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.     

ZipA is required for recruitment of FtsK, FtsQ, FtsL, and FtsN to the septal ring in Escherichia coli

The septal ring in Escherichia coli consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN. Recruitment of FtsK and all downstream components requires the prior localization of FtsA. Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA. The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.     

Probing the catalytic activity of a cell division-specific transpeptidase in vivo with beta-lactams

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of Escherichia coli. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three beta-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the ftsA3(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.     

The distinctive cell division interactome of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis>

Background

Bacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW.

Results

Using a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsA_Ng were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW.

Conclusions

Results from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.

     

Role of FtsEX in cell division of Escherichia coli: viability of ftsEX mutants is dependent on functional SufI or high osmotic strength

In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.     

The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia coli.     

Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET

The bacterial cell division machinery is organized in the so‐called divisome composed of highly dynamic but low abundant interacting (membrane‐bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background‐insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ‐ring and the so‐called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan‐synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto‐ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto‐ring elements.     

FtsI and FtsW Are Localized to the Septum in Escherichia coli

The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in ftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.     

The Escherichia coli FtsK functional domains involved in its interaction with its divisome protein partners

FtsK is a multifunctional protein involved in both cell division and chromosome segregation. As far as its role in cell division is concerned, FtsK is among the first divisome proteins that localizes at mid-cell, after FtsZ, FtsA and ZipA, and is required for the recruitment of the other divisome components. The ability of FtsK to interact with several cell division proteins, namely FtsZ, FtsQ, FtsL and FtsI, by the two-hybrid assay was already shown by our group. In this work, we describe the identification of the protein domain(s) involved in the interaction with the cell division partner proteins. The biological role of some interactions is also discussed.     

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 deficiency in S-adenosylmethionine synthetase interrupts assembly of the septal ring in Escherichia coli K-12

A mutant in which S-adenosylmethionine synthetase is underexpressed makes filaments with no visible septa. Examination with GFP fusions to various septal proteins shows that FtsZ, ZipA and FtsA localize to the septal ring, but FtsQ, FtsW, FtsI or FtsN do not. The requirement for S-adenosylmethionine suggests that some methylation reaction is required before a complete septal ring can be assembled.     

The Escherichia coli Cell Division Protein FtsW Is Required To Recruit Its Cognate Transpeptidase, FtsI (PBP3), to the Division Site

The bacterial cell division protein FtsW has been suggested to perform two functions: stabilize the FtsZ cytokinetic ring, and facilitate septal peptidoglycan synthesis by the transpeptidase FtsI (penicillin-binding protein 3). We show here that depleting Escherichia coli cells of FtsW had little effect on the abundance of FtsZ rings but abrogated recruitment of FtsI to potential division sites. Analysis of FtsW localization confirmed and extended these results; septal localization of FtsW required FtsZ, FtsA, FtsQ, and FtsL but not FtsI. Thus, FtsW is a late recruit to the division site and is essential for subsequent recruitment of its cognate transpeptidase FtsI but not for stabilization of FtsZ rings. We suggest that a primary function of FtsW homologues--which are found in almost all bacteria and appear to work in conjunction with dedicated transpeptidases involved in division, elongation, or sporulation--is to recruit their cognate transpeptidases to the correct subcellular location.     

The monofunctional glycosyltransferase of Escherichia coli localizes to the cell division site and interacts with penicillin-binding protein 3, FtsW, and FtsN

The monofunctional peptidoglycan glycosyltransferase (MtgA) catalyzes glycan chain elongation of the bacterial cell wall. Here we show that MtgA localizes at the division site of Escherichia coli cells that are deficient in PBP1b and produce a thermosensitive PBP1a and is able to interact with three constituents of the divisome, PBP3, FtsW, and FtsN, suggesting that MtgA may play a role in peptidoglycan assembly during the cell cycle in collaboration with other proteins.     

Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls cell septation and mediates the formation of a trimeric complex involving FtsZ, FtsW and PBP3 in mycobacteria

In bacteria, biogenesis of cell wall at the division site requires penicillin-binding protein 3 (PBP3) (or Ftsl). Using pull-down, bacterial two-hybrid, and peptide-based interaction assays, we provide evidence that FtsW of Mycobacterium tuberculosis (FtsWMTB) interacts with PBP3 through two extracytoplasmic loops. Pro306 in the larger loop and Pro386 in the smaller loop of FtsW are crucial for these interactions. Fluorescence microscopy shows that conditional silencing of ftsW in Mycobacterium smegmatis prevents cell septation and positioning of PBP3 at mid-cell. Pull-down assays and conditional depletion of FtsW in M. smegmatis provide evidence that FtsZ, FtsW and PBP3 of mycobacteria are capable of forming a ternary complex, with FtsW acting as a bridging molecule. Bacterial three-hybrid analysis suggests that in M. tuberculosis, the interaction (unique to mycobacteria) of FtsZ with the cytosolic C-tail of FtsW strengthens the interaction of FtsW with PBP3. ftsW of M. smegmatis could be replaced by ftsW of M. tuberculosis. FtsWMTB could support formation of the FtsZ-FtsW-PBP3 ternary complex in M. smegmatis. Our findings raise the possibility that in the genus Mycobacterium binding of FtsZ to the C-tail of FtsW may modulate its interactions with PBP3, thereby potentially regulating septal peptidoglycan biogenesis.     

Structural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.     

The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.     

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re‐direct peptidoglycan biosynthesis to mid‐cell during cell division in polar‐growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid‐cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid‐cell, exhibit GTPase activity and form co‐polymers, only one, FtsZ_AT, is required for cell division. We find that FtsZ_AT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid‐cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ_AT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid‐cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.     

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsW^E289G and FtsI^K211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.     

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.     

FtsN, a late recruit to the septum in Escherichia coli

The localization of FtsN in Escherichia coli was investigated by immunofluorescence microscopy. FtsN is an essential cell division protein with a simple bitopic topology, a short N-terminal cytoplasmic segment fused to a large carboxy periplasmic domain through a single transmembrane domain. FtsN was found to localize to the septum in a ring pattern similar to that observed for FtsZ and FtsA, although the frequency of cells with rings was less. A MalG–FtsN fusion was also localized to the septum, indicating that the information for FtsN localization is supplied by its periplasmic domain. FtsN localization was dependent upon the prior localization of FtsZ and FtsA and required the function of FtsI and FtsQ. Consistent with FtsN functioning after FtsZ, Z rings were observed in a mutant depleted of FtsN.