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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.

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 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.

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.

FtsZ is an essential bacterial protein abundantly studied as a novel and promising target for antimicrobials. FtsZ is highly conserved among bacteria and mycobacteria, and it is crucial for the correct outcome of the cell division process, as it is responsible for the division of the parent bacterial cell into two daughter cells. In recent years, the benzodioxane–benzamide class has emerged as very promising and capable of targeting both Gram-positive and Gram-negative FtsZs. In this study, we explored the effect of including a substituent on the ethylenic linker between the two main moieties on the antimicrobial activity and pharmacokinetic properties. This substitution, in turn, led to the generation of a second stereogenic center, with both erythro and threo isomers isolated, characterized, and evaluated. With this work, we discovered how the hydroxy group slightly affects the antimicrobial activity, while being an important anchor for the exploitation and development of prodrugs, probes, and further derivatives.

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.

Multidrug resistant Staphylococcus aureus is a severe threat, responsible for most of the nosocomial infections globally. This resistant strain is associated with a 64% increase in death compared to the antibiotic-susceptible strain. The prokaryotic protein FtsZ and the cell division cycle have been validated as potential targets to exploit in the general battle against antibiotic resistance. Despite the discovery and development of several anti-FtsZ compounds, no FtsZ inhibitors are currently used in therapy. This work further develops benzodioxane-benzamide FtsZ inhibitors. We seek to find more potent compounds using computational studies, with encouraging predicted drug-like profiles. We report the synthesis and the characterization of novel promising derivatives that exhibit very low MICs towards both methicillin-susceptible and -resistant S. aureus, as well as another Gram positive species, Bacillus subtilis, while possessing good predicted physical-chemical properties in terms of solubility, permeability, and chemical and physical stability. In addition, we demonstrate by fluorescence microscopy that Z ring formation and FtsZ localization are strongly perturbed by our derivatives, thus validating the target.