Elucidating the Function of the Periplasmic Domain of Shape-Determining E. coli Protein RodZ

Harvard Medical School, Boston MA

Investigators

Abstract

Project Summary The peptidoglycan (PG) cell wall surrounds most bacterial cells and is essential for their integrity. In Escherichia coli, two multi-protein complexes synthesize this structure: the Rod system for cell elongation, and the divisome for cell division. The incorporation of new PG must be tightly controlled by regulatory components in these morphogenic complexes to retain cell shape and prevent osmotic lysis. The divisome and Rod system are thought to have a common evolutionary origin and contain proteins with similar functions. RodA-PBP2 (Rod system) and FtsW-FtsI (FtsWI) (divisome) are the essential PG synthases containing a SEDS-family PG glycan polymerase in complex with a class B penicillin-binding protein (bPBP) that crosslinks the strands into the PG matrix. In the divisome, PG synthesis by FtsWI is activated by FtsN through its effect on the FtsQLB complex. FtsN is a bitopic membrane protein with a largely unstructured periplasmic domain containing a small peptide essential for divisome activation and a folded SPOR domain with PG binding activity. The proposed experiments will focus on elucidating the function of Rod system protein RodZ, which I hypothesize is an FtsN analog that activates PG synthesis during cell elongation. Like FtsN, RodZ is a bitopic membrane protein with a periplasmic domain predicted to consist of an unstructured region capped with a folded domain at its C-terminal end (CFD domain). A search for remote homologs of the CFD revealed that it shares features with domains that have glycan binding activity. It therefore may bind PG like the SPOR domain of FtsN. We will test PG binding using co-sedimentation assays and/or fluorescence imaging of purified cell walls mixed with labeled CFD. We will also identify the predicted PG-synthesis activation domain of RodZ. A mutant encoding a hyperactive FtsL division protein, FtsL(E88K), fails to grow at high temperatures and conditions conferring osmotic stress. This phenotype can be rescued by hyperactivating the Rod system to counteract the lethal divisome hyperactivity. We have found that overproduction of RodZ lacking the CFD suppresses the lethality of FtsL(E88K), suggesting that RodZ activates the Rod system and that the CFD may regulate this activity. We will use this phenotype combined with further truncation and mutational analysis to identify the minimal activation domain within RodZ and determine if, like FtsN, it corresponds to a peptide in the unstructured periplasmic region. Inactivation of the PG binding domain of FtsN is synthetically lethal with mutants defective in other aspects of cell wall biogenesis and cell division, revealing the importance of this domain in divisome function. We will therefore use transposon sequencing (Tn-Seq) to identify mutants synthetically lethal with deletion of the CFD or the entire periplasmic domain of RodZ. Such mutations will help define conditions where this domain is essential, providing additional clues to determine its physiological function. Overall, the results from these experiments will elucidate important aspects of RodZ function and its role in Rod system function. The insights gained will also have the potential to enable efforts for targeting this PG synthesis system for antibiotic development.