Reverse diauxie phenotype in biofilm revealed by exometabolomics and label-free proteomics.

Microorganisms enhance fitness by prioritizing catabolism of available carbon sources using a process known as carbon catabolite repression (CCR). Planktonically grown is known to prioritize the consumption of organic acids including lactic acid over catabolism of glucose using a CCR strategy termed "reverse diauxie." is an opportunistic pathogen with well-documented biofilm phenotypes that are distinct from its planktonic phenotypes. Reverse diauxie has been described in planktonic cultures, but it has not been documented explicitly in biofilms. Here a combination of exometabolomics and label-free proteomics was used to analyze planktonic and biofilm phenotypes for reverse diauxie. biofilm cultures preferentially consumed lactic acid over glucose, and in addition, the cultures catabolized the substrates completely and did not exhibit the acetate secreting "overflow" metabolism that is typical of many model microorganisms. The biofilm phenotype was enabled by changes in protein abundances, including lactate dehydrogenase, fumarate hydratase, GTP cyclohydrolase, L-ornithine N(5)-monooxygenase, and superoxide dismutase. These results are noteworthy because reverse diauxie-mediated catabolism of organic acids necessitates a terminal electron acceptor like O, which is typically in low supply in biofilms due to diffusion limitation. Label-free proteomics identified dozens of proteins associated with biofilm formation including 16 that have not been previously reported, highlighting both the advantages of the methodology utilized here and the complexity of the proteomic adaptation for biofilms. Documenting the reverse diauxic phenotype in biofilms is foundational for understanding cellular nutrient and energy fluxes, which ultimately control growth and virulence.