Media component bovine serum albumin facilitates the formation of mycobacterial biofilms in response to reductive stress

Abstract

Background: Mycobacterium tuberculosis (Mtb) forms physiologically relevant biofilms harboring drug-tolerant bacteria. This observation has brought the study of mycobacterial biofilms to the forefront of tuberculosis research. We established earlier that dithiothreitol (DTT) mediated reductive stress induces cellulose-rich biofilm formation in Mtb cultures. The molecular events associated with the DTT-induced biofilm formation are not known. Furthermore, there are only limited tools for monitoring the presence of cellulose in biofilms. Results: To decipher the molecular events associated with DTT-induced biofilm formation, we used Mtb and non-pathogenic, fast-growing Mycobacterium smegmatis (Msm). We observed that DTT induces biofilm formation in Msm cultures. We explored whether media components facilitate biofilm formation in mycobacteria upon exposure to DTT. We observed that media component bovine serum albumin promotes mycobacterial biofilm formation in response to DTT. Furthermore, we analyzed the composition of extracellular polymeric substances of Msm biofilms. We found that, like Mtb biofilms, Msm biofilms are also rich in polysaccharides and proteins. We also developed a novel protein-based molecular probe for imaging cellulose by utilizing the cellulose-binding domain of cellulase CenA from Cellulomonas fimi and fusing it to fluorescent reporter mCherry. Characterization of this new probe revealed that it has a high affinity for cellulose and could be used for visualizing cellulose biosynthesis during the development of Agrobacterium biofilms. Furthermore, we have demonstrated that biological macromolecule cellulose is present in the extracellular polymeric substances of Msm biofilms using this novel probe. Conclusions: This study indicates that DTT-mediated reduction of media component BSA leads to the formation of nucleating foci. These nucleating foci are critical for subsequent attachment of bacterial cells and induction of EPS production. Furthermore, this new tool, IMT-CBD-mC, could be used for monitoring cellulose incorporation in plant cells, understanding cellulose biosynthesis dynamics during biofilm formation, etc.