Role of Penicillin-Binding Proteins in Synthesis and modification of Bacillus subtilis cell envelope

Abstract

The peptidoglycan (PG) layer plays a crucial role in preserving the shape of bacterial cells and facilitating cell division in nearly all Gram-positive bacteria. Composed of glycan chains linked by short peptide fragments, PG creates a mesh-like structure that envelops the cytoplasmic membrane. During cell growth and division, membrane-bound PG synthases, known as penicillin-binding proteins (PBPs). PBPs are essential for building the bacterial cell wall, maintaining cell shape, and supporting bacterial replication. Bacteria exhibit a wide variety of PBPs, suggesting that despite their apparent functional redundancy, there are distinct roles within the PBP family. These seemingly redundant proteins can be vital for helping organisms adapt to and survive environmental stressors. Although the glycosyltransferases PBPs (class A PBPs) have been deleted, the minimal set of PBPs that are required for Bacillus subtilis survival and growth is not yet identified. Additionally, although the transpeptidases PBP2a and PBPH are well identified redundant partners, the way they are regulated and the influence on each other is still elusive. Finally, PBPX is known as penicillin binding protein with endopeptidase activity however its precious function is still not clear specially because it has been linked to Dlt proteins in response to cationic antimicrobial peptides (CAMPs). Here we have systematically deleted PBPs in B. subtilis until we identify strain with minimal PBPs which are PBP2B and PBP2a. The strain was capable of surviving and growing during exponential phase, but the growth was slower compared to the wild type. The strain with minimal sets of PBPs shows elongated shape under microscopy with less rigid cell envelope and changed in peptidoglycan composition but maintain the degree of cross linkage of PG compared to the wild type. We also showed that the deletion of pbpH increases the activity and quantity of PBP2a which was due to the transcriptional regulation between the redundant partners. The PBPH transmembrane domine play a role in the regulation of the redundant partner activity and quantity. Finally, this study shows that the PBPX play a role in modulating the cell surface charge and therefore decrease the sensitivity toward the CAMPs and the deletion of pbpX significantly decreases the d-alanine associated with the wall teichoic acid. The results tighter indicated that the PBPX is the protein that transfer the d-alanine from lipoteichoic acid to the wall teichoic acid.