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Escherichia coli cell division is a complex process involving over 30 proteins that are recruited to the division site. One of the important division proteins is FtsN, which has a C-terminal peptidoglycan (PG) binding region called the SPOR domain. Most SPOR domain proteins are probably involved in bacterial cell division, but their precise role in this process is not known. Although the structure of the FtsN SPOR domain has been solved by NMR, nothing is known about how the domain binds PG. Understanding the SPOR:PG interaction is important because it could lead to novel insights into PG metabolism during cell division. We hypothesize that the SPOR domain from FtsN recognizes and binds septal PG. To test this hypothesis we have conducted a comprehensive mutagenesis of the FtsN SPOR domain to identify amino acid residues critical for septal localization and PG binding. We targeted 33 residues and made a total of 92 point mutants, all of which were tested for septal localization. Our results revealed four amino acids that are critical for septal localization. All four of these residues map in or near the â-sheet, which we now propose is the PG binding site. Further analysis of localization-defective proteins in a PG binding assay led to the realization that the assay measures nonspecific binding to bulk PG rather then specific binding to septal PG. An important priority for the future will be to develop a better PG binding assay.