Distinct Mechanical Roles for Bacteria-Produced Biopolymers in Biofilm Initiation and Strength

Chronic bacterial infections are often in the form of biofilms, which resist antibiotics and evade the immune defense. In biofilms, microbes are embedded in a matrix consisting largely of self-produced extracellular polysaccharides (EPS). Multiple types of EPS can be produced by a single bacterial strain - the reasons for this redundancy are not well-understood. Pseudomonas aeruginosa is an opportunistic human pathogen that produces three types of EPS: PEL, PSL, and alginate. We use a combination of genetic manipulation, microscopy, rheology, and molecular force spectroscopy to show that different polymers confer distinct mechanical and biological properties to single bacteria and to mature biofilms.When individual bacteria attach to a surface, intracellular levels of cyclic-di-GMP increase. Cyclic-di-GMP is required to change gene expression to initiate the transition to the biofilm state. What specific cues control cyclic-di-GMP production were previously unknown - we show that this is controlled by mechanical shear stress, which is primarily impacted by bacterial motility and the EPS coating on bacteria. This opens up the possibility of making a new class of anti-biofilm surface, by using a 2D fluid that cannot sustain a lateral shear stress and thereby preventing activation of the cyclic-di-GMP signal.P. aeruginosa biofilm infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of alginate is well-known to tend to increase during CF infections. More recently, it is becoming recognized that CF infections also evolve to increase PSL production. Alginate chemically protects biofilms, but also makes them softer and weaker. Here, we show that PSL stiffens and strengthens biofilms, and that increased PSL production in biofilms grown from CF clinical isolates completely rescues the mechanical weakening caused by alginate.