A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae.

Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape.IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.

Structural features of the interaction of MapZ with FtsZ and membranes in Streptococcus pneumoniae.

MapZ localizes at midcell and acts as a molecular beacon for the positioning of the cell division machinery in the bacterium Streptococcus pneumoniae. MapZ contains a single transmembrane helix that separates the C-terminal extracellular domain from the N-terminal cytoplasmic domain. Only the structure and function of the extracellular domain is known. Here, we demonstrate that large parts of the cytoplasmic domain is intrinsically disordered and that there are two regions (from residues 45 to 68 and 79 to 95) with a tendency to fold into amphipathic helices. We further reveal that these regions interact with the surface of liposomes that mimic the Streptococcus pneumoniae cell membrane. The highly conserved and unfolded N-terminal region (from residues 17 to 43) specifically interacts with FtsZ independently of FtsZ polymerization state. Moreover, we show that MapZ phosphorylation at positions Thr67 and Thr68 does not impact the interaction with FtsZ or liposomes. Altogether, we propose a model in which the MapZ-mediated recruitment of FtsZ to mid-cell is modulated through competition of MapZ binding to the cell membrane. The molecular interplay between the components of this tripartite complex could represent a key step toward the complete assembly of the divisome.