Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Identification of a region of FtsA required for interaction with FtsZ

The assembly of the Z ring is the earliest step in bacterial cell division. In Escherichia coli this assembly requires either FtsA or ZipA which bind to a conserved, C-terminal 17 amino acid motif in FtsZ and to the membrane. The FtsZ-ZipA interaction is well characterized; however, nothing is known about the region of FtsA involved in the interaction with FtsZ even though the FtsA-FtsZ interaction is nearly ubiquitous in Eubacteria. FtsA is proposed to bind to the membrane through its conserved C-terminal amphiphatic helix before efficiently interacting with FtsZ. Based upon this model we designed a genetic screen to identify mutants specifically impaired for the FtsA-FtsZ interaction. The mutants obtained retain the ability to be targeted to the membrane but fail to be recruited to the Z ring or interact with FtsZ in the yeast two-hybrid system. These mutants do not complement an ftsA-depletion strain. Through this approach we have identified a region of FtsA containing some invariant residues which is required for binding to FtsZ. The results support our model that FtsA is targeted to the membrane before it interacts with FtsZ and demonstrates that this interaction plays an essential role in E. coli cell division.     

Roles of FtsA and FtsZ in Activation of Division Sites

Increasing FtsZ induces the formation of minicells at cell poles but does not increase the frequency or timing of central divisions. A coordinate increase in both FtsZ and FtsA, however, increases the frequency of both polar and central divisions.     

A Rhodanine Derivative CCR-11 Inhibits Bacterial Proliferation by Inhibiting the Assembly and GTPase Activity of FtsZ

A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen-oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC(50)) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC(50) value of 18.1 ± 0.2 μM (～15 × IC(50) of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.     

Inhibition of cell division initiation by an imbalance in the ratio of FtsA to FtsZ.

Elevated levels of FtsA protein block cell division at a very early stage, similar to that caused by inhibition of the action of FtsZ. In contrast, overexpression of FtsA and FtsZ together does not block division. A specific ratio of FtsA to FtsZ protein, therefore, is required for cell division.     

Assembly dynamics of Mycobacterium tuberculosis FtsZ

We have investigated the assembly of FtsZ from Mycobacterium tuberculosis (MtbFtsZ). Electron microscopy confirmed the previous observation that MtbFtsZ assembled into long, two-stranded filaments at pH 6.5. However, we found that assembly at pH 7.2 or 7.7 produced predominantly short, one-stranded protofilaments, similar to those of Escherichia coli FtsZ (EcFtsZ). Near pH 7, which is close to the pH of M. tuberculosis cytoplasm, MtbFtsZ formed a mixture of single- and two-stranded filaments. We developed a fluorescence resonance energy transfer assay to measure the kinetics of initial assembly and the dynamic properties at steady state. Assembly of MtbFtsZ reached a plateau after 60-100 s, about 10 times slower than EcFtsZ. The initial assembly kinetics were similar at pH 6.5 and 7.7, despite the striking difference in the polymer structures. Both were fit with a cooperative assembly mechanism involving a weak dimer nucleus, similar to EcFtsZ but with slower kinetics. Subunit turnover and GTPase at steady state were also about 10 times slower for MtbFtsZ than for EcFtsZ. Specifically, the half-time for subunit turnover in vitro at pH 7.7 was 42 s for MtbFtsZ compared with 5.5 s for EcFtsZ. Photobleaching studies in vivo showed a range of turnover half-times with an average of 25 s for MtbFtsZ as compared with 9 s for EcFtsZ.     

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.     

Timing of FtsZ Assembly in Escherichia coli

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.     

SlmA Antagonism of FtsZ Assembly Employs a Two-pronged Mechanism like MinCD

Assembly of the Z-ring over unsegregated nucleoids is prevented by a process called nucleoid occlusion (NO), which in Escherichia coli is partially mediated by SlmA. SlmA is a Z ring antagonist that is spatially regulated and activated by binding to specific DNA sequences (SlmA binding sites, SBSs) more abundant in the origin proximal region of the chromosome. However, the mechanism by which SBS bound SlmA (activated form) antagonizes Z ring assembly is controversial. Here, we report the isolation and characterization of two FtsZ mutants, FtsZ-K190V and FtsZ-D86N that confer resistance to activated SlmA. In trying to understand the basis of resistance of these mutants, we confirmed that activated SlmA antagonizes FtsZ polymerization and determined these mutants were resistant, even though they still bind SlmA. Investigation of SlmA binding to FtsZ revealed activated SlmA binds to the conserved C-terminal tail of FtsZ and that the ability of activated SlmA to antagonize FtsZ assembly required the presence of the tail. Together, these results lead to a model in which SlmA binding to an SBS is activated to bind the tail of FtsZ resulting in further interaction with FtsZ leading to depolymerization of FtsZ polymers. This model is strikingly similar to the model for the inhibitory mechanism of the spatial inhibitor MinCD.     

Regulation of C-type Lectin Antimicrobial Activity by a Flexible N-terminal Prosegment

Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIγ and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity.The gastrointestinal tracts of mammals are heavily colonized with vast symbiotic microbial communities and are also a major portal of entry for bacterial pathogens. To cope with these complex microbial challenges, intestinal epithelial cells produce a diverse repertoire of protein antibiotics from multiple distinct protein families (1). These proteins are secreted apically into the luminal environment of the intestine where they play a pivotal role in protecting against enteric infections (2, 3) and may also function to limit opportunistic invasion by symbiotic bacteria (4).We previously identified lectins as a novel class of secreted antibacterial proteins in the mammalian intestine. RegIIIγ is a member of the RegIII subgroup of the C-type lectin family and is expressed in the small intestine in response to microbial cues (5), stored in epithelial cell secretory granules, and released into the small intestinal lumen (5). Similarly, HIP/PAP (hepatointestinal pancreatic/pancreatitis-associated protein; the human ortholog of RegIIIγ)⁶ is expressed in the human intestine (6) and is up-regulated in patients with inflammatory bowel disease (7). These proteins are produced in multiple epithelial lineages, including enterocytes and Paneth cells (5, 6). Both RegIIIγ and HIP/PAP are directly bactericidal at low micromolar concentrations for Gram-positive bacteria (5), revealing a previously unappreciated biological function for mammalian lectins. The antibacterial functions of RegIIIγ and HIP/PAP are dependent upon binding bacterial targets through interactions with peptidoglycan (5). As peptidoglycan is localized on surfaces of Gram-positive bacteria but is buried in the periplasmic space of Gram-negative bacteria, this binding activity provides a molecular explanation for the Gram-positive specific bactericidal effects of these lectins. Although the mechanism of lectin-mediated antibacterial activity remains unclear, RegIIIγ and HIP/PAP have been shown to elicit extensive damage to the cell surfaces of targeted bacteria (5).In this study, we show that C-type lectin bactericidal activity is under stringent post-translational control. RegIIIγ and HIP/PAP each undergo in vivo proteolytic removal of a flexible anionic N-terminal prosegment that maintains the proteins in a biologically inactive state. NMR spectroscopy suggests that the prosegment functions by controlling a two-state conformational switch between the biologically active and inactive states of the protein. We propose that this regulatory mechanism allows the host to restrict expression of RegIII lectin antibacterial activity to the intestinal lumen. Together, our findings represent a unique example of post-translational control of C-type lectin biological activity, and provide novel insight into the regulation of lectin-mediated innate immunity in the mammalian intestine.     

Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly

Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division.Chloroplasts evolved from an ancient cyanobacterium through endosymbiosis (Gould et al., 2008; Keeling, 2013). Like their prokaryotic relatives, chloroplasts replicate by binary fission, which is driven by a dynamic macromolecular complex located at the middle of the organelle (Falconet, 2011; Miyagishima et al., 2011; Osteryoung and Pyke, 2014). The major contractile components of the division complex include the FtsZ ring (Z ring), which assembles on the stromal surface of the inner envelope membrane (IEM; McAndrew et al., 2001; Vitha et al., 2001), and the DYNAMIN-RELATED PROTEIN 5B (DRP5B; also called ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 [ARC5]) ring, which assembles on the cytosolic surface of the outer envelope membrane (OEM; Gao et al., 2003; Miyagishima et al., 2003; Yoshida et al., 2006). In green algae and land plants, the Z ring is composed of the tubulin-like, heteropolymer-forming proteins FtsZ1 and FtsZ2, which are both required for normal Z-ring function (Schmitz et al., 2009; TerBush and Osteryoung, 2012). DRP5B is a member of the dynamin family of membrane fission proteins, which polymerize into collar-like structures to mediate a variety of membrane fission processes in eukaryotes (Morlot and Roux, 2013). The Z ring and DRP5B ring function together to drive the simultaneous constriction of the IEM and OEM during chloroplast division.The assembly and constriction of the inner Z ring and outer DRP5B ring are coordinated across the two membranes by the activities of midplastid-localized envelope membrane proteins whose functions have been studied in Arabidopsis (Arabidopsis thaliana). ARC6 (Pyke et al., 1994) is a bitopic IEM protein of cyanobacterial origin that is conserved throughout green-lineage chloroplasts (Koksharova and Wolk, 2002; Vitha et al., 2003; Osteryoung and Pyke, 2014). Its N-terminal region extends into the stroma, where it interacts directly and specifically with FtsZ2 (Maple et al., 2005). As FtsZ1 and FtsZ2 are soluble (McAndrew et al., 2001), this interaction probably serves both to tether the Z ring to the IEM and to promote FtsZ polymerization at the division site (Vitha et al., 2003). The C-terminal region of ARC6 protrudes into the intermembrane space (IMS) and interacts with the IMS region of the plant-specific bitopic OEM protein PLASTID DIVISION2 (PDV2). ARC6-PDV2 interaction is required for the localization of PDV2 to the midplastid (Glynn et al., 2008). PDV2 and its paralog PDV1, also in the OEM, in turn recruit DRP5B from a cytosolic pool to the OEM (Miyagishima et al., 2006), probably through direct interaction with their cytosolic regions (Holtsmark et al., 2013). Thus, interactions between FtsZ2 and ARC6 in the stroma, ARC6 and PDV2 in the IMS, and PDV2 (and PDV1) and DRP5B in the cytosol connect and coordinate the FtsZ and DRPB5B rings across the IEM and OEM.Previously, we showed that, despite the fact that an interaction between the IMS regions of ARC6 and PDV1 could not be detected, ARC6 was nevertheless required for the equatorial localization of PDV1 as well as PDV2, suggesting the existence of a factor that acted downstream of ARC6 to position PDV1 (Glynn et al., 2008). This downstream factor was subsequently shown to be the nucleus-encoded chloroplast division protein PARALOG OF ARC6 (PARC6; Glynn et al., 2009), also called CDP1 (Zhang et al., 2009) and ARC6H (Ottesen et al., 2010). parc6 mutants exhibited mislocalization of PDV1 but not PDV2, demonstrating a specific role for PARC6 in PDV1 positioning. PARC6 is restricted to vascular plants, suggesting that it arose by the duplication and divergence of ARC6 following separation of the nonvascular and vascular lineages. As suggested by its name, PARC6 shares significant sequence similarity with ARC6 and is similarly imported to the chloroplast by a cleavable N-terminal transit peptide and localized in the IEM. However, whereas ARC6 has a single transmembrane domain (TMD), PARC6 is predicted to bear two, and while a portion of its N terminus was clearly shown to reside in the stroma, its full topology has not been established (Glynn et al., 2009). Furthermore, genetic analysis suggested that, unlike ARC6, which positively regulates FtsZ assembly (Vitha et al., 2003), PARC6 functions partly as a negative regulator of FtsZ assembly. Interaction assays provided evidence that this negative regulation may be mediated by interaction of the N terminus of PARC6 with the stromal division protein ARC3 (Pyke et al., 1994; Shimada et al., 2004; Maple et al., 2007), a Z-ring positioning factor recently shown to inhibit Z-ring assembly and/or promote FtsZ filament and Z-ring destabilization (TerBush and Osteryoung, 2012; Zhang et al., 2013; Johnson et al., 2015). Although the interaction of PARC6 with FtsZ was not detected previously, the significance of this finding has remained uncertain in the absence of definitive data on PARC6 topology (Glynn et al., 2009).Here, we report a detailed topological analysis of Arabidopsis PARC6, investigate its interactions with other division factors, and assess the effect of PARC6 on chloroplast FtsZ assembly. Our findings provide evidence that the negative effect of PARC6 on Z-ring assembly results from its interaction with ARC3 and reveal a role for PARC6 in coordinating the inner Z ring and outer DRP5B ring partially analogous to the role of ARC6.     

Recruitment of ZipA to the Septal Ring of Escherichia coli Is Dependent on FtsZ and Independent of FtsA

Cell division in prokaryotes is mediated by the septal ring. In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.     

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.     

The pH Dependence of Polymerization and Bundling by the Essential Bacterial Cytoskeltal Protein FtsZ

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution—it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)—it stablizes associations of protofilaments.     

Bacterial Community Affects Toxin Production by Gymnodinium catenatum

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell⁻¹) was similar to the parent cultures (169–206 fmol STX cell⁻¹), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell⁻¹) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell⁻¹). Specific toxin production rate (fmol STX day⁻¹) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell⁻¹ day⁻¹) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.     

Assembly of archaeal cell division protein FtsZ and a GTPase-inactive mutant into double-stranded filaments

We have studied the assembly and GTPase of purified FtsZ from the hyperthermophilic archaeon Methanococcus jannaschii, a structural homolog of eukaryotic tubulin, employing wild-type FtsZ, FtsZ-His6 (histidine-tagged FtsZ), and the new mutants FtsZ-W319Y and FtsZ-W319Y-His6, with light scattering, nucleotide analyses, electron microscopy, and image processing methods. This has revealed novel properties of FtsZ. The GTPase of archaeal FtsZ polymers is suppressed in Na+-containing buffer, generating stabilized structures that require GDP addition for disassembly. FtsZ assembly is polymorphic. Archaeal FtsZ(wt) assembles into associated and isolated filaments made of two parallel protofilaments with a 43 A longitudinal spacing between monomers, and this structure is also observed in bacterial FtsZ from Escherichia coli. The His6 extension facilitates the artificial formation of helical tubes and sheets. FtsZ-W319Y-His6 is an inactivated GTPase whose assembly remains regulated by GTP and Mg2+. It forms two-dimensional crystals made of symmetrical pairs of tubulin-like protofilaments, which associate in an antiparallel array (similarly to the known Ca2+-induced sheets of FtsZ-His6). In contrast to the lateral interactions of microtubule protofilaments, we propose that the primary assembly product of FtsZ is the double-stranded filament, one or several of which might form the dynamic Z ring during prokaryotic cell division.