Silvia Zorrilla

The conserved tubulin homolog FtsZ assembles into GTP-dependent protofilaments that organize the bacterial cell division machinery. Purified Escherichia coli FtsZ (FtsZ _Ec ) can form biomolecular condensates in the absence of GTP in macromolecular crowding conditions, but FtsZ condensates have not yet been confirmed in vivo . FtsZ from the Alphaproteobacterium Agrobacterium tumefaciens (FtsZ _At ) localizes to distinct polar foci during growth and contains an intrinsically disordered region much longer than FtsZ _Ec , suggesting a greater tendency to form condensates. Here, we show that purified FtsZ _At readily formed dynamic condensates in vitro under molecular crowding conditions, and the addition of GTP induced the formation of filaments radiating from the FtsZ _At droplets. Notably, FtsZ _At formed similar foci and filaments in vivo that dynamically assembled and coalesced, supported by fluorescence recovery after photobleaching analysis of FtsZ _At and another Alphaproteobacterial FtsZ. These results suggest that FtsZ _At forms polar condensates in A. tumefaciens cells, perhaps to sequester FtsZ _At before cell division as an adaptation to slow generation times. IMPORTANCE: Alphaproteobacteria are highly diverse; however, most orthologs of the essential cell division protein FtsZ from this class feature an unusually long intrinsically disordered internal domain compared with other bacterial FtsZs. As proteins harboring such domains have an increased tendency to assemble into phase-separated biomolecular condensates, we investigated the condensate-forming properties of Alphaproteobacterial FtsZ proteins in vivo and in vitro . We found that Agrobacterium tumefaciens FtsZ has a strong tendency to form condensates in vitro and in vivo as part of its cell cycle, as does FtsZ of Caulobacter crescentus, a distantly related Alphaproteobacterium. This behavior contrasts with FtsZ from Escherichia coli, which has a much shorter internal disordered domain and forms condensates in combination with partner proteins under more stringent conditions. Our results suggest that Alphaproteobacterial FtsZs form condensates during normal growth, perhaps serving as part of a conserved mechanism of cell cycle control in this class of bacteria.

Antimicrobial resistance (AMR) is nowadays widely recognized as one of the most challenging problems in human health, with deep impact also in animals and the environment, highlighting the interconnected vulnerabilities shared across the One Health spectrum

Cytoskeletal structures shape and confer resistance to cells. The intermediate filament protein vimentin forms versatile structures that play key roles in cytoskeletal crosstalk, in the integration of cellular responses to a variety of external and internal cues, and in the defense against stress. Such multifaceted roles can be fulfilled thanks to the vast variety of vimentin proteoforms, which in turn arise from the combinations of a myriad of tightly regulated posttranslational modifications. Diverse vimentin proteoforms will differentially shape its polymeric assemblies, underlying vimentin ability to organize in filaments, bundles, squiggles, droplets, cell surface-bound and/or various secreted forms. Interestingly, vimentin dots or droplets have been lately categorized as biomolecular condensates. Biomolecular condensates are phase-separated membraneless structures that are critical for the organization of cellular components and play important roles in pathophysiology. Recent findings have unveiled the importance of low complexity sequence domains in vimentin filament assembly. Moreover, several oxidants trigger the transition of vimentin filaments into phase-separated biomolecular condensates, a reversible process that may provide clues on the role of condensates as seeds for filament formation. Revisiting previous results in the light of recent knowledge prompts the hypothesis that vimentin condensates could play a role in traffic of filament precursors, cytoskeletal crosstalk and cellular responses to stress. Deciphering the “vimentin PTM code”, that is, the structure-function relationships of vimentin proteoforms, constitutes a major challenge to understand the regulation of vimentin behavior and its multiple personalities. This will contribute to unveil essential cellular mechanisms and foster novel opportunities for drug discovery.

The widespread emergence of antimicrobial resistance (AMR) is a serious threat to global public health and among Gram-positive cocci, Streptococcus pneumoniae constitutes a priority in the list of AMR-threatening pathogens. To counteract this fundamental problem, the bacterial cell division cycle and the crucial proteins involved in this process emerged as novel attractive targets. FtsZ is an essential cell division protein, and FtsZ inhibitors, especially the benzamide derivatives, have been exploited in the last decade. In this work, we identified, for the first time, some benzodioxane–benzamide inhibitors capable of targeting FtsZ in Streptococcus pneumoniae, in addition to their previously demonstrated activity against other bacteria. These promising benzamides, with minimal inhibitory concentrations (MICs) ranging from 25 to 80 µg/mL, demonstrated bactericidal activity against S. pneumoniae. This was evidenced by their ability to dramatically affect growth and viability, further supported by the morphological changes observed through microscopy. Moreover, the compounds were characterized in vitro, combining turbidity measurements and confocal imaging, and significant alteration of a GTP-induced FtsZ assembly was found, in line with our previous data from other microorganisms.

An increasing number of proteins involved in bacterial cell cycle events have been recently shown to undergo phase separation. The resulting biomolecular condensates play an important role in cell cycle protein function and may be involved in development of persister cells tolerant to antibiotics. Here we report that the E. coli chromosomal Ter macrodomain organizer MatP, a division site selection protein implicated in the coordination of chromosome segregation with cell division, forms biomolecular condensates in cytomimetic systems. These condensates are favored by crowding and preferentially localize at the membrane of microfluidics droplets, a behavior probably mediated by MatP-lipid binding. Condensates are negatively regulated and partially dislodged from the membrane by DNA sequences recognized by MatP ( matS ), which partition into them. Unexpectedly, MatP condensation is enhanced by FtsZ, a core component of the division machinery previously described to undergo phase separation. Our biophysical analyses uncover a direct interaction between the two proteins, disrupted by matS sequences. This binding might have implications for FtsZ ring positioning at mid-cell by the Ter linkage, which comprises MatP and two other proteins that bridge the canonical MatP/FtsZ interaction. FtsZ/MatP condensates interconvert with bundles in response to GTP addition, providing additional levels of regulation. Consistent with discrete foci reported in cells, MatP biomolecular condensates may facilitate MatP’s role in chromosome organization and spatiotemporal regulation of cytokinesis and DNA segregation. Moreover, sequestration of MatP in these membraneless compartments, with or without FtsZ, could promote cell entry into dormant states that are able to survive antibiotic treatments.

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.