Cell division in Escherichia coli
requires approximately a dozen proteins, all of which localize to a ring structure at the division site (Fig. ) (for a recent review, see reference 15
). The division proteins localize in a defined order, which starts with assembly of the tubulin homolog FtsZ into a contractile ring at the midcell. Subsequent recruitment of the other division proteins is thought to result in the assembly of a complex that mediates inward growth of the cell envelope. According to the current model, the last four proteins recruited to the septal ring are FtsW, FtsI, FtsN, and AmiC (in that order) (4
). The precise roles of FtsW and FtsN in septum assembly are not yet known. FtsI is a transpeptidase that introduces peptide cross-linking into the peptidoglycan cell wall in the division septum (6
). AmiC is a periplasmic amidase that hydrolyzes peptide cross-links and contributes to the separation of daughter cells after division (25
FIG. 1. (A) Recruitment of proteins to the septal ring of E. coli. Assembly of the ring starts with formation of the FtsZ ring at the midcell. The remaining proteins are then recruited in the order indicated, with AmiC being the last. (B) Domain structure of (more ...)
gene encodes a protein of 588 amino acids, but a proteolytic processing event removes 11 residues from the C terminus (32
). Processing is not required for FtsI function (22
). The membrane topology of FtsI has been studied with protein fusions, which revealed that the protein can be divided into three domains: an amino-terminal cytoplasmic domain (23 amino acids), a single transmembrane helix (17 residues), and a large periplasmic domain (537 residues) (7
). The periplasmic domain appears to comprise two parts, a noncatalytic domain of unknown function and a catalytic domain that is directly responsible for introducing cross-links into septal peptidoglycan (reviewed in reference 33
). A cartoon of the domain structure of FtsI is shown in Fig. .
Several reports have implicated the cytoplasmic domain and membrane-spanning segment in targeting of FtsI to the septal ring (12
). The most direct evidence comes from analysis of hybrid proteins in which these domains have been replaced with the corresponding parts of other membrane proteins. Such hybrid proteins fail to complement temperature-sensitive and null alleles of ftsI
and also fail to localize to the septal ring (20
The catalytic domain extends from residues 237 to 577 and is the site of the transpeptidase activity responsible for cross-linking the peptidoglycan cell wall (1
). The catalytic domain exhibits homology to other transpeptidases involved in peptidoglycan metabolism (18
). In particular, the active site contains a universally conserved serine residue (S307) that forms a covalent bond with the peptide substrate during transpeptidation, a reaction that proceeds via an acyl enzyme intermediate. The catalytic domain also binds β-lactam antibiotics, which mimic a transpeptidase substrate and serve as suicide inhibitors by forming a long-lived covalent adduct with the catalytic serine (28
). Thus, FtsI is also known as penicillin-binding protein 3 (PBP3).
The noncatalytic domain extends from residues 41 to 236. This domain was first recognized as a distinct structural element when sequence comparisons revealed that it was conserved among a subset of transpeptidases called the class B high-molecular-mass penicillin-binding proteins (16
). It has been proposed that the noncatalytic domain interacts with other proteins of the septal ring and that these interactions are important for septal localization of FtsI, recruitment of downstream proteins such as FtsN, and regulation of the transpeptidase activity of FtsI (33
). Marrec-Fairley and coworkers have characterized several mutants with properties consistent with these roles. Most notably, they have suggested that changing D58 to V prevents localization of FtsI to the septal ring and changing both R210 and R213 to Q (a double mutation) interferes with a protein-protein interaction but not septal localization per se (29
). These interpretations were based on the recessive nature of the D58V lesion and the dominant nature of the R210Q-R213Q lesions. The abilities of the mutant proteins to localize and/or interact with other division proteins were not tested directly.
The three-dimensional structure of an FtsI homolog, PBP2x from Streptococcus pneumoniae
, has been solved by X-ray crystallography (Fig. ) (13
). The catalytic domain looks like a typical penicillin-binding domain, except that the active site is at the center of a long groove that is not seen in other penicillin-binding enzymes. This groove presumably accommodates the two peptides to be joined in the cross-linking reaction. The noncatalytic domain has a strikingly elongated shape. One end fits into a pocket in the catalytic domain, and two long arms extend into solution, creating a large central cavity that could accommodate another protein in vivo. This arrangement suggests that the noncatalytic domain regulates the catalytic domain and that the arms might engage in protein-protein interactions (35
). This arrangement also suggests why the noncatalytic domain appears to be required for folding of the catalytic domain (17
); these domains interact so extensively that neither is likely to be stable alone.
To identify sequences in FtsI that are needed for localization to the septal ring and for interaction with other division proteins, we have isolated and characterized mutant forms of the protein that fail to support cell division but appear to be normal for insertion into the membrane and catalytic activity (penicillin binding). These mutants are described in this paper. Single-amino-acid substitutions that severely impaired localization invariably mapped to the membrane-spanning segment rather than the noncatalytic domain. We also found several mutant proteins that were defective in recruitment of FtsN to the septal ring. These proteins had single-amino-acid substitutions in the noncatalytic domain and might define a site of FtsI-FtsN interaction. Finally, we found one lesion in the noncatalytic domain that renders FtsI incapable of supporting cell division, but the protein appears essentially normal in all of our assays.