Identification of Potential Regulatory Domains Within the MreC and MreD Components of the Cell Elongation Machinery

Cell shape in bacteria is largely determined by the cell wall structure that surrounds them. The multiprotein machine called the Rod system (elongasome) has long been implicated in rod shape determination in bacilli. ABSTRACT The bacterial peptidoglycan (PG) cell wall maintains cell shape and prevents osmotic lysis. During growth of rod-shaped cells, PG is incorporated along the cell cylinder by the RodA-PBP2 synthase of the multiprotein Rod system (elongasome). Filaments of the actin-like MreB protein orient synthesis of the new PG material. They are connected to the RodA-PBP2 synthase in part through the RodZ component. MreC and MreD are other conserved components of the system, but their function is not well understood. Amino acid changes in RodA-PBP2 that bypass a requirement for MreC and MreD function were recently identified, suggesting that the Mre proteins act as activators of the synthase. To further investigate their function, we developed a genetic strategy to identify dominant negative alleles of mreC and mreD in Escherichia coli. Residues essential for Rod system function were identified at the junction of two subdomains within MreC and in a predicted ligand-binding pocket of MreD. Additionally, we found that although the proline-rich C-terminal domain of MreC is nonessential, substitutions within this region disrupt its function. Based on these results, we propose that the C terminus of MreC and the putative ligand-binding domain of MreD play regulatory roles in controlling Rod system activity. IMPORTANCE Cell shape in bacteria is largely determined by the cell wall structure that surrounds them. The multiprotein machine called the Rod system (elongasome) has long been implicated in rod shape determination in bacilli. However, the functions of many of its conserved components remain unclear. Here, we describe a new genetic system to dissect the function of these proteins and how we used it to identify potential regulatory domains within them that may modulate the function of the shape-determining machinery.