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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 bottom-up reconstitution of proteins for their modular engineering into synthetic cellular systems can reveal hidden protein functions in vitro. This is particularly evident for the bacterial Min proteins, a paradigm for self-organizing reaction-diffusion systems that displays an unexpected functionality of potential interest for bioengineering: the directional active transport of any diffusible cargo molecule on membranes. Here, the MinDE protein system is reported as a versatile surface patterning tool for the rational design of synthetically assembled 3D systems. Employing two-photon lithography, microswimmer-like structures coated with tailored lipid bilayers are fabricated and demonstrate that Min proteins can uniformly pattern bioactive molecules on their surface. Moreover, it is shown that the MinDE system can form stationary patterns inside lipid vesicles, which allow the targeting and distinctive clustering of higher-order protein structures on their inner leaflet. Given their facile use and robust function, Min proteins thus constitute a valuable molecular toolkit for spatially patterned functionalization of artificial biosystems like cell mimics and microcarriers.

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.

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.

In most bacteria, division depends on a cytoskeletal structure, the FtsZ ring, that functions as a scaffold to recruit additional proteins, with which it forms the machinery responsible for division, the divisome. The detailed architecture of the ring, in particular the mechanisms of assembly, stabilization, and disassembly, are still largely unknown. Here, we highlight the role of FtsZ-associated proteins (Zaps) that stabilize the FtsZ ring by crosslinking the filaments. Among Zap proteins, ZapD binds the C-terminal domain of FtsZ, which serves as a hub for its regulation. We demonstrate that ZapD crosslinks FtsZ filaments into ring-like structures formed by a discontinuous arrangement of short filaments. Using cryo-electron tomography combined with biochemical analysis, we reveal the three-dimensional organization of the ring-like structures and shed light on the mechanism of FtsZ filament crosslinking by ZapD. Together, our data provide a model of how FtsZ-associated proteins can stabilize FtsZ filaments into discontinuous ring-like structures reminiscent of that existing in the bacterial cell.

German Rivas is an executive editor of the IUPAB Biophysical Reviews journal based in Spain. As the head of the Department of Structural and Chemical Biology at the Center for Biological Research (CIB) Margarita Salas (one of the largest research institutes devoted to life sciences of the Spanish National Research Council (CSIC)), he leads a research program aimed at understanding the structure function relationship of large macromolecular complexes (involved in bacterial cell division) when placed in physiologically complex and "crowded" media toward their reconstitution from the bottom up in cell-like compartments. In this "Meet the Editors'" piece, he briefly describes his research interests and history.

Taking into account the presence of the crowded environment of a macromolecule has been an important goal of biology over the past 20 years. Molecular crowding affects the motions, stability and the kinetic behaviour of proteins. New powerful approaches have recently been developed to study molecular crowding, some of which make use of the synchrotron radiation light. The meeting “New Frontiers in Molecular Crowding” was organized in July 2022at the European Synchrotron Radiation facility of Grenoble to discuss the new frontiers of molecular crowding. The workshop brought together researchers from different disciplines to highlight the new developments of the field, including areas where new techniques allow the scientists to gain unprecedently expected information. A key conclusion of the meeting was the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives that could let the molecular crowding field flourish further.

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.

Microfluidically fabricated polymer microgels are used as an experimental platform to analyze protein–DNA interactions regulating bacterial cell division. In particular, we focused on the nucleoid-associated protein SlmA, which forms a nucleoprotein complex with short DNA binding sequences (SBS) that acts as a negative regulator of the division ring stability in Escherichia coli. To mimic the bacterial nucleoid as a dense DNA region of a bacterial cell and investigate the influence of charge and permeability on protein binding and diffusion in there, we have chosen nonionic polyethylene glycol and anionic hyaluronic acid as precursor materials for hydrogel formation, previously functionalized with SBS. SlmA binds specifically to the coupled SBS for both types of microgels while preferentially accumulating at the microgels’ surface. We could control the binding specificity by adjusting the buffer composition of the DNA-functionalized microgels. The microgel charge did not impact protein binding; however, hyaluronic acid-based microgels exhibit a higher permeability, promoting protein diffusion; thus, they were the preferred choice for preparing nucleoid mimics. The approaches described here provide attractive tools for bottom-up reconstitution of essential cellular processes in media that more faithfully reproduce intracellular environments.

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.

The cellular interior is composed of a variety of microenvironments defined by distinct local compositions and composition-dependent intermolecular interactions.We review the various types of nonspecific interactions between proteins and between proteins and other macromolecules and supramolec ular structures that influence the state of association and functional prop erties of a given protein existing within a particular microenvironment at a particular point in time. The present state of knowledge is summarized, and suggestions for fruitful directions of research are offered.

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

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.

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.