapted for NMR analysis of metabolites from a wide range of biological samples including plasma, urine, synovial fluid, cartilage and aortic tissue.

Case study 2: Solving the structure of a protein involved in metastasis