Microcolony formation by the opportunistic pathogen Pseudomonas aeruginosa requires pyruvate and pyruvate fermentation.

A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that microcolony formation is associated with stressful, oxygen-limiting but electron-rich conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation, while addition of pyruvate partly restored microcolony formation in mifR mutant biofilms. In contrast, expression of ldhA in mifR::Mar fully restored microcolony formation by this mutant strain. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation of the P. aeruginosa biofilm environment.