Putative role of cellulosomal protease inhibitors in Clostridium cellulovorans based on gene expression and measurement of activities

Abstract

This study is the first to demonstrate the activity of putative cellulosomal protease/peptidase inhibitors (named cyspins) of Clostridium cellulovorans, using the Saccharomyces cerevisiae display system. Cyspins exhibited inhibitory activities against several representative plant proteases. This suggests that these inhibitors protect their microbe and cellulosome from external attack by plant proteases.

Fig. 1.

Constructed plasmids and immunofluorescence staining of cyspin-displaying yeast cells. (a) Plasmid map of the pULD1-cyspin 1, 2, and 3 plasmids for display of cyspins on the yeast cell surface. Amp^r, ampicillin resistance gene for bacterial selection; 2μm, replication origin of yeast; ori, replication origin of Escherichia coli; URA3, uracil marker gene; leu2-d, leucine marker gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GA s.s., glucoamylase secretion signal. (b) Immunofluorescence staining for cell surface display of cyspins with mouse monoclonal anti-FLAG M2 antibody as the primary antibody and Alexa Fluor 488 goat anti-mouse IgG as the secondary antibody. Scale bar, 5 μm.

Fig. 2.

Inhibition activity of cyspin-displaying yeast cells against papain, ficin, and bromelain. Closed, open, and stippled bars indicate the remaining protease activity rates against papain, ficin, and bromelain, respectively. Z-Phe-Arg-MCA was used as the substrate. A value of 1 for relative protease activity (in relative fluorescence units [RFU]/min) means that there was no inhibitory activity. The initial rates of activity against papain, ficin, and bromelain were 1.80, 0.715, and 0.739 RFU/min. Each data set represents averages and standard deviations from three independent experiments.

Fig. 3.

Putative role of cellulosomal cyspins in C. cellulovorans. Cellulosomal protease/peptidase inhibitors protect their microbe and cellulosome. PP, plant proteases; CE, various cellulosomal enzymes.