Recently, it was discovered that the monofunctional class B enzymes PBP2x and PBP2b operate in conjunction with FtsW and RodA, two newly discovered transglycosylases belonging to the SEDS family proteins ( shape, elongation, division and sporulation) 8, 9. The class A PBPs, PBP1a, PBP2a, PBP1b, perform both reactions, while the class B PBPs, PBP2b and PBP2x, only have transpeptidase activity. pneumoniae contain six penicillin binding proteins (PBPs), five of which participate in building the cell wall via transglycosylase and transpeptidase reactions. The cell wall synthesis machineries of S. The divisome synthesizes the septal crosswall, while extension of the lateral cell body is carried out by the elongasome 6, 7. In ovoid bacteria, such as the important human pathogen Streptococcus pneumoniae, two modes of cell wall synthesis occur. During cell division and growth, the peptidoglycan synthesis machineries add new material into the existing cell wall. The peptidoglycan sacculus also serves as an anchoring device for surface proteins and other cell wall components such as teichoic acids and extracellular polysaccharides 2, 3, 4, 5.
In most bacteria, the cytoplasmic membrane is surrounded by a peptidoglycan layer, which gives the cell its shape and provides resistance to internal turgor pressure 1. Furthermore, we found that the EloR/KhpA complex co-localizes with FtsZ throughout the cell cycle. In support of this conclusion, we found that localization of KhpA to the pneumococcal mid-cell region depends on its interaction with EloR. Of note, loss of KhpA homodimerization did not give rise to this phenotype, demonstrating that the EloR/KhpA complex is crucial for regulating the activity of the elongasome. pneumoniae, we were able to show that disruption of EloR/KhpA heterodimerization makes the elongasome redundant in S. In contrast, both homo- and heterodimerization were lost when I61 was substituted with tyrosine.
When substituting I61 with phenylalanine, KhpA lost the ability to homodimerize, while it still interacted clearly with EloR. Site directed mutagenesis identified isoleucine 61 (I61) as crucial for KhpA homodimerization. Protein-protein interaction studies demonstrated that KhpA forms homodimers and that KhpA in addition binds to the KH-II domain of EloR. In the present study, we used 3D modeling and site directed mutagenesis in combination with protein crosslinking to further study the relationship between EloR and KhpA. It has been shown previously in formaldehyde crosslinking experiments that EloR co-precipitates with KhpA, indicating that they form a complex in vivo.
Loss of either protein reduces cell length, and makes the essential elongasome proteins PBP2b and RodA dispensable. The RNA binding proteins EloR and KhpA are important components of the regulatory network that controls and coordinates cell elongation and division in S.