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Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.

The influence of potassium content (at neutral pH and millimolar Mg(2+)) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5'-(¿,ß-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring, Depto. de Química Física, Fac. de Ciencias Químicas, TRUE, pub

The conserved process of cell division in bacteria has been a long-standing target for antimicrobials, although there are few examples of potent broad-spectrum compounds that inhibit this process. Most currently available compounds acting on division are directed towards the FtsZ protein, a self-assembling GTPase that is a central element of the division machinery in most bacteria. Benzodioxane-benzamides are promising candidates, but poorly explored in Gram-negatives. We have tested a number of these compounds on E. coli FtsZ and found that many of them significantly stabilized the polymers against disassembly and reduced the GTPase activity. Reconstitution in crowded cell-like conditions showed that FtsZ bundles were also susceptible to these compounds, including some compounds that were inactive on protofilaments in dilute conditions. They efficiently killed E. coli cells defective in the AcrAB efflux pump. The activity of the compounds on cell growth and division generally showed a good correlation with their effect in vitro, and our experiments are consistent with FtsZ being the target in vivo. Our results uncover the detrimental effects of benzodioxane-benzamides on permeable E. coli cells via its central division protein, implying that lead compounds may be found within this class for the development of antibiotics against Gram-negative bacteria.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies i

Cytokinesis is a fundamental process for bacterial survival and proliferation, involving the formation of a ring by filaments of the GTPase FtsZ, spatio-temporally regulated through the coordinated action of several factors. The mechanisms of this regulation remain largely unsolved, but the inhibition of FtsZ polymerization by the nucleoid occlusion factor SlmA and filament stabilization by the widely conserved cross-linking protein ZapA are known to play key roles. It was recently described that FtsZ, SlmA and its target DNA sequences (SlmA-binding sequence (SBS)) form phase-separated biomolecular condensates, a type of structure associated with cellular compartmentalization and resistance to stress. Using biochemical reconstitution and orthogonal biophysical approaches, we show that FtsZ-SlmA-SBS condensates captured ZapA in crowding conditions and when encapsulated inside cell-like microfluidics microdroplets. We found that, through non-competitive binding, the nucleotide-dependent FtsZ condensate/polymer interconversion was regulated by the ZapA/SlmA ratio. This suggests a highly concentration-responsive tuning of the interconversion that favours FtsZ polymer stabilization by ZapA under conditions mimicking intracellular crowding. These results highlight the importance of biomolecular condensates as concentration hubs for bacterial division factors, which can provide clues to their role in cell function and bacterial survival of stress conditions, such as those generated by antibiotic treatment.

Bacterial division is driven by a contractile ring consisting of a multiprotein complex, which accurately assembles at midcell. For its formation, the polymerization of the GTPase protein FtsZ is required as scaffold for the rest of the proteins involved. The control mechanisms underlying division ring positioning remain largely unknown. However, the nucleoid occlusion protein SlmA and the cross-linking protein ZapA are known to be relevant. FtsZ, SlmA and its DNA sequence have shown to form biomolecular condensates, structures emerging as a mechanism to organize intracellular space, linked to stress tolerance., We have found that FtsZ-SlmA-SBS condensates are able to recruit ZapA in crowding conditions in bulk and also when encapsulated inside cell-like microfluidics microdroplets. Our combined biophysical and reconstitution approach showed that the ZapA/SlmA ratio controls FtsZ condensate/polymer interconversion through non-competitive binding and that FtsZ polymer stabilization by ZapA is favored under crowding conditions. These findings underline the significance of condensates as concentrated hubs of proteins, which can shed light on cell survival under stress conditions, such as those induced by antibiotic treatment.

Bacterial division is an essential process orchestrated by a protein machinery, the divisome [1], forming a ring in the crowded, heterogeneous and compartmentalized bacterial cytoplasm. This ring, the scaffold of which are the GTP-induced polymers of the conserved tubulin homolog FtsZ, is anchored to the membrane through specific tethering proteins, and constrained at the cell centre by the coordinated action of various mechanisms. One of these mechanisms is the nucleoid occlusion that protects the chromosome from the guillotining action of the division machinery [2]. In E. coli, nucleoid occlusion is mediated by SlmA, a protein that binds specific DNA sequences distributed over the chromosome, except for the replication terminus region. In addition, SlmA directly interacts with FtsZ, antagonizing its GTP-triggered assembly [3]. We have studied the effects of macromolecular crowding on the polymerization of FtsZ and on its dynamic distribution and localization in model two-phase crowding systems mimicking cell compartmentalization. Analysis was conducted by fluorescence measurements in bulk and through reconstruction inside microfluidics microdroplets stabilized by a lipid boundary. We have also studied the impact of crowding on the complexes formed by FtsZ, SlmA and the specific DNA sequences recognized by the latter by fluorescence confocal microscopy and turbidity. We have found that these nucleoprotein complexes form reversible dynamic biomolecular condensates that evolve towards polymers upon addition of GTP. Furthermore, under defined crowding conditions favouring its oligomerization, FtsZ was also found to assemble into homotypic biomolecular condensates. These analyses show that crowding and phase separation phenomena may have a significant impact on the regulation of cell division, a crucial process for bacterial proliferation.

Dynamic biomolecular condensates formed by liquid–liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

Antimicrobial resistance is a serious worldwide health threat. The identification of novel potential antibiotic targets is one of the ways to slow down its worsening. FtsZ, one of the bacterial cell division machinery proteins, emerged in the last decade for its crucial role in bacterial replication and viability [1]. Benzamide compounds are the most studied and promising FtsZ inhibitors developed so far, due to their high anti-staphylococcal activity, their low cytotoxicity and the interesting results obtained in association with other antibiotic classes [2]. Along these lines, here we report our recent findings on a class of FtsZ inhibitors, containing a 2,6-difluoro-benzamide scaffold linked to a hydrophobically substituted 1,4-benzodioxane ring [3-6]. We firstly validated a robust computational model, which drove us to identify the structural features the 1,4-benzodioxane moiety and the alkoxy linker should possess, in order to perfectly fit the FtsZ binding pocket. We thus developed several interesting compounds, having submicromolar antibacterial activities and showing comparable inhibitory activities towards both Gram-positive (Staphylococcus aureus and Bacillus subtilis) [3,5] and Gram-negative (Escherichia coli) FtsZ. Nevertheless, these derivatives proved to be substrates of E. coli efflux pump AcrAB, thus affecting their potencies [4]. These surprising and novel results confirmed how a single molecule can target both species while maintaining potent antimicrobial activity. We set-up and performed different assays, to firstly validate FtsZ as the target of our class of compounds. Morphometric analysis and fluorescence microscopy let us evaluate the typical alterations of cell division and FtsZ inhibition, as well as the effects on FtsZ localization [6].Moreover, we took advantages of fluorescence anisotropy to investigate and assess the impact of our derivatives on the kinetics of disassembly of the GTP triggered FtsZ polymers. Furthermore, we used confoca

Biomolecular condensation through phase separation may be a novel mechanism to regulate bacterial processes,including cell division. Previous work revealed that FtsZ, a protein essential for cytokinesis in most bacteria,forms biomolecular condensates with SlmA, a protein that protects the chromosome from damage inflicted by the division machinery in Escherichia coli. The absence of condensates composed solely of FtsZ under the conditions used in that study suggested this mechanism was restricted to nucleoid occlusion by SlmA or to bacteria containing this protein. Here we report that FtsZ alone, under physiologically relevant conditions, can demix into condensates in bulk and when encapsulated in synthetic cell-like systems generated by microfluidics. Condensate assembly depends on FtsZ being in the GDP-bound state and on conditions mimicking the crowded environment of the cytoplasm that promote its oligomerization. Condensates are dynamic and reversibly convert into filaments upon GTP addition. Notably, FtsZ lacking its C-terminal disordered region, a structural element likely to favor biomolecular condensation, also forms condensates, albeit less efficiently. The inherent tendency of FtsZ to form condensates susceptible to modulation by physiological factors, including binding partners, suggests that such mechanisms may play a more general role in bacterial division than initially envisioned.

FtsZ is an essential and central protein for cell division in most bacteria. Because of its ability to organize into dynamic polymers at the cell membrane and recruit other protein partners to form a “divisome”, FtsZ is a leading target in the quest for new antibacterial compounds. Strategies to potentially arrest the essential and tightly regulated cell division process include perturbing FtsZ’s ability to interact with itself and other divisome proteins. Here, we discuss the available methodologies to screen for and characterize those interactions. In addition to assays that measure protein-ligand interactions in solution, we also discuss the use of minimal membrane systems and cell-like compartments to better approximate the native bacterial cell environment and hence provide a more accurate assessment of a candidate compound’s potential in vivo effect. We particularly focus on ways to measure and inhibit under-explored interactions between FtsZ and partner proteins. Finally, we discuss recent evidence that FtsZ forms biomolecular condensates in vitro, and the potential implications of these assemblies in bacterial resistance to antibiotic treatment.

Protection of the chromosome from scission by the division machinery during cytokinesis is critical for bacterial survival and fitness. This is achieved by nucleoid occlusion, which, in conjunction with other mechanisms, ensures formation of the division ring at midcell. In Escherichia coli, this mechanism is mediated by SlmA, a specific DNA binding protein that antagonizes assembly of the central division protein FtsZ into a productive ring in the vicinity of the chromosome. Here, we provide evidence supporting direct interaction of SlmA with lipid membranes, tuned by its binding partners FtsZ and SlmA binding sites (SBS) on chromosomal DNA. Reconstructions in minimal membrane systems that mimic cellular environments show that SlmA binds to lipid-coated microbeads or locates at the edge of microfluidic-generated microdroplets, inside which the protein is encapsulated. DNA fragments containing SBS sequences do not seem to be recruited to the membrane by SlmA but instead compete with SlmA's ability to bind lipids. The interaction of SlmA with FtsZ modulates this behavior, ultimately triggering membrane localization of the SBS sequences alongside the two proteins. The ability of SlmA to bind lipids uncovered in this work extends the interaction network of this multivalent regulator beyond its well-known protein and nucleic acid recognition, which may have implications in the overall spatiotemporal control of division ring assembly.IMPORTANCE Successful bacterial proliferation relies on the spatial and temporal precision of cytokinesis and its regulation by systems that protect the integrity of the nucleoid. In Escherichia coli, one of these protectors is SlmA protein, which binds to specific DNA sites around the nucleoid and helps to shield the nucleoid from inappropriate bisection by the cell division septum. Here, we discovered that SlmA not only interacts with the nucleoid and septum-associated cell division proteins but also binds directly to cytomimetic lipid mem

Here we have summarized several strategies to reconstruct complexes containing the FtsZ protein, a central element of the cell division machinery in most bacteria, and to test their functional organization in minimal membrane systems and cell-like containers, as vesicles and droplets produced by microfluidics. These synthetic systems have been devised to mimic elements of the intracellular complexity, as excluded volume effects due to natural crowding, and macromolecular condensation resulting from biologically regulated liquid-liquid phase separation, in media of known and controllable composition. This integrative approach has allowed to demonstrate that macromolecular phase separation and crowding may also help to dynamically organize FtsZ in the intracellular space thus modulating its functional reactivity in cell division.

Division ring formation at midcell is controlled by various mechanisms in Escherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipids in vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in the E. coli inner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands., IMPORTANCE The division of an E. coli cell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integrated in vivo and in vitro analysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in the E. coli inner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

Macromolecular condensation resulting from biologically regulated liquid-liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding-induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ-rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP-driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.

There is growing interest in analyzing the effect of microenvironments, which may be mimicked through liquid-liquid phase separation (LLPS), on the reactivity of biological macromolecules. We report the encapsulation by microfluidics of the division protein FtsZ and a LLPS system inside microdroplets and their conversion into permeable vesicles (allowing ligand uptake), with higher yield, homogeneity and biomolecular compatibility than those previously described.

ZipA protein from Escherichia coli is one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein. A sedimentation assay was used to measure the equilibrium binding of FtsZ-GDP and FtsZ-GTP to ZipA immobilized at controlled densities on the surface of microbeads coated with a phospholipid mixture resembling the composition of E. coli membrane. We found that for both nucleotide-bound species, the amount of bound FtsZ exceeds the monolayer capacity of the ZipA immobilized beads at high concentrations of free FtsZ. In the case of FtsZ-GDP, equilibrium binding does not appear to be saturable, whereas in the case of FtsZ-GTP equilibrium binding appears to be saturable. The difference between the two modes of binding is attributed to the difference between the composition of oligomers of free FtsZ-GDP and free FtsZ-GTP formed in solution.

We report a microfluidic approach to generate aqueous droplets in oil of different dimensionality,stabilized by a lipid monolayer, to systematically probe the polymerization of bacterial cell-divisionprotein FtsZ intofibrous networks as a function of the concentrations of crowding agent, FtsZ, and GTP.FtsZ bundles confined in droplets were dynamic, and their distribution depended on the intrinsicproperties of the system and restrictions imposed by the spatial boundaries.

The division of Escherichia coli is an essential process strictly regulated in time and space. It requires the association of FtsZ with other proteins to assemble a dynamic ring during septation, forming part of the functionally active division machinery, the divisome. FtsZ reversibly interacts with FtsA and ZipA at the cytoplasmic membrane to form a proto-ring, the first molecular assembly of the divisome, which is ultimately joined by the rest of the division-specific proteins. In this review we summarize the quantitative approaches used to study the activity, interactions, and assembly properties of FtsZ under well-defined solution conditions, with the aim of furthering our understanding of how the behavior of FtsZ is controlled by nucleotides and physiological ligands. The modulation of the association and assembly properties of FtsZ by excluded-volume effects, reproducing in part the natural crowded environment in which this protein has evolved to function, will be described. The subsequent studies on the reactivity of FtsZ in membrane-like systems using biochemical, biophysical, and imaging technologies are reported. Finally, we discuss the experimental challenges to be met to achieve construction of the minimum protein set needed to initiate bacterial division, without cells, in a cell-like compartment. This integrated approach, combining quantitative and synthetic strategies, will help to support (or dismiss) conclusions already derived from cellular and molecular analysis and to complete our understanding on how bacterial division works.

The concerted formation of a narrow distribution of oligomeric FtsZ species in the presence of GTP or a GTP analogue under close to physiological conditions (neutral pH and 0.5 M K+) has been characterized recently by various biophysical methods [Monterroso, B., et al. (2012) Biochemistry51, 4541–4550]. An equilibrium model may semiquantitatively account for the results of this study; in the model, FtsZ self-associates in a noncooperative fashion to form linear fibrils, that upon increasing to a certain size exhibit an increasing tendency to form closed cyclic fibrils, as previously suggested [González, J. M., et al. (2005) Proc. Natl. Acad. Sci. U.S.A.102, 1895–1900]. The closed cyclic fibrils are formed when the natural curvature and flexibility of a linear oligomer bring the ends of a linear fiber sufficiently close to overcome the entropic barrier to loop closure. The size distribution of cyclic oligomers is thus a reflection of the tendency toward curvature of linear fibrils of FtsZ under the conditions used in these experiments.

Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solid-state NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.