Evolution and regulation of microbial secondary metabolism

Microbes have disproportionate impacts on the macroscopic world. This is in part due to their ability to grow to large groups and cooperatively secrete massive amounts of secondary metabolites that impact their environment. Yet, the conditions enabling secondary metabolism without compromising primary needs remain unclear. Here we investigated the biosynthesis of thamnolipids, a secondary metabolite that Pseudomonas aeruginosa makes to decrease the surface tension of surrounding liquid. Using a combination of genomics, metabolomics, transcriptomics, and mathematical modeling we show that biosynthesis of rhamnolipids from glycerol varies inconsistently across the phylogenetic tree; instead, non-producer lineages are also those worse at reducing the oxidative stress of primary glycerol metabolism. The link to oxidative stress explains the inconsistent distribution across the P. aeruginosa tree, adding a new layer to the regulation of rhamnolipids—a microbial secondary metabolite important for fitness in natural and clinical settings.
Methods
Metabolite extraction. All P. aeruginosa strains were grown until the end of exponential phase of growth in glycerol minimal medium. Bacteria was then loaded into 0.25 μm nylon membranes (Millipore) using vacuum, transferred to pre-warmed hard agar plates with the same medium composition and incubated at 37ºC during 2.5 h. The filters were then passed to 35 mm polystyrene dishes (Falcon) with 1 mL of 2:2:1 methanol:acetonitrile:H2O quenching buffer and incubated there during 15 minutes on dry ice. Cells were removed by scraping and the lysate containing quenching buffer was transferred to 1.5 mL tubes and centrifuged at 16000 rpm for 10 minutes at 4ºC. Supernatant transferred to fresh tubes and stored at -80ºC.