Characterization of the Porphyromonas gingivalis FtsZ Containing a Novel GTPase Activity

The FtsZ protein is a GTPase that is essential for cell division. We have cloned, sequenced, and expressed the FtsZ (PgFtsZ) gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease. The PgFtsZ gene consisted of 1374 bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da. The deduced amino acid sequence exhibited a significant homology with E. coli FtsZ (54% identical residues). Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG) and hydrolysis (NLDFADV). When PgFtsZ was overexpressed in E. coli, cell division was inhibited. Recombinant PgFtsZ was purified to homogeneity and characterized. The purified PgFtsZ exhibited GTPase activity even in the absence of Mg²⁺, and completely retained its activity with EDTA. Furthermore, Na⁺ and K⁺ ions inhibited its GTPase activity in a dose-dependent manner. These results suggest that PgFtsZ contains an atypical GTPase activity that has not been previously described. Received: 25 May 2001 / Accepted: 8 August 2001     

FtsZ ring: the eubacterial division apparatus conserved in archaebacteria

FtsZ is a tubulin-like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.     

FtsZ and the division of bacterial cell

In this review we have tried to describe proteins and supermolecular structures which take part in the division of bacterial cell. The principal cell division protein of the most of prokaryotes is FtsZ, a homologue of eukaryotic tubulin. FtsZ just as tubulin is capable to bind and hydrolyze GTP. The division of bacterial cell begins with forming of so called divisome. The basis of such divisome is a contractile ring (Z ring); the ring encircles the cell about midcell. Z ring consists of a bundle of laterally bound protofilaments, which have been formed as a result of FtsZ polymerization. Z ring is rigidly bounded to cytozolic side of inner membrane with participation of FtsA, ZipA, FtsW and many other cell division proteins of divisome. The ring directs the process of cytokinesis transmitting power of constriction to membrane. Primary structures of members of the family of prokaryotic FtsZs differ from eukaryotic tubulines significantly except the region, where the site of GTP binding is placed. There is high degree of homology between structures of these proteins in the region. FtsZ is one of the most conservative proteins in prokaryotes, but ftsZ genes have not been found in completely sequenced genomes of several species of microorganisms. There are 2 species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea among them. So these organisms divide without FtsZ. There are many open reading frames which encode proteins with unknown functions in genomes of U. parvum and M. mobile. The comparison of primary structures of these hypothetical proteins with structures of cell division proteins did not allow researchers to find similar proteins among them. We suppose that the process of cell division of these organisms should recruit proteins with function similar to FtsZ and having homologous with FtsZ or other cell division proteins spatial structures.     

Peptidase activity of dipeptidyl aminopeptidase IV produced by Porphyromonas gingivalis is important but not sufficient for virulence

Porphyromonas gingivalis is a pathogen associated with adult periodontitis, which is a chronic inflammatory disease characterized by breakdown of the periodontal tissue. Dipeptidyl aminopeptidase IV (DPPIV) produced by P. gingivalis has been considered to be a potential virulence factor based on the finding that the virulence was reduced by disruption of the gene (dpp ) coding for DPPIV. In the present study, we constructed a shuttle vector that is mobilized from Escherichia coli to P. gingivalis and is maintained stably in both bacteria, and we showed that the virulence was restored by introducing the cloned wild-type dpp gene into the null mutant of P. gingivalis using our vector system. To assess the implications of the peptidase activity in the virulence, mutant DPPIV with the catalytic Ser mutagenized to Ala (DPPSA) was produced. The P. gingivalis strain expressing DPPSA exhibited an intermediate virulence between the strain expressing wild-type DPPIV and the strain harboring a vector. From these results, it is suggested that peptidase activity is very important but not sufficient for virulence.     

Glutamate-induced assembly of bacterial cell division protein FtsZ

The polymerization of FtsZ is a finely regulated process that plays an essential role in the bacterial cell division process. However, only a few modulators of FtsZ polymerization are known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and extent of polymerization of FtsZ in a concentration-dependent manner; approximately 90% of the protein was sedimented as polymer in the presence of 1 m glutamate. Electron micrographs of glutamate-induced polymers showed large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers; interestingly, although 1 m glutamate produced a larger light-scattering signal than produced by 10 mm calcium, the amount of polymer sedimented in the presence of 1 m glutamate and 10 mm calcium was similar. Thus, the increased light scattering in the presence of glutamate must be due to its ability to induce more extensive bundling of FtsZ polymers than calcium. The data suggest that calcium and glutamate might induce FtsZ polymerization by different mechanisms.     

FtsZ, a tubulin homologue in prokaryote cell division

Sequence alignments convincingly demonstrate that FtsZ is a prokaryotic homologue of eukaryotic tubulins. FtsZ appears to be universal in eubacteria and archaebacteria and has also been identified in chloroplasts. Like tubulin, it appears to have a cytoskeletal role as both proteins assemble into two types of characteristic polymers in vitro - protofilament sheets and mini-rings. Recent advances in immunofluorescence and the use of green fluorescent protein have provided clear images of FtsZ localized in a ring at the septation site in bacteria, and new insights into assembly of this Z-ring.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

ZipA is a MAP-Tau homolog and is essential for structural integrity of the cytokinetic FtsZ ring during bacterial cell division

The first visible event in prokaryotic cell division is the assembly of the soluble, tubulin-like FtsZ GTPase into a membrane-associated cytokinetic ring that defines the division plane in bacterial and archaeal cells. In the temperature-sensitive ftsZ84 mutant of Escherichia coli, this ring assembly is impaired at the restrictive temperature causing lethal cell filamentation. Here I present genetic and morphological evidence that a 2-fold higher dosage of the division gene zipA suppresses thermosensitivity of the ftsZ84 mutant by stabilizing the labile FtsZ84 ring structure in vivo. I demonstrate that purified ZipA promotes and stabilizes protofilament assembly of both FtsZ and FtsZ84 in vitro and cosediments with the protofilaments. Furthermore, ZipA organizes FtsZ protofilaments into arrays of long bundles or sheets that probably represent the physiological organization of the FtsZ ring in bacterial cells. The N-terminal cytoplasmic domain of membrane-anchored ZipA contains sequence elements that resemble the microtubule-binding signature motifs in eukaryotic Tau, MAP2 and MAP4 proteins. It is postulated that the MAP-Tau-homologous motifs in ZipA mediate its binding to FtsZ, and that FtsZ-ZipA interaction represents an ancient prototype of the protein-protein interaction that enables MAPs to suppress microtubule catastrophe and/or to promote rescue.     

The polymerization mechanism of the bacterial cell division protein FtsZ

Translin and Trax are components of an RNA binding complex initially detected in extracts of brain and testes. Although other tissues appear to contain much lower or negligible levels of the Translin/Trax gel-shift complex, we found, unexpectedly, that several of these peripheral tissues express Translin and Trax proteins at levels comparable to those present in brain. In this study, we demonstrate that the paradoxically low levels of the Translin/Trax complex in kidney and other peripheral tissues are due to masking of these sites by endogenous RNA. Thus, these findings indicate that the Translin/Trax complex is involved in RNA processing in a broader range of tissues than previously recognized.     

Magnesium-induced linear self-association of the FtsZ bacterial cell division protein monomer. The primary steps for FtsZ assembly

The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg(2+) bound, it shows GTPase activity, and requires GTP and Mg(2+) to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg(2+) concentrations, at pH 7.5, is a compact and globular monomer. Mg(2+) induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg(2+) and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg(2+) for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear single-stranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.     

FtsZ regulates frequency of cell division in Escherichia coli.

Cell division is regulated so that it occurs only once per cell cycle. In Escherichia coli, a rod-shaped bacterium, division normally takes place at the center of the long axis of the cell; however, in the minicell mutant, division can also take place at the cell pole. Such divisions take place at the expense of normal divisions, resulting in an overall increase in nucleated cell length. We report here that increasing the level of FtsZ can completely suppress the cell length of the minicell mutant by increasing the frequency at which cell division events take place. This result suggests that the level of FtsZ controls the frequency of cell division in E. coli.     

Interaction between FtsZ and inhibitors of cell division.

The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system. An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains. This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA. No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system. However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD. The implications of these findings for cell division are discussed.     

Spatial separation of FtsZ and FtsN during cell division

The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex known as ‘the divisome’. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super‐complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.     

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.     

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.     

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

P. gingivalis, an important periodontal pathogen associated with adult periodontitis and a likely contributing factor to atherosclerosis and cardiovascular disease, traffics in endothelial cells via the autophagic pathway. Initially, P. gingivalis rapidly adheres to the host cell surface followed by internalization via lipid rafts and incorporation of the bacterium into early phagosomes. P. gingivalis activates cellular autophagy to provide a replicative niche while suppressing apoptosis. The replicating vacuole contains host proteins delivered by autophagy that are used by this asaccharolytic pathogen to survive and replicate within the host cell. When autophagy is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to the phagolysosome where it is destroyed and degraded. Therefore, the survival of P. gingivalis depends upon the activation of autophagy and survival of the endothelial host cell, but the mechanism by which P. gingivalis accomplishes this remains unclear.