Activation of the Bacterial Cell Elongation Machinery

Abstract

Bacteria expand their peptidoglycan cell wall in order to grow and divide while maintaining shape and withstanding turgor pressure. A broadly conserved multiprotein complex called the Rod system performs peptidoglycan synthesis during cell elongation. The complex is organized by the actin-like MreB protein and includes the RodA-PBP2 cell wall synthase. Although the components of the Rod system were identified some time ago, the regulatory mechanisms governing the synthetic enzymes have remained poorly defined. To better understand Rod system function, I developed a genetic system to identify Escherichia coli mutants with a defective Rod system and suppressor alleles that overcome their malfunction. This analysis led to the identification of an amino acid substitution in the transpeptidase PBP2 that increases Rod system activity by directly stimulating the RodA polymerase. The substitution is located in a non-enzymatic domain that undergoes a conformational change upon association with the conserved Rod system subunit MreC. My results suggest that the Rod system is activated through an MreC-induced change in PBP2 that, in turn, allosterically activates RodA. This proposed regulatory cascade provides a mechanism for coupling cell wall polymerase activity and crosslinking during cell elongation. Next, I used my genetic system to identify critical residues within the Rod system components MreC and MreD. Surprisingly, certain amino acid variants within the C-terminal domain of MreC disrupt Rod system activity, even though this domain as a whole is dispensable for Rod system function. These data suggest that this domain may have an inhibitory role under certain conditions, perhaps in fine-tuning Rod system activity and coordinating it with other physiological processes. It is unclear whether Rod system activity is modulated in response to external signals. A clue to a potential stimulus came from the finding that certain Rod system defects can be suppressed by increasing the rate of outer membrane synthesis. Conversely, blocking cell wall synthesis impairs outer membrane assembly. Although the mechanistic details are unclear, we infer that growth of the cell wall peptidoglycan and outer membrane envelope layers may be coordinated in order to properly build the multi-layered Gram-negative cell envelope.