Cellulase

We used clonal ancestral and evolved samples to assay the ability of two distinct hydrolytic enzymes, cellulase (Tokyo Chemical Industries Co. Ltd., Tokyo (Japan)) and proteinase K (Thermo Scientific^TM), to inhibit/reduce multicellular macroscopic clustering. Specifically, we streaked the clones derived from their respective cryo-stocks on Luria agar and used a single colony to inoculate liquid cultures in six different environments: 5 ml LB with habitual or high salinity containing either cellulase (~386 U (22.7 mg)), proteinase K (~12 U (400 µg)), or no hydrolytic enzyme. We documented the phenotype at the level of cell collectives (macroscopic multicellular clusters versus turbid planktonic growth) at saturation.

Vanillin, vanillic acid, 3.4-dihydroxy-5-methoxybenzaldehyde (Wako Pure Chemical Industries, Osaka, Japan), 2-methoxyhydroquinone (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), protocatechuic aldehyde (Sigma-Aldrich, St. Louis, MO, USA), and protocatechuic aldehyde (Nacalai Tesque, Kyoto, Japan) were purchased and used as inhibitors of yeast growth in cultures. Cellulase (mainly containing endo-β-1,4-glucanase and β-1,4-glucosidase) from A. niger [54 (link), 55 (link)] was obtained from Tokyo Chemical Industry Co., Ltd., and its activity was 29,500 unit/g. Rice straw was grown and harvested on the farm at Meijo University (Aichi, Japan). The straw was cut, dried at 45 °C for 3 h, and milled to a particle size of 1 mm, followed by washing at a weight ratio of 1:20 of rice straw to distilled deionized water. The washed straw was dried at 45 °C for 24 h and used for subsequent experiments.