Recruitment of the TolA Protein to Cell Constriction Sites in Escherichia coli via Three Separate Mechanisms, and a Critical Role for FtsWI Activity in Recruitment of both TolA and TolQ.

The Tol-Pal system of Gram-negative bacteria helps maintain the integrity of the cell envelope and ensures that invagination of the envelope layers during cell fission occurs in a well-coordinated manner. In Escherichia coli, the five Tol-Pal proteins (TolQ, -R, -A, and -B and Pal) accumulate at cell constriction sites in a manner that normally requires the activity of the cell constriction initiation protein FtsN. While septal recruitment of TolR, TolB, and Pal also requires the presence of TolQ and/or TolA, the latter two can recognize constriction sites independently of the other system proteins. What attracts TolQ or TolA to these sites is unclear. We show that FtsN indirectly attracts both proteins and that PBP1A, PBP1B, and CpoB are dispensable for their septal recruitment. However, the β-lactam aztreonam readily interferes with the septal accumulation of both TolQ and TolA, indicating that FtsN-stimulated production of septal peptidoglycan by the FtsWI synthase is critical to their recruitment. We also discovered that each of TolA's three domains can separately recognize division sites. Notably, the middle domain (TolAII) is responsible for directing TolA to constriction sites in the absence of other Tol-Pal proteins and CpoB, while recruitment of TolAI requires TolQ and that of TolAIII requires a combination of TolB, Pal, and CpoB. Additionally, we describe the construction and use of functional fluorescent sandwich fusions of the ZipA division protein, which should be more broadly valuable in future studies of the E. coli cell division machinery. IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction will eventually occur. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We studied how two such proteins, TolA and TolQ, which also play a role in maintaining the integrity of the outer membrane, are recruited to the machinery. We find that TolA can be recruited by three separate mechanisms and that both proteins rely on the activity of a well-studied cell division enzyme for their recruitment.