FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.In Escherichia coli, the division septum forms via the coordinated inward growth of all three layers of the cell envelopethe cytoplasmic membrane, the peptidoglycan wall, and the outer membrane. To date, about a dozen E. coli genes are known to be specifically required for septation (3, 11). These genes share two important properties: (i) loss of function mutations result in the formation of long, aseptate filaments with regularly spaced nucleoids (the fts, or filamentation temperature-sensitive phenotype), and (ii) the proteins encoded by these genes localize to the division site. Because cell division genes are generally essential and because lesions in many housekeeping genes can affect cell division indirectly, there have not been any exhaustive screens for division mutants. Thus, it seems likely that more division genes remain to be described.A number of mutant hunts, starting with the pioneering work of Hirota and coworkers in the 1960s, suggested that there is an important cell division gene located at about 76 min on the E. coli chromosome (30). This locus was originally designated ftsE. One interesting property of ftsE mutants is that many are salt remedial, meaning that viability is restored by inclusion of NaCl in the growth medium. The amount of salt required for rescue is strain dependent, but generally in the range of 0.5%. Studies by Salmond and colleagues in the 1980s revealed that "ftsE" comprised two genes, which were then designated ftsE and ftsX (13). Moreover, the sequence of these genes revealed clear homology to ABC transporters; FtsE is the ATP-binding cassette (ABC) component, while FtsX is the membrane component. ABC transporters use energy from ATP to transport a wide variety of sub...

FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring. FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain. The last two domains reside in the periplasm. We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened ϳ7,500 transformants for gfp-ftsI alleles that failed to complement an ftsI null mutant. Western blotting and penicillin-binding assays were then used to weed out proteins that were unstable, failed to insert into the cytoplasmic membrane, or were defective in catalysis. The remaining candidates were tested for septal localization and ability to recruit another division protein, FtsN, to the septal ring. Mutant proteins severely defective in localization to the septal ring all had lesions in one of three amino acids-R23, L39, or Q46-that are in or near the transmembrane helix and implicate this region of FtsI in septal localization. Mutant FtsI proteins defective in recruitment of FtsN all had lesions in one of eight residues in the noncatalytic domain. The most interesting of these mutants had lesions at G57, S61, L62, or R210. Although separated by ϳ150 residues in the primary sequence, these amino acids are close together in the folded protein and might constitute a site of FtsI-FtsN interaction.Cell division in Escherichia coli requires approximately a dozen proteins, all of which localize to a ring structure at the division site ( Fig. 1A) (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 crosslinking into the peptidoglycan cell wall in the division septum (6, 37). AmiC is a periplasmic amidase that hydrolyzes peptide cross-links and contributes to the separation of daughter cells after division (25).The ftsI 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 cataly...

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FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.The use of fluorescence microscopy to visualize proteins in bacteria has revealed that many proteins are not distributed randomly but instead localize to specific subcellular sites, such as the midcell or pole(s) (22, 37). Moreover, proteins that are targeted to specific sites often fail to function properly if they are mislocalized. Despite the importance of proper localization, little is known about how targeting information is encoded in the amino acid sequences of bacterial proteins. In this report, we describe a small peptide from a bacterial cell division protein, FtsI, that is sufficient to target green fluorescent protein (GFP) to the division site in Escherichia coli. Interestingly, this peptide is a transmembrane helix (TMH). These findings help to clarify how targeting information is encoded in FtsI's primary sequence and demonstrate that a bacterial TMH can serve as a targeting signal.FtsI, also known as penicillin-binding protein 3 (PBP3), is a transpeptidase needed for cross-linking septal peptidoglycan (1,3,38). Previous studies from a number of laboratories have shown that FtsI is one of over a dozen proteins that localize to the division site, where they form a structure called the septal ring (for recent reviews, see references 12 and 43). As division proceeds, the ring constricts so as to remain at the leading edge of the developing septum. The septal ring is thought to be a multiprotein complex that mediates cell division. Studies of septal ring assembly in various mutant backgrounds have revealed that, at least in E. coli, the division proteins are recruited to the ring in a sequential fashion. In this hierarchy, FtsI is one of the last proteins to join the ring; localization of FtsI appears to depend upon the prior localization of FtsZ, FtsA, ZipA, FtsEX (though this is a leaky requirement), FtsK, FtsQ, FtsBL, and FtsW. This scheme suggests that FtsI is recruited to the septal ring by a cascade of protein-protein interactions involved in the assembly of a multiprotein complex. Moreover, FtsI might localize by binding to ...