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14 p.-4 fig.-2 tab. ; We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the dynamic septal ring and, hence, for bacterial division. It was earlier demonstrated that high concentrations of inert polymers like Ficoll 70, used to mimic the crowded cellular interior, favor the assembly of FtsZ into bundles with slow depolymerization. We have found, by fluorescence anisotropy together with light scattering measurements, that the presence of protein crowders increases the tendency of FtsZ to polymerize at micromolar magnesium concentration, being the effect larger with ovomucoid, a negatively charged protein. Neutral polymers and a positively charged protein also diminished the critical concentration of assembly, the extent of the effect being compatible with that expected according to pure volume exclusion models. FtsZ polymerization was also observed to be strongly promoted by a negatively charged polymer, DNA, and by some unrelated polymers like PEGs at concentrations below the crowding regime. The influence of mixed crowders mimicking the heterogeneity of the intracellular environment on the tendency of FtsZ to assemble was also studied and nonadditive effects were found to prevail. Far from exactly reproducing the bacterial cytoplasm environment, this approach serves as a simplified model illustrating how its intrinsically crowded and heterogeneous nature may modulate FtsZ assembly into a functional Z-ring. ; This work was supported by Spanish government BIO2011-28941-C03 (GR and SZ) and BFU 2014-52070-C2-2-P (GR) (www.mineco.gob.es), European Commission HEALTH-F3-2009-223432 (GR) (http://ec.europa.eu/), and Human Frontiers Science Program RGP0050/2010-C102 (GR) (www.hfsp.org). ; Peer reviewed

9 p.-6 fig. ; 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. ; This work was supported by the Spanish government through grants BFU2014-52070-C2-2-P and BFU2016-75471-C2-1-P (to G.R.). G.R. is member of the CIB Intramural Program 'Macromolecular Machines for Better Life' (MACBET). Research of A.P.M. is supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH. ; Peer reviewed

4 p.-2 fig. ; 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. ; This work was supported by the Spanish government (BFU2014-52070-C2-2-P and BFU2016-75471-C2-1-P, G. R.) and by the National Science Foundation (MCB-1244180, C. D. K.). ; Peer reviewed

13 p.-8 fig. ; The influence of membrane-free microcompartments resulting from crowding-induced liquid/liquid phase separation (LLPS) on the dynamic spatial organization of FtsZ, the main component of the bacterial division machinery, has been studied using several LLPS systems. The GTP-dependent assembly cycle of FtsZ is thought to be crucial for the formation of the septal ring, which is highly regulated in time and space. We found that FtsZ accumulates in one of the phases and/or at the interface, depending on the system composition and on the oligomerization state of the protein. These results were observed both in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets. The visualization of the droplets revealed that both the location and structural arrangement of FtsZ filaments is determined by the nature of the LLPS. Relocation upon depolymerization of the dynamic filaments suggests the protein may shift among microenvironments in response to changes in its association state. The existence of these dynamic compartments driven by phase transitions can alter the local composition and reactivity of FtsZ during its life cycle acting as a nonspecific modulating factor of cell function. ; This work was supported by the Spanish government through grants BIO2011-28941-C03 (G.R. and S.Z.) and BFU 2014-52070-C2-2-P (G.R.); by the European Commission through contract HEALTH-F3-2009-223432(G.R.); by Human Frontiers Science Program through grant RGP0050/2010-C102 (G.R.); and by the National Science Foundation through grant MCB-1244180 (C.D.K.). ; Peer reviewed

11 p.-7 fig. ; The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,β)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection. ; This work was supported, in whole or in part, by National Institutes of Health Grant GM61074 (to W. M.). This work was also supported by Human Frontier Science Program Grant RGP0050/2010 (to G. R. and W. M.) and Spanish Government Grant BIO2011-28941-C03 (to G. R. and S. Z.). ; Peer reviewed

15 p.-5 fig.-3 tab. ; Protein modification by lipid derived reactive species or lipoxidation is increased during oxidative stress, a common feature observed in many pathological conditions. Biochemical and functional consequences of lipoxidation include changes in the conformation and assembly of the target proteins, altered recognition of ligands and/or cofactors, changes in the interactions with DNA or protein-protein interactions, modifications in membrane partitioning and binding and/or subcellular localization. These changes may impact, directly or indirectly, signaling pathways involved in the activation of cell defense mechanisms, but when these are overwhelmed they may lead to pathological outcomes. Mass spectrometry provides state of the art approaches for the identification and characterization of lipoxidized proteins/residues and the modifying species. Nevertheless, understanding the complexity of the functional effects of protein lipoxidation requires the use of additional methodologies. Herein, biochemical and biophysical methods used to detect and measure functional effects of protein lipoxidation at different levels of complexity, from in vitro and reconstituted cell-like systems to cells, are reviewed, focusing especially on macromolecular interactions. Knowledge generated through innovative and complementary technologies will contribute to comprehend the role of lipoxidation in pathophysiology and, ultimately, its potential as target for therapeutic intervention. ; This work was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement number 675132 (http://cordis.europa.eu/project/rcn/198275_en.html), and by grants from the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER, http://www.mineco.gob.es/portal/site/mineco/idi) SAF2015-68590-R to DPS and BFU2016-75471-C2-1-P to GR, and RETIC Aradyal from ISCIII/FEDER (RD16/0006/0021) to DPS. ; Peer reviewed

9 p.-3 fig. ; Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein purification, both to eliminate contaminating divalent cations and to inhibit protease activity. For a number of subsequent applications EDTA needs to be exhaustively removed. Most purification methods rely in extensive dialysis and/or gel filtration in order to exchange or remove protein buffer components, including metal chelators. We report here that dialysis protocols,even as extensive as those typically employed for protein refolding, may not effectively remove EDTA, which is reduced only by approximately two-fold and it also persists after spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably, the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually undetectable. These results highlight a potentially widespread source of experimental variability affecting free divalent cation concentrations in protein applications. ; This work has been funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklowdowska-Curie grant agreement number 675132 (http://cordis.europa.eu/project/rcn/198275_en.html), and by grants from the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER, http://www.mineco.gob.es/ portal/site/mineco/idi) SAF2015-68590R to DPS and CTQ2015-64597-C2-2-P to FJC. ; Peer reviewed

12 p.-8 fig. ; The interaction of MinC with FtsZ and its effects on FtsZ polymerization were studied under close to physiological conditions by a combination of biophysical methods. The Min system is a widely conserved mechanism in bacteria that ensures the correct placement of the division machinery at midcell. MinC is the component of this system that effectively interacts with FtsZ and inhibits the formation of the Z-ring. Here we report that MinC produces a concentration-dependent reduction in the size of GTP-induced FtsZ protofilaments (FtsZ-GTP) as demonstrated by analytical ultracentrifugation, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. Our experiments show that, despite being shorter, FtsZ protofilaments maintain their narrow distribution in size in the presence of MinC. The protein had the same effect regardless of its addition prior to or after FtsZ polymerization. Fluorescence anisotropy measurements indicated that MinC bound to FtsZ-GDP with a moderate affinity (apparent KD ∼10 μM at 100 mm KCl and pH 7.5) very close to the MinC concentration corresponding to the midpoint of the inhibition of FtsZ assembly. Only marginal binding of MinC to FtsZ-GTP protofilaments was observed by analytical ultracentrifugation and fluorescence correlation spectroscopy. Remarkably, MinC effects on FtsZ-GTP protofilaments and binding affinity to FtsZ-GDP were strongly dependent on ionic strength, being severely reduced at 500 mM KCl compared with 100 mM KCl. Our results support a mechanism in which MinC interacts with FtsZ-GDP, resulting in smaller protofilaments of defined size and having the same effect on both preassembled and growing FtsZ protofilaments. ; This work was supported in part by Human Frontier Science Program Grant RGP0050/2010 (to G. R. and W. M.), European Commission Contract HEALTH-F3-2009-223431 (to G. R.), and Spanish Government Grants BIO2008-04478-C03 (to G. R.), CSICPIE-201020I001 (to C. A.), BFU2010- 14910 (to S. Z.), and BIO2011-28941-C03 (to G. R. and S. Z ; Peer reviewed

13 p.-6 fig. ; 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. ; This work was supported by the Fondo Europeo de Desarrollo Regional (FEDER) and the Agencia Estatal de Investigación (AEI); by the Spanish government (BFU2014-52070-C2-2-P and BFU2016-75471-C2-1-P, G. R.); by the National Institutes of Health (GM61074, W. M.); and by the National Science Foundation (MCB-1715984,C. D. K.). M.L.-A. was supported by the European Social Fund (ESF 2014-2020). ; Peer reviewed

21 p.-7 fig.-1 tab. ; Division site selection is achieved in bacteria by different mechanisms, one of them being nucleoid occlusion, which prevents Z-ring assembly nearby the chromosome. Nucleoid occlusion in E. coli is mediated by SlmA, a sequence specific DNA binding protein that antagonizes FtsZ assembly. Here we show that, when bound to its specific target DNA sequences (SBS), SlmA reduces the lifetime of the FtsZ protofilaments in solution and of the FtsZ bundles when located inside permeable giant vesicles. This effect appears to be essentially uncoupled from the GTPase activity of the FtsZ protofilaments, which is insensitive to the presence of SlmA·SBS. The interaction of SlmA·SBS with either FtsZ protofilaments containing GTP or FtsZ oligomers containing GDP results in the disassembly of FtsZ polymers. We propose that SlmA·SBS complexes control the polymerization state of FtsZ by accelerating the disassembly of the FtsZ polymers leading to their fragmentation into shorter species that are still able to hydrolyze GTP at the same rate. SlmA defines therefore a new class of inhibitors of the FtsZ ring different from the SOS response regulator SulA and from the moonlighting enzyme OpgH, inhibitors of the GTPase activity. SlmA also shows differences compared with MinC, the inhibitor of the division site selection Min system, which shortens FtsZ protofilaments by interacting with the GDP form of FtsZ. ; This work was supported by the Spanish government through grants BIO2011-28941-C03 (to MV, GR and SZ); by the European Commission through contract HEALTH-F3-2009-223432 DIVINOCELL (to MV and GR), and by Human Frontier Science Program through grant RGP0050/2010-C102 (to MV and GR). ; Peer reviewed

14 p.-5 fig. ; 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. ; This work was supported by the Spanish government through grants BFU2014-52070-C2-2-P and BFU2016-75471-C2-1-P (to G. R.) and by the Dutch government through grant NWO ALW open program no. 822.02.019 (to N.Y.M.). M.R.-R was supported by the Agencia Estatal de Investigación and the European Social Fund through grant BES-2017-082003. ; Peer reviewed

SAFO study group and the Spanish Network for Research in Infectious Diseases (REIPI). ; [Introduction] Methicillin-susceptible Staphylococcus aureus (MSSA) bacteraemia is a frequent condition, with high mortality rates. There is a growing interest in identifying new therapeutic regimens able to reduce therapeutic failure and mortality observed with the standard of care of beta-lactam monotherapy. In vitro and small-scale studies have found synergy between cloxacillin and fosfomycin against S. aureus. Our aim is to test the hypothesis that cloxacillin plus fosfomycin achieves higher treatment success than cloxacillin alone in patients with MSSA bacteraemia. ; [Methods] We will perform a superiority, randomised, open-label, phase IV–III, two-armed parallel group (1:1) clinical trial at 20 Spanish tertiary hospitals. Adults (≥18 years) with isolation of MSSA from at least one blood culture ≤72 hours before inclusion with evidence of infection, will be randomly allocated to receive either cloxacillin 2 g/4-hour intravenous plus fosfomycin 3 g/6-hour intravenous or cloxacillin 2 g/4-hour intravenous alone for 7 days. After the first week, sequential treatment and total duration of antibiotic therapy will be determined according to clinical criteria by the attending physician. Primary endpoints: (1) Treatment success at day 7, a composite endpoint comprising all the following criteria: patient alive, stable or with improved quick-Sequential Organ Failure Assessment score, afebrile and with negative blood cultures for MSSA at day 7. (2) Treatment success at test of cure (TOC) visit: patient alive and no isolation of MSSA in blood culture or at another sterile site from day 8 until TOC (12 weeks after randomisation). We assume a rate of treatment success of 74% in the cloxacillin group. Accepting alpha risk of 0.05 and beta risk of 0.2 in a two-sided test, 183 subjects will be required in each of the control and experimental groups to obtain statistically significant difference of 12% (considered clinically significant). ; [Ethics and dissemination] Ethical approval has been obtained from the Ethics Committee of Bellvitge University Hospital (AC069/18) and from the Spanish Medicines and Healthcare Product Regulatory Agency (AEMPS, AC069/18), and is valid for all participating centres under existing Spanish legislation. The results will be presented at international meetings and will be made available to patients and funders. ; [Trial registration number] The protocol has been approved by AEMPS with the Trial Registration Number EudraCT 2018-001207-37. ClinicalTrials.gov Identifier: NCT03959345; Pre-results. ; The SAFO trial is supported by a competitive grant awarded by the Fondo de Investigaciones Sanitarias at the Spanish government's National Institute of Health Research, Instituto de Salud Carlos III (ISCIII), (FIS PI17/01116). This study was supported by Plan Nacional de I+D+i 2017–2021 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0005). ; Peer reviewed