Berkovich, R. | bioRxiv Science

bioRxiv2020

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for studying biofilm formation. In order to elucidate the role of the bacteria flagella in biofilm formation, we developed a new tool for flagella bio-tracking. We have site-specifically labeled the bacterial flagella by incorporating an unnatural amino acid into the flagella monomer via genetic code expansion. This enabled us to label and track the bacterial flagella during biofilm maturation. Direct, live imaging revealed for the first-time presence and synthesis of flagella throughout the biofilm lifecycle. To ascertain the possible role of the flagella in the strength of a biofilm we produced a "flagella knockout" strain and compared its biofilm to that of the wild type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison to the flagella knockout strain. This suggests a newly discovered structural role for bacterial flagella in biofilm structure, possibly acting as a scaffold. Based on our findings we suggest a new model for biofilm maturation dynamic and underscore the importance of direct evidence from within the biofilm.

microbiology↗

bioRxiv2018

Reversible two-state folding of the ultrafast protein gpW under mechanical force

Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy (AFM) experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (~1 kBT). Applying pulling forces of only 5 pN we maintain gpW in quasi-equilibrium near its mechanical unfolding midpoint, and detect how it interconverts stochastically between the folded and an extended state. This binary pattern indicates that, under an external force, gpW (un)folds over a significant free energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single molecule force spectroscopy.

biophysics↗