Bacterial membrane dynamics: Compartmentalization and repair

In every bacterial cell, the plasma membrane plays a key role in viability as it forms a selective barrier between the inside of the cell and its environment. This barrier function depends on the physical state of the lipid bilayer and the proteins embedded or associated with the bilayer. Over the past decade or so, it has become apparent that many membrane‐organizing proteins and principles, which were described in eukaryote systems, are ubiquitous and play important roles in bacterial cells. In this minireview, we focus on the enigmatic roles of bacterial flotillins in membrane compartmentalization and bacterial dynamins and ESCRT‐like systems in membrane repair and remodeling.


| MEMB R ANE DYNAMIC S: REG UL ATION OF FLU ID IT Y
Membrane lipids undergo specific interactions with other lipids and proteins.These interactions depend on the nature of the lipid and also on external factors such as temperature or solvents.At low temperatures, the membrane is typically in a solid ordered (S o ) phase (also called gel phase).In this phase, lipids are highly ordered and have little lateral mobility.Upon increasing temperature, the membrane melts, leading to an increase in lateral mobility, or fluidity, of the lipids.This phase is called liquid disordered (Ld) or liquid crystalline phase.Lipids in the Ld phase are characterized by higher diffusion coefficients.The interaction of planar lipids such as cholesterol (mainly in eukaryotes) or hopanoids (in bacteria) organizes the membrane into a liquid ordered (Lo) phase.This phase is usually attributed to be the phase of the so-called lipid rafts.Importantly, biological membrane can exist in more than one phase (usually Ld and Lo phase), and hence, phase separation can help to compartmentalize the membrane (Strahl & Errington, 2017).
The study of the regulation of membrane fluidity, which is critical to correct function, has primarily focused on the adaptation of membrane lipids in organisms to changes in growth temperature (or pH, pressure) with the aim of keeping the membrane in the liquidcrystalline phase-so-called homo-viscous adaptation (Mansilla & de Mendoza, 2019;Siliakus et al., 2017;Sinensky, 1974).This adaptation prevents a phase transition to the gel phase (S o ) at lower temperatures, where the membrane would rigidify and be much less dynamic, and phase transition to a fully fluid phase at higher temperatures, which would result in a loss of the barrier function of the membrane.
A systematic study on Bacillus subtilis grown over a range of temperatures showed that the bacterium increases the average length of fatty acid acyl chains, their degree of saturation, and the ratio of iso-and anteiso branched fatty acids with temperature, with the net result of keeping proton permeability of the membrane constant over the range of growth temperatures (van de Vossenberg et al., 1999).Various systems exist to regulate fatty acid synthesis to control fluidity, such as temperature sensitivity of enzymes involved in fatty acid synthesis, as well as a two-component signaling system (Des) that directly senses membrane thickness and controls transcription of desaturases that introduce double bonds in fatty acid chains (Cybulski et al., 2010;Mansilla & de Mendoza, 2019).Recently, an important study described the effect of tuning membrane fluidity by synthetically lowering fluidity in engineered Escherichia coli and B. subtilis strains, which resulted in the phase separation in the membranes into gel phase and fluid like lipid domains (Gohrbandt et al., 2022).Strikingly, this large-scale phase separation was not lethal, indicating that despite the presence of gel phase domains in the membrane, the permeability function of the membrane remained intact.Nevertheless, many cellular processes were affected due to the crowding of membrane proteins in the fluid like regions, and the membrane potential of these cells was significantly lowered (Gohrbandt et al., 2022).
Although overall fluidity is controlled to keep the membrane in a liquid crystalline phase, recent work has shown that local demixing of membranes into Liquid ordered (Lo) and Liquid disordered (Ld) phases occurs in model systems, provided that the membranes are composed of a mix of fatty acids with saturated and unsaturated acyl chains combined with hopanoids, pentacyclic compounds from bacterial membranes with a function analogous to cholesterol in eukaryotic membranes (Mangiarotti et al., 2019).Evidence for local phase separation in nanoscale Lo domains was also found in live B. subtilis cells using neutron scattering, with the domains being up to 40 nm in diameter (Nickels et al., 2017).These domains are too small to image by conventional light microscopy, which has been used to study asymmetric distribution of lipids and membrane fluidity in bacterial cells.One of the earliest dyes to be used to study specific lipid distributions was nonyl acridine orange (NAO).The NAO fluorescence shift toward the red spectrum was thought to be specific for cardiolipin (Mileykovskaya & Dowhan, 2000), but it was later shown that this effect is unspecific upon binding to anionic phospholipids (Oliver et al., 2014;Pogmore et al., 2018).Still, NAO staining identified polar domains in many bacterial species (Strahl & Errington, 2017), and the enrichment of cardiolipin in polar regions was independently confirmed by analysis of the lipid content of E. coli minicells (Koppelman et al., 2001), which fits the idea that the conical shape of cardiolipin is a driver for accumulation at the more strongly curved poles of bacterial cells.It is assumed but not proven that the accumulation of cardiolipin and anionic phospholipids at the cell pole serves specific functions, for example, in transertion (below), in chemoreception, or in cell division which generates new cell poles after division.
Staining with DiI-C12, a membrane stain with a preference for fluid areas (Ld domains), showed that there are specific areas in the bacterial membrane distributed along the entire cell surface, where fluidity is increased (Figure 1) (Oswald et al., 2016;Strahl et al., 2014).
These areas colocalize with, and are organized by, the peripheral membrane protein MreB and were termed Regions of Increased Fluidity (RIFs) (Strahl et al., 2014).MreB is a critical component of the peptidoglycan synthesis machinery, coordinating the "elongasome" which is a protein complex involved in synthesis of peptidoglycan in the lateral wall of nonspherical bacteria (Egan et al., 2020).These regions are dynamic, and MreB dynamics is directly linked to peptidoglycan synthesis activity (Dominguez-Escobar et al., 2011;Garner et al., 2011;van Teeffelen et al., 2011).Peptidoglycan synthesis requires the presence of Lipid II, the peptidoglycan precursor that is embedded in membranes via an isoprenoid chain and that has been shown to prefer a more fluid membrane environment and in fact induces membrane disorder (Ganchev et al., 2006;Jia et al., 2011).
Whether the increased fluidity in RIFs is a direct consequence of the presence of Lipid II, or that RIFs are created to produce a favorable environment for Lipid II and peptidoglycan synthesis, remains to be established.In the absence of MreB and elongasome function, peptidoglycan synthesis in the lateral wall is mediated by class aPBPs, bifunctional peptidoglycan synthases, but the orientation of synthesis by aPBPs is random rather than directional (Dion et al., 2019).
It may be that the presence of a more fluid membrane (RIF) allows rapid vectorial movement of the elongasome driven by MreB polymerization (Figure 1).
The nanometer-scale regions of decreased fluidity (Lo domains) observed by neutron scattering in B. subtilis are similar in size to membrane patches formed by bacterial flotillin homologs, of which B. subtilis has two (FloA and FloT) (Nickels et al., 2017;Schneider et al., 2015).Flotillins, in eukaryotes, are found in membrane domains known as "lipid rafts," which are enriched in sphingolipids and cholesterol.Eukaryotic flotillins are bound to the membrane via palmitoyl-or myristoyl-membrane anchors and contain an N-terminal SPFH (Stromatin, Prohibitin, Flotillin, and HflK/C) domain that associates with the membrane, and a flotillin domain that contains coiled-coil structures that mediate homo-oligomerization.
Flotillin homologs are widespread in bacteria, but are anchored to the membrane via an N-terminal hairpin loop or a transmembrane segment, instead of via attached lipids (Bach & Bramkamp, 2015;Bramkamp & Lopez, 2015;Lopez & Koch, 2017).Also, the SPFH domain in bacterial flotillins aids in oligomerization rather than in membrane attachment (Bach & Bramkamp, 2015).Bacterial flotillins, like their eukaryotic counterparts, are enriched in fractions of the membrane that are more resistant to detergent solubilization (detergent resistant membrane, DRM) in fractionation studies and have therefore been associated with a more rigid membrane structure (Lingwood & Simons, 2007).However, it is commonly accepted now that detergent extraction at low temperatures likely induces changes in the membrane organization and DRM fractions thus do not represent lipid and protein assemblies as they occur in the native membrane structure.Analysis of membrane fractions from B. subtilis and Staphylococcus aureus showed that specific lipids, negatively charged phospholipids and carotenoids, respectively, are enriched in the detergent resistant membrane fraction (DRM) and, in the case of S. aureus staphyloxanthin, directly interact with flotillins (Donovan & Bramkamp, 2009;García-Fernández et al., 2017).This indicates that the flotillin oligomers, also known as Functional Membrane Microdomains (FMMs), may interact with specific lipids (Figure 1), as has been shown for several eukaryotic SPFH-domain containing proteins that directly bind cholesterol (Huber et al., 2006).Deletion of flotillins results in an overall increase of membrane rigidity and loss of membrane heterogeneity (Bach & Bramkamp, 2013;Zielińska et al., 2020).Interestingly, similar to the deletion of flotillins, loss of yisP, required for the production of farnesol (Feng et al., 2014), leads to an increase in membrane rigidity (Bach & Bramkamp, 2013).Thus, a yisP deletion phenocopies a flotillin deletion and both phenotypes can be partially restored by adding carotenoids to cells, for example, staphyloxanthin, which was found to be associated with flotillins in S. aureus (Bach & Bramkamp, 2013;García-Fernández et al., 2017;Lopez & Kolter, 2010).

| What is the relation between DRMs and FMMs?
Following the initial description of bacterial flotillins and the finding that several proteins found in DRMs are no longer found in DRMs when flotillins are absent (Donovan & Bramkamp, 2009;Lopez & Kolter, 2010), the protein composition of DRMs was characterized in various studies, combined with pull-down or native PAGE to enrich proteins physically associated with flotillins (Bach & Bramkamp, 2013;García-Fernández et al., 2017;Schneider et al., 2015;Yepes et al., 2012).This identified a large set of membrane proteins involved in various processes such as signaling, cell division, protein secretion, and peptidoglycan synthesis, which fit the rather pleiotropic phenotypes observed with Flotillin deletions.Careful super-resolution microscopy analysis of membrane protein dynamics revealed that many of the proteins identified as FMM constituents/Flotillin binding partners were in fact not, or only transiently, associated with Flotillins, with the exception of NfeD2 (Dempwolff et al., 2016).Also, when the surface area occupied by FloA and FloT foci is calculated in a B. subtilis cell (Schneider et al., 2015), the FMMs occupy less than 2% of the total membrane surface area.It is therefore likely that the DRM fraction contains a much larger fraction of the membrane than only FMMs.Another intriguing question is why deletion of genes involved in the synthesis of carotenoids, which organize Lo domains, leads to the overall rigidification of membranes and the reduction (based on protein content) of DRM fractions (Bach & Bramkamp, 2013;Lopez & Kolter, 2010).

| What is the function of flotillins?
The transient association with a plethora of membrane proteins that function in (homo)oligomeric complexes, many of which function suboptimally when flotillins are absent, led to the hypothesis that flotillins act as oligomerization platforms that ensure correct formation of these membrane protein complexes (Lopez & Koch, 2017).Native PAGE experiments identified such a role in the formation of cell wall synthesis complexes in S. aureus (García-Fernández et al., 2017), but this was not confirmed in a similar analysis of cell wall synthesis proteins from B. subtilis (Zielińska et al., 2020).Flotillins themselves cluster in high-molecular-weight oligomers (12 to 50 mers), which corresponds with their appearance in distinct patches in the membrane (Bach & Bramkamp, 2013;Dempwolff et al., 2016;Schneider et al., 2015).In vitro, flotillins have been shown to fluidize the membranes, and the membrane rigidification observed in the absence of flotillins in vivo results in a reduced dynamics of MreB, which can be restored when a chemical fluidizer is added to cells (Zielińska et al., 2020).MreB dynamics are directly coupled to elongasomemediated cell wall synthesis-which, in B. subtilis, becomes upregulated in the absence of the aPBP1 (Patel et al., 2020).This explains the observed growth defect of strains that lack both aPBP1 and flotillins: The elongasome, which is now the principal source of peptidoglycan, cannot move fast enough through the membrane (Zielińska et al., 2020).It is not unlikely that the observed rigidification also impacts other membrane processes linked to flotillins (Bramkamp & Lopez, 2015;Lopez & Koch, 2017), such as division, sporulation, signaling, biofilm formation, and protein secretion.Various important questions remain, such as the biophysical mechanism by which flotillins fluidize the membrane and how flotillins interact with other membrane proteins and lipids.An important clue may be found in the recently described structures of the HflK/C-FtsH protease complex (Daumke & Lewin, 2022;Ma et al., 2022;Qiao et al., 2022).HflK and HflC are both proteins with a single transmembrane segment and an SPFH domain, and 12 subunits of each protein together were found to form a large membrane embedded ring that forms a cage like structure on top of the membrane outer leaflet which encloses four FtsH hexamers (Ma et al., 2022;Qiao et al., 2022).The SPFH domains interact at the base of this cage, close to the membrane, with long α-helices forming the barrel of the cage, which is closed on top by a β-barrel formed of β-strands from the individual subunits.It is quite likely that the oligomers formed by flotillins, through interaction of their SPFH domains (Bach & Bramkamp, 2015), can form a similar cage like structure at the inner leaflet of the membrane, anchored to the membrane by hairpin loops or transmembrane segments.Such a ring-like structure would create a circular microdomain, and the extensive interactions could act as a scaffold structure for Liquid-Liquid Phase Separation (Azaldegui et al., 2021;Daumke & Lewin, 2022) to create a microdomain to which, for example, specific lipids, such as hopanoids, could be recruited.Various protein classes containing SPFH domains interact with proteases, such as the HflK/C-FtsH combination (Kihara et al., 1996).Flotillins are often found in genetic organization, but also interacting and colocalizing with a cognate NfeD protein that often contains a serine protease domain and is anchored to the membrane via several transmembrane helices (Dempwolff, Moller, & Graumann, 2012;Hinderhofer et al., 2009).The association with proteases could be a hint that flotillins provide microdomains where membrane-anchored proteases can perform their function in cellular quality control.
Recruitment of specific lipids (such as hopanoids) to these microdomains may facilitate the formation of Ld domains (with Lipid II) elsewhere in the membrane.Interestingly, the effect of flotillins on membrane fluidity was only observed in cells growing fast on rich medium (Zielińska et al., 2020).This suggests that specific lipids, produced when cells grow fast, can be found in these patches-even though the in vitro data strongly suggest that flotillins do not need a specific lipid to fluidize a membrane (Zielińska et al., 2020).Alternatively, the higher rate of Lipid II synthesis at fast growth rates may necessitate a better controlled organization of the membrane to allow efficient peptidoglycan synthesis.A puzzling observation is that expression of flotillin genes in B. subtilis is regulated during growth, with at least one flotillin always present in sufficient amounts and one flotillin upregulated in poor medium, or stress conditions that trigger developmental processes like sporulation and biofilm formation-where at least an effect of flotillins on bulk fluidity cannot be detected (Nicolas et al., 2012;Schneider et al., 2015;Zielińska et al., 2020).Thus, the exact function(s) of flotillins are far from known and may well combine the creation of a favorable membrane environment to allow dynamic processes, as well as controlling protease activity and possibly protein oligomerization.
Finally, the limited number of organisms in which flotillin function has been studied so far makes it difficult to make general statements about the role of these proteins.

| MEMB R ANE COMPA RTM ENT ALI Z ATI ON IN MYCOBAC TERIA
Recently, a very specific case of membrane compartmentalization was described in Mycobacteria, where the synthesis and membrane insertion of the peptidoglycan precursor Lipid II was shown to be spatially separated from the incorporation of Lipid II into peptidoglycan (García-Heredia et al., 2021).The latter process occurs at the tip of the cell, which, like other actinobacteria, grows from the pole.Again, a separation of the membrane in fractions with different biochemical properties, in this case using density centrifugation or solubilization, had revealed that lipid biosynthesis occurs in a distinct membrane fraction and that the two membrane fractions identified had a different proteome and lipidome composition (Morita et al., 2005).The lipid biosynthesis fraction, termed IMD (for intracellular membrane domain-please note that this domain is part of the plasma membrane), is the place where Lipid II synthesis takes place.These IMDs are located just after the tip of the cell where actual peptidoglycan synthesis occurs, and these two domains are clearly separate as demonstrated by microscopy (García-Heredia et al., 2021).The organization of these domains is dependent on the polar organizing protein DivIVA, which localizes to the convex cytoplasmic leaflet of the membrane (Lenarcic et al., 2009).DivIVA is required for polar cell wall synthesis in actinobacteria, such as mycobacteria, corynebacteria, and streptomyces (Howell & Brown, 2016).
Again, membrane fluidity seems to play an important role in the separation of these domains as treatment of mycobacteria with a chemical fluidizer delocalized peptidoglycan synthesis from the poles to the sidewall (García-Heredia et al., 2021).

| MEMB R ANE COMPA RTM ENT ALI Z ATI ON IN TR AN S ERTI ON ZONE S
The coupled transcription and translation at the membrane and the subsequent insertion of membrane proteins into the lipid bilayer at these sites has been termed "transertion" (Binenbaum et al., 1999;Matsumoto et al., 2015;Norris, 1995;Norris & Madsen, 1995).The hypothesis of transertion sites as a membrane organizing principle has been formulated about 30 years ago (Norris, 1995;Norris & Madsen, 1995;Woldringh et al., 1995).Cotranslational biogenesis of membrane proteins via the signal recognition particle receptor FtsY that couples the ribosome to the SecYEG/YidC apparatus is a wellknown process in bacteria (Rapoport et al., 2017).However, we still have very limited data about transertion processes that also include localization of the respective genes to the membrane.A recent study suggests that the expression and assembly of the type III secretion system (T3SS2) in Vibrio parahaemolyticus occurs via a transertion mechanism (Kaval et al., 2023).A two-step transertion mechanism is proposed in which a membrane bound receptor complex composed of VtrA/C is activated by bile salts and recruits the target gene vtrB to the membrane.VtrB is a transmembrane protein, acts as transcriptional activator of the T3SS2 genes, and activates transcription and assembly of the T3SS2 needle apparatus (Kaval et al., 2023).
Similarly, under inducing conditions, the gene encoding the lactose permease LacY shifts toward the membrane, indicating that LacY could be inserted into the membrane by a transertion mechanism as well (Libby et al., 2012).Negatively charged phospholipids like cardiolipin (CL) and phosphatidylglycerine (PG) have been connected with transertion (Fishov & Norris, 2012;Matsumoto et al., 2015).
Cardiolipin enriches at curved membrane regions due to is molecular geometry (Koppelman et al., 2001;Mileykovskaya & Dowhan, 2000;Mukhopadhyay et al., 2008), and hence, several transport proteins have been shown to localize in a CL dependent manner to cell pole regions (Romantsov et al., 2010).However, it remains unclear whether transertion requires specific lipid environments or whether the process itself would induce membrane compartmentalization.
Conventional imaging methods do not provide sufficient resolution, and the highly dynamic nature of protein-membrane interactions hampers analysis using nanometer scale resolution with electron microscopy.Recent advances in nanoscopy techniques will likely provide useful insights into these processes.

| MEMB R ANE DYNAMI C S: REPAIR AND REMODELING
The membrane is a vital structure for cellular integrity.Rupture of the plasma membrane in bacteria is usually considered a sign of cell death.Experimentally this is exploited in the classical live/ dead staining, using propidium iodine that is not membrane permeable and only intercalates with DNA inside the cell when membrane lesions exist.Ruptures within the membrane, even at small scale, will lead to a rapid loss of ion gradients and de-energize the cell.It is therefore plausible that repair and maintenance systems evolved that help to repair membrane lesions and ensure tight sealing of the cell interior.Interestingly, most of the systems that we currently know have been identified and studied in eukaryotes for a long time.These systems include the membrane fusion and fission catalysts of the dynamin protein family (Bramkamp, 2012;Praefcke & McMahon, 2004) and members of the ESCRT-III/PspA/ Vipp protein family (Barisch et al., 2023;Isono, 2021;Jimenez et al., 2014).We focus here on membrane repair in the sense of sealing of membrane rupture.Phospholipid repair via the LplT/Aas system is not discussed (Lin et al., 2018); however, repair of individual phospholipid molecules contributes to membrane integrity and fluidity control.

| FUN C TION OF DYNAMIN -LIKE PROTEIN S IN BAC TERIA
Membrane remodeling in eukaryotes is often mediated by large GTPases of the dynamin family of proteins (Praefcke & McMahon, 2004).The name-giving dynamin is the paradigm of these enzymes and crucially involved in scission of endocytic vesicles, where it forms a ring-like collar around the neck of the budding vesicle (Perrais, 2022).Assembly stimulated GTP hydrolysis leads to a constriction of the dynamin collar squeezing the membrane until the vesicle pinches off (Schmid & Frolov, 2011).There are many related proteins, sharing a similar domain setup with dynamin, which we collectively call dynamin-like proteins (DLPs) (Praefcke & McMahon, 2004).A hallmark of all DLPs is their low affinity for guanine nucleotides (~10-100 μM) and their relatively high basal activity (~0.4-1 min −1 ) (Song & Schmid, 2003).They all self-assemble into larger oligomeric structures, bound to membranes (Ford & Chappie, 2019).Consistent with their membrane-binding capacities, DLPs are involved in scission and fusion of membranes.These activities are in particular well studied in membrane dynamics of organelles, such as mitochondria, chloroplasts, and the endoplasmic reticulum (Ford & Chappie, 2019;Praefcke & McMahon, 2004).DLPs share a conserved molecular architecture, a globular, GTP-binding domain (G-domain), a neck (also termed bundle signaling element, BSE), and an extended stalk (or trunk) (Bramkamp, 2012;Praefcke & McMahon, 2004).There are variations in the arrangement of the helical bundle between different DLPs.However, a common theme is that at the tip of the stalk domain, DLPs contain a lipid binding domain.This domain can differ significantly, from a pleckstrin homology domain as in dynamin to a membrane binding loop, merely having amino acids with hydrophobic side chains, poking into one membrane leaflet, usually stabilized by electrostatic interactions coming from positively charged amino acids (Low et al., 2009;Low & Löwe, 2006).
Despite early reports about homologous DLPs in bacteria (Leipe et al., 2002), the function of bacterial dynamin like proteins (BDLPs) has been unclear until very recently.Structures of BDLP reveal their canonical DLP domain architecture (Low et al., 2009, Low & Löwe, 2006).Lipid binding is mediated by a paddle domain.In the Nostoc punctiforme BDLP, a phenylalanine and two leucine residues insert into the membrane.
BDLPs have been implicated with several cellular functions, including cytokinesis (Bürmann et al., 2011;Schlimpert et al., 2017), membrane surveillance and repair (Sawant et al., 2016), vesicle formation (Michie et al., 2014), and phage defense (Guo et al., 2021;Sawant et al., 2016).A common theme is that these functions are all associated with cell membrane dynamics.In line with a membrane-associated function of BDLPs, a double deletion of dynamin and flotillin has been shown to impair cell motility in B. subtilis (Dempwolff & Graumann, 2014;Dempwolff, Wischhusen, et al., 2012).Consistent with functions of eukaryotic DLPs in membrane fusion and fission, in vitro studies with the B. subtilis BDLP DynA revealed that DynA tethers membranes in trans and catalyzes membrane fusion (Bürmann et al., 2011;Guo & Bramkamp, 2019).Lipid mixing, for example, the fusion of the outer leaflet of two membranes, is an intermediate step during both fusion and fission.
DynA is capable to mediate content mixing in liposomes, indicating full membrane fusion (Guo & Bramkamp, 2019).In vitro fusion processes were GTP independent; however, they were slow processes and it can be assumed that in vivo membrane fusion is faster (Guo & Bramkamp, 2019).Fusion activity is well in line with function of BDLPs in membrane repair.Indeed, it was shown that BDLPs in various organisms react after treatment of bacteria with antibiotics (Sawant et al., 2016).The membrane surveillance function has been best addressed in the B. subtilis DynA protein.In unstressed cells, DynA is mainly cytosolic; however, a significant fraction is associated to the membrane (Sattler & Graumann, 2022;Sawant et al., 2016).The membrane-associated fraction diffuses along the inner leaflet.In response to cell membrane stress, DynA assembles into larger, membrane-associated complexes that are basically immobile (Guo et al., 2021;Sattler & Graumann, 2022;Sawant et al., 2016) (Figure 2).Importantly, DynA reacts exclusively to stresses that induce membrane pores or membrane deformations.This includes pore-forming antimicrobials such as nisin (Sawant et al., 2016) (Figure 2a-c).This is in line with the findings that deletion of dynA renders cells more sensitive toward nisin.Nisin induces membrane deformations in B. subtilis that can be stained by specific membrane dyes, such as nile red, due to their increase in fluidity.
DynA localizes to these areas where it forms stable foci.Similarly, in Mycobacterium smegmatis, the BDLP protein IniA was shown to localize to membrane ruffles and is induced by treatment with the anti-tuberculosis drug isoniazide (INH) (Wang et al., 2019).INH inhibits mycolic acid synthesis required for the outer membrane in Mycobacteria and other mycolic acid containing Corynebacterineae.
A direct effect on the plasma membrane is not reported for INH, but antibiotics interfering with the cell wall or cell envelope in general often cause plasma membrane stress due to the tight coupling of the different envelope layers.
Lack of DynA impairs membrane integrity in B. subtilis under membrane stress conditions as shown by live/dead staining techniques (Sawant et al., 2016).The molecular mechanism by which DynA supports membrane integrity in vivo is not entirely clear.
Based on the in vitro membrane fusion activity, it is tempting to speculate that DynA fuses membrane lesions or excises defective membrane areas (Figure 2a-c).Additionally, it was shown that DynA directly influences lipid diffusion (Sawant et al., 2016).Cells lacking DynA show a reduced lipid dynamic and, hence, DynA might contribute to membrane repair by fluidizing the membrane that in turn facilitates fusion processes.BDLP-promoted membrane integrity plays also a role in population-wide resistance against phage predation.B. subtilis cells infected with phages (or in which prophages have been activated) lyse significantly later and less disruptive, compared to dynA mutant strains (Guo et al., 2021).This delays and restricts the spreading of phage progeny, thereby providing a last line of defense in phage infection (Figure 2a-c).Although membrane tethering and fusion by DynA was GTP independent in vitro, phage resistance is strictly dependent on a functional GTPase activity (Guo et al., 2021).Recent phylogenetic data show that genes encoding BDLPs are often in the vicinity of defense islands, gene loci coding for anti-phage systems, further lending support to the notion that BDLPs are involved in phage defense (Millman et al., 2022).
The protein pair DynA/DynA in Streptomces have been implicated with stabilization of the main division protein FtsZ and may also be involved in membrane fusion at the end of cytokinesis (Schlimpert et al., 2017).In vegetative hyphae of S. coelicolor, a cell wall deficient, membrane cross wall is formed, independent of FtsZ function (Yague et al., 2016).Currently, it is unclear whether the Streptomyces-specific dynamins are involved in the formation of these membrane structures.(c, left).DynA is also involved in prevention of progeny egress upon bacterial infection with phages (a, right).Cell lysis that is triggered by phage encoded murein hydrolases leads to bulging of the plasma membrane through holes in the peptidoglycan (b, right).DynA seems to stabilize these sites and prevents explosive disruption of the membrane and therefore restricts the release of phage progeny (c, right).This results in a protective effect on the population level.Right panel: The ESCRT-III-like protein PspA is recruited to the membrane by the tripartite complex YdjGHI (d).YdjI is a member of the SPFH-domain family of proteins and likely forms oligomeric assemblies like other members of this family.The Ydj complex senses membrane damage and recruits PspA, which in turn assembles at the sites of membrane lesions (e).Oligomerization of PspA likely seals off and cleaves off the damaged membrane area (f).

| E SCRT-LIK E SYS TEMS
Another conserved family of proteins involved in membrane remodeling is related to the endosomal sorting complex required for transport (ESCRT).ESCRT proteins are ubiquitously distributed in pro-and eukaryotes (Isono, 2021;Siebenaller & Schneider, 2023).
The core component of the eukaryotic membrane remodeling complex is the ESCRT-III protein that was shown to have membrane scission activities.The ESCRT-III complex has been implicated with several cellular processes that require membrane dynamics, including cytokinetic abscission (Samson et al., 2008), exosome release (Colombo et al., 2013), vesicle formation (Cruz Camacho et al., 2023), and viral budding (Ju et al., 2021).However, in recent years, it became also clear that ESCRT contributes to cell membrane integrity.In eukaryotic cells, it has been observed that ESCRT-III proteins are specifically recruited to sites of damaged membranes and accumulate there until membrane closure (Jimenez et al., 2014).This process requires calcium ions and several associated proteins, and ESCRT-mediated repair is achieved by membrane budding and subsequent shedding of damaged membranes.Interestingly, ESCRT-III functions in membrane repair also antagonized programmed cell death induced by gasdermins (Ruhl et al., 2018), thereby restricting the inflammatory response.
The precise function of ESCRT-III complexes is not entirely clear and a "dome" and "buckling" model are discussed.Similar in both models, the main function is to release budding vesicles away from the cytoplasm in eukaryotes.
In contrast to the ESCRT function in eukaryotes, little is known about the functions in prokaryotic cells.In archaea, ESCRT-III systems are involved in cytokinesis, likely driving the cell membrane dynamics required to separate the daughter cells (Samson et al., 2008).Bacteria encode various ESCRT-III homologs.The best known is the phage shock protein A (PspA).PspA was discovered in E. coli where large amounts of this protein are produced in response to stresses such as infection by filamentous phages, elevated temperature, ethanol addition, and osmotic shock (Brissette et al., 1990(Brissette et al., , 1991)).A common denominator of all these stresses is their immediate effect on membrane integrity.The formation of PspA carpet-like oligomers has been proposed to seal membrane leakage (Kobayashi et al., 2007).The structures of PspA and its cyanobacterial homolog Vipp1 were recently solved and revealed their canonical helical fold with a helix-loop-helix structure forming a central coiled-coil (Gupta et al., 2021;Junglas et al., 2021;Liu et al., 2021).While PspA functions on the plasma membrane, Vipp1 is required for thylakoid membrane repair.Both proteins form stacked oligomers that bind to membranes via amphipathic helices.Although the overall fold of PspA and Vipp1 is similar to ESCRT-III, their membrane interaction is different (Gupta et al., 2021;Junglas et al., 2021;Liu et al., 2021).While ESCRT-III deforms membranes away from its polymer, PspA and Vipp1 deform membranes toward the polymer, leading to membrane tubulation and eventually to membrane fusion and scission events (Deo & Prinz, 2021).
A recent detailed bioinformatic analysis has shown that PspA homologs are widely distributed in bacteria and archaea (Popp et al., 2022).Among the most conserved proteins of the Psp system is the core subunit PspA, while associated proteins vary drastically in their distribution.This suggests that PspA proteins might interact with so far unknown complex partners in various bacteria.Under nonstressed conditions, E. coli PspA forms a complex with PspF, thereby inhibiting PspF-induced transcriptional activation of the psp operon (Joly et al., 2009).Genetic regulation of the pspA-ydjGHI operon is under control of the extra cytoplasmic function sigma factor σ W that acts in response to cell envelope stress.Upon membrane stress, the membrane integral components PspB and PspC disassemble the complex and release PspF and initiate PspA oligomerization, likely at sites of membrane damage.In several Gram-negative pathogens, the PspA system has also been linked to virulence and infection, suggesting roles for Psp proteins apart from membrane repair (DeAngelis et al., 2019).Fluorescently labeled PspA forms foci under membrane stress conditions (Scholz et al., 2021).
The B. subtilis PspA requires the SPFH domain protein YdjI and the two membrane integral proteins YdjG and YdjH for focus formation (Scholz et al., 2021) (Figure 2d-f).The YdjGHI complex is thought to form a membrane integral sensory complex that reacts to membrane stress.PspA foci do not always co-localize with YdjGHI complexes, suggesting that the Ydj complex does not remain associated with the PspA assemblies during the process of membrane repair.Unlike the other proteins with SPFH domains in bacteria, such as FloT and FloA, YdjI resides in fluid membrane domains and is therefore mutually exclusive with FloT.However, deletion of YdjI also decreases overall membrane fluidity, suggesting a conserved function of SPFH domain proteins in membrane fluidizing (Scholz et al., 2021).Deletion of the membrane integral components YdjG and YdjH results in severe morphology defects, due to mislocalization of the cell wall synthetic machinery, showing how sensitive the cell wall synthesis machinery reacts to impaired cell membrane stress response (Scholz et al., 2021).The genetic link between PspA proteins and SPFH domain proteins (also termed BAND7) is found not only in Firmicutes but also in other phyla (Popp et al., 2022).
Since PspA binds membranes via its N-terminal amphipathic helix, it can be speculated that binding of PspA is facilitated in fluid membrane regions.
Similarly, the PspA homolog LiaH from B. subtilis is involved in cell envelope stress response induced by specific antibiotics such as nisin, daptomycin, and vancomycin.A weaker induction is also observed in presence of ethanol, detergents, and alkaline shock (Dominguez-Escobar et al., 2014).Although the presence of the lia operon does not refer resistance, nor does deletion cause significantly increased sensitivity, it is believed that the system acts in a membrane protective manner.However, LiaH has been linked to auto-resistance against a B. subtilis produced antimicrobial peptide, YydF.YydF is a prepro-peptide that gets proteolytically activated and exported, where it acts as a membrane active antimicrobial compound.The Lia system is strongly induced by the active peptide and counteracts membrane pertubations (Popp et al., 2020).

| CON CLUDING REMARK S
As is clear, a lot of progress has been made over the past years in the study of various protein families that regulate bacterial membrane dynamics.Many of these families were first described in eukaryotes and there are similarities, but also clear differences, in the way these proteins work in prokaryotes.What is now needed are advanced microscopy techniques that allow the study of the formation of FMMs, PspA carpets, and other events with sufficient temporal and spatial resolution to elucidate their function and regulation.Also, biophysical and structural studies are required to reveal the molecular detail of the protein-protein and protein-lipid interactions involved.
Finally, it is becoming more and more clear that physico-chemical properties of the membrane, such as fluidity, regulate various processes, like peptidoglycan synthesis, which were thought to be regulated through protein-protein interactions (almost) exclusively.

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I G U R E 1 Membrane regions with increased and decreased fluidity.Left: Zones of MreB/SEDS/bPBP mediated peptidoglycan synthesis correlate with Liquid disordered (Ld) membranes.Lipid II prefers Ld membrane zones and is translocated by MurJ and inserted into a nascent peptidoglycan strand via the concerted action of a SEDS family glycosyl transferase (RodA) and a Class B Penicillin Binding Protein (bPBP) transpeptidase.Right: Flotillins oligomerize on the membrane, hypothetically forming cage-like structures as seen for the SPFH proteins HflK/C.Putatively, these structures organize Liquid ordered (Lo) domains in the membrane, the so-called Functional Membrane Microdomains (FMMs).Associated with these FMMs are specific lipids such as carotenoids (dark yellow) and hopanoids (dark blue), which could either be on the outside of or enclosed by (or both) the flotillin ring.The SPFH domain cage could provide a space for specific flotillin-associated proteins, such as the NfeD proteases.Insert (b): View of the hypothetical flotillin cage from the cytoplasmic side, with phospholipid headgroups in green.

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Function of dynamin-and ESCRT-III-like proteins in bacterial membrane repair.Left panel: The dynamin-like protein DynA from B. subtilis is involved in repair of membrane pores.Exemplarily, pore formation is shown by the antimicrobial compound nisin that forms lipid-II-dependent membrane pores (a, left).DynA accumulates at these sites (b, left) and likely cleaves off parts of damaged cell membrane