Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly

Cell division in Escherichia coli requires the concerted action of at least 10 essential proteins. One of these proteins, FtsQ, is physically associated with multiple essential division proteins, including FtsK, FtsL, FtsB, FtsW, and FtsI. In this work we performed a genetic analysis of the ftsQ gene. Our studies identified C-terminal residues essential for FtsQ's interaction with two downstream proteins, FtsL and FtsB. Here we also describe a novel screen for cell division mutants based on a wrinkled-colony morphology, which yielded several new point mutations in ftsQ. Two of these mutations affect localization of FtsQ to midcell and together define a targeting role for FtsQ's alpha domain. Further characterization of one localization-defective mutant protein [FtsQ(V92D)] revealed an unexpected role in localization for the first 49 amino acids of FtsQ. Finally, we found a suppressor of FtsQ(V92D) that was due to a point mutation in domain 1C of FtsA, a domain previously implicated in the recruitment of divisome proteins. However, despite reports of a potential interaction between FtsA and FtsQ, suppression by FtsA(I143L) is not mediated via direct contact with FtsQ. Rather, this mutation acts as a general suppressor of division defects, which include deletions of the normally essential genes zipA and ftsK and mutations in FtsQ that affect both localization and recruitment. Together, these results reveal increasingly complex connections within the bacterial divisome.     

Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of Escherichia coli

The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.     

The FtsQ protein of Escherichia coli: membrane topology, abundance, and cell division phenotypes due to overproduction and insertion mutations.

The ftsQ gene is one of several genes thought to be specifically required for septum formation in Escherichia coli. Published work on the cell division behavior of ftsQ temperature-sensitive mutants suggested that the FtsQ product is required throughout the whole process of septum formation. Here we provide additional support for this hypothesis based on microscopic observations of the cell division defects resulting from insertional and temperature-sensitive mutations in the ftsQ gene, and constitutive overexpression of its gene product. On the basis of the published, predicted amino acid sequence of the FtsQ protein and our analysis of fusion proteins of the FtsQ protein to bacterial alkaline phosphatase, we conclude that FtsQ is a simple cytoplasmic membrane protein with a approximately 25-amino-acid cytoplasmic domain and a approximately 225-amino-acid periplasmic domain. We estimate that the FtsQ protein is present at about 22 copies per cell.     

The β-Lactam Resistance Protein Blr,a Small Membrane Polypeptide,Is a Component of the Escherichia coli Cell Division Machinery

In Escherichia coli, cell division is performed by a multimolecular machinery called the divisome, made of 10 essential proteins and more than 20 accessory proteins. Through a bacterial two-hybrid library screen, we identified the E. coli β-lactam resistance protein Blr, a short membrane polypeptide of 41 residues, as an interacting partner of the essential cell division protein FtsL. In addition to FtsL, Blr was found to associate with several other divisomal proteins, including FtsI, FtsK, FtsN, FtsQ, FtsW, and YmgF. Using fluorescently tagged Blr, we showed that this peptide localizes to the division septum and that its colocalization requires the presence of the late division protein FtsN. Although Blr is not essential, previous studies have shown that the inactivation of the blr gene increased the sensitivity of bacteria to β-lactam antibiotics or their resistance to cell envelope stress. Here, we found that Blr, when overproduced, restores the viability of E. coli ftsQ1(Ts) cells, carrying a thermosensitive allele of the ftsQ gene, during growth under low-osmotic-strength conditions (e.g., in synthetic media or in Luria-Bertani broth without NaCl). In contrast, the inactivation of blr increases the osmosensitivity of ftsQ1(Ts) cells, and blr ftsQ1 double mutants exhibit filamentous growth in LB broth even at a moderate salt concentration (0.5% NaCl) compared to parental ftsQ1(Ts) cells. Altogether, our results suggest that the small membrane polypeptide Blr is a novel component of the E. coli cell division apparatus involved in the stabilization of the divisome under certain stress conditions.     

Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts).

A new cell division gene, ftsN, was identified in Escherichia coli as a multicopy suppressor of the ftsA12(Ts) mutation. Remarkably, multicopy ftsN suppressed ftsI23(Ts) and to a lesser extent ftsQ1(Ts); however, no suppression of the ftsZ84(Ts) mutation was observed. The suppression of ftsA12(Ts), ftsI23(Ts), and ftsQ1(Ts) suggests that FtsN may interact with these gene products during cell division. The ftsN gene was located at 88.5 min on the E. coli genetic map just downstream of the cytR gene. ftsN was essential for cell division, since expression of a conditional null allele led to filamentation and cell death. DNA sequence analysis of the ftsN gene revealed an open reading frame of 319 codons which would encode a protein of 35,725 Da. The predicted gene product had a hydrophobic sequence near its amino terminus similar to the noncleavable signal sequences found in several other Fts proteins. The presumed extracellular domain was unusual in that it was rich in glutamine residues. A 36-kDa protein that was localized to the membrane fraction was detected in minicells containing plasmids with the ftsN gene, confirming that FtsN was a membrane protein.     

Septal Localization of FtsQ, an Essential Cell Division Protein in Escherichia coli

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.     

Regulation of cell division in Escherichia coli K-12: probable interactions among proteins FtsQ, FtsA, and FtsZ.

In Escherichia coli, the FtsQ, FtsA, and FtsZ proteins are believed to play essential roles in the regulation of cell division. Of the three proteins, FtsZ has received the most attention, particularly because of its interactions with SfiA. Double mutants which carry mutations located in the ftsQ, ftsA, or ftsZ gene in combination with the lon-1 mutation were constructed. In the presence of the lon-1 mutation, which is known to stabilize SfiA, the ftsQ1 mutant cells were not capable of forming colonies on a rich agar medium, whereas mutant cells harboring either one of the mutations grew well on this medium. Examination of lon-1 fts double-mutant cells for sensitivity to UV light revealed that those carrying the ftsA10 allele were resistant. It was also observed that in the presence of a multicopy plasmid containing a wild-type ftsZ gene, the ftsQ1 mutant filamented markedly following a nutritional shift-up and that the division rate of ftsZ84 mutant cells was slightly reduced when they harbored a wild-type ftsQ-containing plasmid. The possibility that the Fts proteins are interacting with one another and forming a molecular complex is discussed.     

FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division

During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.     

Contribution of the FtsQ transmembrane segment to localization to the cell division site

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.     

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.     

Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.     

Evidence for functional overlap among multiple bacterial cell division proteins: compensating for the loss of FtsK

In Escherichia coli, at least 12 proteins colocalize to the cell midpoint, assembling into a membrane-associated protein machine that forms the division septum. Many of these proteins, including FtsK, are essential for viability but their functions in cell division are unknown. Here we show that the essential function of FtsK in cell division can be partially bypassed. Cells containing either the ftsA R286W mutation or a plasmid carrying the ftsQAZ genes suppressed a ftsK44(ts) allele efficiently. Moreover, ftsA R286W or multicopy ftsQAZ, which can largely bypass the requirement for the essential cell division gene zipA, allowed cells with a complete deletion of ftsK to survive and divide, although many of these ftsK null cells formed multiseptate chains. Green fluorescent protein (GFP) fusions to FtsI and FtsN, which normally depend on FtsK to localize to division sites, localized to division sites in the absence of FtsK, indicating that FtsK is not directly involved in their recruitment. Cells expressing additional ftsQ, and to a lesser extent ftsB and ftsN, were able to survive and divide in the absence of ftsK, although cell chains were often formed. Surprisingly, the cytoplasmic and transmembrane domains of FtsQ, while not sufficient to complement an ftsQ null mutant, conferred viability and septum formation in the absence of ftsK. These findings suggest that the N-terminal domain of FtsK is normally involved in stability of the division protein machine and shares functional overlap with FtsQ, FtsB, FtsA, ZipA and FtsN.     

A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region

Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB. FtsQ, in turn, requires FtsK for its localization. Here, we show that FtsL, FtsB and FtsQ form a complex in vivo. Strikingly, this complex forms in the absence of FtsK, which is required for the localization of all three proteins to the mid-cell. These findings indicate that the FtsL, FtsB, FtsQ interactions can take place in cells before movement to the mid-cell and that migration to this position might occur only after the formation of the complex. Evidence indicating the regions of the three proteins involved in complex formation is presented. These findings provide the first example of preassembly of a subcomplex of cell division proteins before their localization to the septal region.     

A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli

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.     

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 beta-strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsADelta1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two-hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsADelta1C are therefore incomplete and do not support division. The production of high levels of FtsADelta1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12-S13 strands of domain 2B generates a protein (FtsADeltaS12-13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild-type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsADeltaS12-13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid-free cells. The S12-S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.     

The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.

Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of the ftsZ, ftsA, or ftsQ genes. We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ. Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA. Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ. In addition, although wild-type strains were relatively insensitive to overexpression of ftsQ, we observed that cell division was sensitized to ftsQ overexpression in ftsI, ftsA, and ftsZ mutants. Among these, the ftsI mutant was the most sensitive. These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.