SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis

Bacteria often form multicellular, organized communities known as biofilms, which protect cells from a variety of environmental stresses. During biofilm formation, bacteria secrete a species-specific matrix; in Bacillus subtilis biofilms, the matrix consists of protein polymers and exopolysaccharide. Many domesticated strains of B. subtilis have a reduced ability to form biofilms, and we conducted a two-month evolution experiment to test whether laboratory culturing provides selective pressure against biofilm formation in B. subtilis.

Bacteria grown in two-month-long batch culture rapidly diversified their biofilm-forming characteristics, exhibiting highly diverse colony morphologies on LB plates in the initial ten days of culture. Generally, this diversity decreased over time; however, multiple types of colony morphology remained in our final two-month-old populations, both under shaking and static conditions. Notably, while our final populations featured cells that produce less biofilm matrix than did the ancestor, cells overproducing biofilm matrix were present as well. We took a candidate-gene approach to identify mutations in the strains that overproduced matrix and found point mutations in the biofilm-regulatory gene sinR. Introducing these mutations into the ancestral strain phenocopied or partially phenocopied the evolved biofilm phenotypes.

Our data suggest that standard laboratory culturing conditions do not rapidly select against biofilm formation. Although biofilm matrix production is often reduced in domesticated bacterial strains, we found that matrix production may still have a fitness benefit in the laboratory. We suggest that adaptive specialization of biofilm-forming species can occur through mutations that modulate biofilm formation as in B. subtilis.