| dc.description.abstract | Lytic polysaccharide monooxygenases (LPMOs), traditionally recognized for degrading recalcitrant polysaccharides such as cellulose and chitin, constitute a superfamily with diverse substrate specificities and biological roles. This study examines the structural and functional properties of the previously uncharacterized BsLPMO10A from Bacillus spizizenii along with the chitin-active BlLPMO10A from the closely related Bacillus licheniformis, focusing on their potential roles in bacterial cell wall remodeling and endospore germination.
Initially, the study investigated the expression of BlLPMO10A using a reporter gene system, fusing its promoter to mCherry within a vector transformed into B. licheniformis to potentially infer promoter activity. The findings revealed no detectable mCherry signal under various conditions, including growth in different media and during sporulation, suggesting an absence of LPMO expression under the tested conditions.
The activity and affinity of LPMOs for chitin were analyzed by mass spectrometry and binding assays, which confirmed the chitinolytic activity of BsLPMO10A but suggested a relatively low chitin affinity for both LPMOs, which may hint to alternative biological functions.
The potential impact of LPMOs on bacterial cell walls was assessed by monitoring changes in optical density (OD600), indicative of cell lysis. While the data suggested that these LPMOs could potentially enhance lysozyme-mediated peptidoglycan breakdown in vegetative cells, any definitive conclusions were not drawn, and the potential impact of LPMO mediated accumulation of reactive oxygen species (ROS) on cell integrity was considered.
Genomic investigations suggested a potential involvement of family 18 glycoside hydrolases in endospore germination in the parent strains of these LPMOs, substantiating the hypothesis that LPMOs may assist in this process. The potential impact of these Bacillus-derived LPMOs on endospore germination was assessed by monitoring the release of dipicolinic acid (DPA) and OD600. Despite complications in interpreting the results, an observed increase in DPA release in LPMO-treated endospores suggested possible LPMO involvement in endospore germination. Supporting these observations, phase contrast microscopy revealed that LPMO-treated endospores appeared phase bright and with size increases similar to what is observed in initial stages of germination, offering further indication of the involvement of LPMOs in this process.
Further investigations are necessary to explore how these LPMOs might contribute to endospore germination and to determine whether this potential function is widespread. | |
