Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress

1The cell envelope is a multilayered outer barrier that protects the cell from a changing environment. Cell envelope stress responses (CESRs) are regulatory pathways that sense threats and mount a protective response, often involving modification of lipopolysaccharides (in Gram-negative bacteria), teichoic acids (Gram-positive bacteria), peptidoglycan, and the inner membrane. Here, we focus on Bacillus subtilis as a Gram-positive model for the role of CESRs in membrane homeostasis.

2The cell membrane is a dynamic, fluid mosaic comprising a lipid bilayer and associated proteins (Figure 1). In B. subtilis, the major lipid species are phospholipids, glucolipids, and the lipoteichoic acids (LTA). The membrane proteome includes proteins for transport and signaling, as well as membrane synthesis, remodeling, and protection. As the innermost and last line of defense, the cell membrane is critical for viability. In B. subtilis, for example, collapsing the proton motive force activates autolysins resulting in rapid cell lysis. Membrane-active compounds such as detergents, antimicrobial peptides, and pore-forming compounds often trigger stress responses that modify the lipidome and membrane proteome to confer resistance. Membrane stress responses can modify the cell membrane, by (i) modulating the length, branching, and saturation of the fatty acid (FA) acyl chains, (ii) altering membrane lipid composition, or (iii) synthesizing proteins that modify or protect the membrane (Table 1).

3Most bacteria utilize a type II FA synthase that catalyzes repeated cycles of acyl chain elongation. The committed step, catalyzed by acetyl-CoA carboxylase (ACC), generates malonyl-CoA and then malonyl-ACP to serve in FA chain initiation by FabH and elongation by FabF. B. subtilis has two isoforms of FabH, and both preferentially synthesize branched chain FAs (BCFAs). Acylation of glycerol-3-phosphate by the PlsX/PlsY/PlsC acyltransferase system with long chain FAs generates phosphatidic acid, the precursor to all other phospholipids.

4FapR is the key transcriptional regulator of membrane lipid synthesis in B. subtilis and clinically relevant pathogens such as Staphylococcus aureus, Bacillus anthracis, and Listeria monocytogenes, and modulates the overall rate of membrane synthesis in response to precursor availability. B. subtilis FapR represses genes for FA and phospholipid synthesis, and this repression is relieved by allosteric interactions with malonyl-CoA or malonyl-ACP.

5As a branchpoint enzyme, ACC is often under complex regulation. In B. subtilis, ACC is regulated in part by YqhY, a conserved DUF322/Asp23 protein which is highly expressed and often encoded together with ACC subunits as part of an accB-accC-yqhY operon. The namesake, S. aureus Asp23, is a membrane-associated protein originally linked to alkaline shock. Loss of Asp23/YqhY causes cell wall stress and poor growth. In B. subtilis, yqhY null mutants acquire suppressors that decrease ACC activity, but this selective pressure is alleviated in medium supplemented with acetate. We suggest that ACC-dependent depletion of acetyl-CoA may contribute to wall stress by negatively affecting synthesis of UDP-N-acetylglucosamine needed for peptidoglycan synthesis. A key challenge for future research will be to understand the precise role of YqhY/Asp23 proteins and how they control ACC activity to balance FA synthesis with other cellular needs.

6Tuning of FA composition provides one way in which the cell can optimize membrane properties in response to a changing environment. Even under non-stressed conditions, B. subtilis membranes contain ∼7 distinct FAs varying in length from C14 to C18 (indicating the number of carbon atoms) and include both branched (∼24% iso and 66% anteiso) and straight chain (∼10%) FAs. Since membrane phospholipids and glucolipids each contain 2 FA chains, the lipidome contains a complex mix of species (Figure 1B), with a preponderance containing one C15 and one C17 FA chain.

7Modifications of FAs are important for regulating membrane fluidity in a process known as homeoviscous adaptation. In B. subtilis, temperature downshift induces a FA desaturase (Des) controlled by the DesKR two-component system (TCS). Des modifies existing membrane lipids, and is thereby suited for rapid adaptation. DesK is one of the better understood TCS sensors, with both kinase and phosphatase activity. DesK lacks an extracellular sensor domain, but has multiple transmembrane segments that sense changes in the membrane physical state. DesK phosphorylates the DesR response regulator, which induces des, encoding a FA Δ5 desaturase. The resultant unsaturated FAs increase bilayer fluidity, which restores DesK phosphatase activity in a negative feedback loop. Longer term adaptation to low temperatures relies on an isoleucine-dependent switch to primarily anteiso-FAs. Since anteiso-FAs perturb the lateral interactions between adjacent lipids to a greater extent than iso-FAs (Figure 1B), this shift increases membrane fluidity. This shift may result from a cold-dependent change in FabH activity.

8Membranes must also adapt to conditions that increase fluidity. In B. subtilis, the ECF σ factor σW is activated by detergents, antibiotics, and bacteriocins active on the membrane. A σW promoter within the fabHA-fabF operon plays a major role in homeoviscous adaptation. Activation of σW leads to a decrease in FabHA levels, resulting in increased reliance on FabHB and an increase in straight chain FAs (from ∼10 to 30%). Elevated expression of the FabF elongation enzyme leads to increased FA chain length. The combined effect is a membrane with longer acyl chains and less BCFA. This increased membrane rigidity serves to protect cells against detergents and antimicrobial peptides. The activation of σW is controlled by regulated proteolysis of its membrane-bound anti-σW factor (RsiW). However, the mechanisms by which membrane stressors trigger σW activation remain unclear.

9To better understand the role of FA heterogeneity in controlling membrane properties, it would be desirable to study bacteria with chemically simple membranes. This has been achieved in B. subtilis by feeding exogenous FAs to cells with de novo FA synthesis blocked by cerulenin and a mutation to inhibit FA degradation. Growth can be rescued with only two FA species: a straight-chain C16 FA (high melting) and an anteiso C15 FA (low melting). Even with only these two FA species, four distinct arrangements are possible upon acylation of glycerol-3-phosphate to generate phosphatidic acid. Cells compensate for this reduced FA complexity by altering the distribution of phospholipid headgroups, a modest induction of the DesRK system, apparent downregulation of the σW stress response, and an increase in isoprenoid lipids. These results highlight the remarkable adaptability of bacterial membranes, and the interconnection between diverse stress responses.

10One of the persistent challenges in membrane biology is to define the roles of the diverse constituent lipids. Although membranes have a complex and adaptable composition (the lipidome), cells are remarkably resilient to genetic alterations that remove lipid species. Because of its single membrane and ease of genetic manipulation, B. subtilis presents an attractive model system. The B. subtilis lipidome comprises ∼70% phospholipids and ∼30% neutral glucolipids. The major phospholipids are phosphatidylglycerol (PhG) and phosphatidylethanolamine (PE), with minor contributions from cardiolipin and lysylphosphatidylglycerol (LPG). Variations in phospholipid headgroup size and charge modulate membrane properties (Figure 1B). Membranes also contain LTA anchored to neutral glucolipids, which together with peptidoglycan-linked wall teichoic acid (WTA) can account for up to 60% of the dry weight of the cell wall. However, LTA fractionates with the wall during membrane lipid extraction, and is often not considered in lipidome measurements.

11The only essential phospholipid in B. subtilis is PhG. Remarkably, the membrane can be simplified to contain close to 100% PhG with no glucolipids. Despite a greatly simplified membrane, such mutants can grow rapidly, albeit with a highly abnormal coiled filament morphology. Genetic perturbations of membrane composition can lead to resistance to cationic antimicrobial peptides (CAMP). For example, gain-of-function mutations in mprF, encoding the LPG synthase/flippase, can confer daptomycin resistance possibly by reducing surface charge. Consistently, mprF null mutants have increased daptomycin sensitivity and overexpression decreases sensitivity in B. subtilis. Daptomycin resistance also results from pgsA mutations that decrease PhG levels.

12In addition to the dominant phospholipids and glucolipids, membranes contain numerous other lipid species. Most prominent are the isoprenoid lipids synthesized by polymerization of C5 isoprene units (Figure 1C). The key intermediate farnesyl-PP (C15) can be joined (head-to-head) to generate squalene (C30), a precursor of cholesterol and other sterols in eukaryotes and of structurally related hopanoid lipids in many bacteria. One major hopanoid is diploterol (Figure 1C), with five fused rings that can be further modified in a variety of ways. Farnesyl-PP can also be extended by UppS, which sequentially adds eight isopentenyl units to generate undecaprenyl-PP, the C55 carrier lipid that supports cell wall synthesis (Figure 1C). Alternatively, the HepST complex can extend farnesyl-PP to generate heptaprenyl (C35)-PP, an isoprenoid used as a lipid anchor for menaquinone (MK-7), the electron carrier for respiration. In B. subtilis, this same precursor can be processed to polycyclic C35-sesquarterpenoids, which may be functionally similar to C30 hopanoids. This process is initiated by YtpB, which generates tetraprenyl-β-curcumene, and then SqhC (a homolog of squalene-hopene cyclases) to generate the C35 tetracyclic product known as baciterpenol A. Although initially described in spores, and named “sporulenes”, these sesquarterpenoids are found in vegetative cells. Finally, heptaprenyl-PP can be coupled to glycerol-1-phosphate by PcrB, and then further processed by an unidentified phosphatase and the YvoF acetyltranferase to generate an ether linked lipid of unknown function.

13In eukaryotes, cholesterol is associated with the generation of functional membrane microdomains (FMM), also called lipid rafts. These regions have relatively low membrane fluidity (a liquid-ordered, or Lo phase) and are associated with flotillins. B. subtilis also encodes flotillin homologs, regulated by σW. These proteins, subsequently renamed FloA and FloT, are implicated in FMM formation (Figure 1A). The notion of FMMs in bacteria received strong impetus from the finding that yisP mutants, lacking a putative squalene synthase, was defective in biofilm formation. Together with the finding of a punctate localization for FloT, and chemical inhibition studies with compounds that affect sterol synthesis, this led to the proposal that bacteria harbor FMMs. However, subsequent work revealed that YisP is a farnesyl-PP phosphatase that generates farnesol (Figure 1C), rather than squalene. Moreover, farnesol itself complements the biofilm defect of the yisP mutant, suggesting that this long chain alcohol may have an ordering effect on FMMs provided in other systems by hopanoids or carotenoids. B. subtilis FMMs are enriched in flotillins (FloA and FloT) and their associated signaling complexes, with FMM formation apparently stabilized by farnesol (YisP product). No role for the C35 isoprenoid lipids has yet been demonstrated in biofilm formation or in FMM formation or function.

14Lateral heterogeneity, including FMMs, is likely a feature of most bacterial membranes. However, the lipid species that are required to form FMMs are still poorly understood, but likely include carotenoids, hopanoids, and other polycyclic isoprenoid lipids. Hopanoids are structurally diverse and fulfill a broad range of functions in bacterial membranes. The hopanoid diplopterol (Figure 1C) orders saturated lipids and glycolipids in the outer membrane of Methylobacterium extorquens, and deficient mutants are impaired in multidrug transport. Hopanoids and other polycyclic isoprenoids are present in many Gram-positive bacteria as well, suggestive of a role in the plasma membrane. In methicillin-resistant S. aureus, the carotenoid staphyloxanthin (Figure 1C) colocalizes in FMMs with FloA, and disruption of these domains with isoprenoid synthesis inhibitors interferes with the function of the penicillin-binding protein required for β-lactam resistance (PBP2a). The formation and function of FMMs, in both the inner (plasma) and outer membrane, remains an important area for future research.

15Bacteria generally have a negatively charged membrane, which contributes to their susceptibility to CAMPs, bacteriocins, and antimicrobials. In B. subtilis, membrane composition and properties are regulated by ECF σ factors. Because of their overlapping activation and promoter recognition properties, these CESRs are intertwined and referred to as an σECF stress response. In B. subtilis, activation of σX reduces the net negative charge of the membrane by increasing zwitterionic PE levels. The net negative charge of the cell wall can be further reduced by D-alanylation of teichoic acids, activated by σX and σV, a lysozyme-responsive CESR. In S. aureus, surface membrane charge is modified by the induction of mprF by the GraRS TCS, thereby increasing LPG levels. In B. anthracis, the membrane-active compound targocil activates the EdsRS TCS, which induces expression of a cardiolipin synthase. Thus, many different stimuli can trigger changes in the membrane lipidome.

16Bacillus subtilis σECF factors also control other membrane-related functions, although the effects are not yet understood. For example, σM activates the ytpAB operon. The YtpA lysophospholipase cleaves FAs from the 2 position of phospholipids resulting in a lysophospholipid (bacilysocin) suggested to function as an antibiotic. However, it is unclear if bacilysocin is ever released at levels sufficient to serve as an antibiotic, and it may instead modify membrane properties or be an intermediate in lipid remodeling. As noted above, YtpB initiates synthesis of baciterpenol (Figure 1C;;; ). Genetic studies have revealed only modest phenotypes for ytpAB mutants, including effects on antibiotic sensitivity, sporulation, and germination. In the case of ytpB, the observed phenotype (bacitracin sensitivity) was due to the accumulation of the substrate (heptaprenyl-PP) rather than a loss of baciterpenol.

17Genetic perturbations of membrane composition can also trigger CESRs. For example, deletion of LTA synthases induces σECF factors. An ltaS mutation upregulates σM, which then activates expression of the alternate LTA synthase LtaSa. The absence of both ltaS and ltaSa leads to activation of additional σECF factors. The depletion of PhG, a building block of LTA, also activates σM and to a lesser extent σV. The effects of mutations that affect glucolipids have been particularly challenging to understand. Glucolipids produced by UgtP are important membrane lipids and also serve as the lipid anchor of LTA. ugtP mutants lacking glucolipids are shorter and rounder, have abnormal localization of MreB, and altered assembly of FtsZ. Whether this abnormal morphology is due, in part, to the loss of glucolipids is unclear. Mutation of ugtP activates a σECF stress response and can be suppressed by production of monoglycosyldiacylglycerol (MGlcDG) using a heterologous synthase. Since this product does not function as an LTA anchor lipid, this suggests that it is the loss of glucolipids that induces the σECF response. The mechanistic basis for activation of σECF factors in the absence of glucolipids is unclear, but at least for σV does not require intramembrane proteolysis of the anti-σ factor. One hypothesis is that glucolipids might regulate folding and function of intramembrane proteins.

18In addition to modulating lipid composition, CESRs also induce proteins that function in membrane protection and remodeling. In B. subtilis, these proteins include two flotillin homologs (FloA, FloT), two members of the phage shock protein family (LiaH, PspA), as well as antibiotic specific detoxification modules. The roles of these proteins in stabilizing and repairing the membrane are increasingly appreciated, although the precise mechanisms remain controversial.

19Flotillins are members of the widely conserved stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins. Flotillins localize to FMMs and are thought to be required for FMM function. In S. aureus, FloA colocalizes with staphyloxanthin in FMMs. In other systems, flotillins and FMMs are associated with flagellar function and chemotaxis, type VII secretion, signaling, and interaction with the host during infection. Ongoing efforts strive to track the mobility, oligomerization state, and interaction partners of flotillins in living cells.

20Bacillus subtilis FloA and FloT are oligomeric, integral membrane proteins implicated in the formation of FMMs, and regulated by σW. FloA and FloT are thought to help partition the membrane into low fluidity FMM regions that are spatially distinct from more fluid regions. A direct role for flotillins in FMM formation has been challenged, however, since B. subtilis FloA and FloT do not always colocalize, and form separated foci of ∼100 nm in diameter that appear spatially distinct from FMMs. Counter-intuitively, flotillins appear to be required for regions of increased fluidity (RIFs), which are the counterpart to the FMMs. A lack of flotillins leads to a decrease in membrane fluidity and a concomitant reduction in activity of the MreB-directed elongasome complex that synthesizes peptidoglycan. This loss of membrane fluidity can be chemically complemented with fluidizing agents such as benzoyl alcohol.

21Flotillins also functionally interact with DynA, a constitutively expressed dynamin homolog. Dynamins are membrane-associated GTPases implicated in membrane remodeling, fusion and fission, and lipid mixing. DynA may help repair damaged membrane regions, and contribute to resistance against antibiotics that bind membrane components, including nisin, bacitracin, and daptomycin. Our understanding of flotillins and dynamins, and their roles in bacterial physiology is still incomplete and rapidly evolving.

22Cell envelope stress responses also support membrane stability through induction of PspA proteins, including two paralogs in B. subtilis: PspA an LiaH. Originally defined as part of the phage-shock protein response in Escherichia coli, PspA proteins comprise a conserved family including the vesicle-inducing protein in plastids (VIPP1/IM30) and mammalian ESCRT III. PspA proteins have a conserved N-terminal amphipathic helix required for membrane binding, which seems to depend on anionic lipid content and regions with unfavorable packing geometries creating stored curvature elastic stress. Structural studies reveal that VIPP1 forms oligomeric rings of various symmetries that stack together to form domes. These rings are dynamic, and are thought to stabilize membranes during budding, tubulation, and fusion. However, the role of these oligomeric structures has been questioned. An alternative model suggests that these rings dissociate, and the resultant intrinsically disordered monomers interact with the membrane surface to form a protective protein “carpet” to stabilize the membrane and suppress proton leakage.

23Although PspA proteins are assumed to function in membrane protection and repair, their regulation differs markedly. B. subtilis PspA is regulated by σW, whereas the paralog LiaH is regulated by the LiaRS TCS. Both paralogs localize to the membrane in response to stress and protect against membrane-damaging antibiotics. In the case of LiaH, membrane association is mediated by interaction with the integral membrane protein LiaI. LiaH may also protect the membrane against proton leakage during the export of proteins through the twin-arginine translocation (TAT) system. While the B. subtilis LiaRS regulon is rather limited in scope, LiaRS orthologs (e.g., S. aureus VraRS) play an important role in stress resistance in many Gram-positive pathogens, and mutations in these regulators are associated with clinical antibiotic resistance. In Mycobacterium tuberculosis, the PspA ortholog is also under control of the ECF σ factor σE, whereas E. coli pspA requires the σ54 RNAP and PspF activator. A common theme in these systems is that PspA-like proteins are often regulated by a specific CESR; they can accumulate to high levels in stressed cells, and they seem to protect the membrane against disruptions that can dissipate the proton gradient.

24Bacillus subtilis, like many soil bacteria, can synthesize a wide range of antimicrobial compounds and also encodes diverse resistance mechanisms. Many antimicrobial peptides induce the B. subtilis LiaRS stress response that protects cells through induction of LiaH. Induction of σW also leads to expression of the SppA membrane-localized protease and its regulatory protein SppI, which function to clear the membrane of embedded peptides to protect against lantibiotics. Other antimicrobial peptides induce specific detoxification machinery, often including ABC transporters that either export the peptide antibiotic or disassemble membrane-bound peptide complexes.

25A prototype for such systems is the BceRS TCS, which regulates the bacitracin-specific induction of the BceAB ABC transporter. Bacitracin is a peptide antibiotic made by Bacillus spp. that inhibits cell wall synthesis by binding to undecaprenyl-PP. The BceAB system appears to act in disassembly of bacitracin complexes to confer resistance. In addition, BceAB interacts with the BceRS TCS to allow sensing of bacitracin. The BceRS-AB system provides a first line of defense against bacitracin, with higher levels of antibiotic activating the protective responses mediated by the LiaRS and σECF regulons. The detailed study of the B. subtilis bacitracin stress response has provided lessons relevant to the understanding of other antibiotic detoxification modules. Similar genetic modules, encoding both TCS and ABC transporter/sensors have been described for several other antimicrobial peptides. Since induction can be quite specific, these systems provide a basis for antibiotic-inducible gene expression systems.

26Bacillus subtilis also encodes and responds to many other secondary metabolites that can induce membrane stress. For example, the toxic peptide YydF∗ is encoded by the yydFGHIJ operon, together with a radical-SAM epimerase (YydG), protease (YydH), and ABC transporter (YydIJ). Transposon insertions in the presumptive efflux pump lead to the upregulation of the LiaRS stress system. Subsequent studies revealed that YydF is post-translationally processed to convert two L-amino acids to D-amino acids. The resulting epipeptide, YydF∗, induces LiaRS-regulated LiaH and the FloT flotillin. The modified YydF∗ peptide kills B. subtilis cells by dissipating the membrane potential via membrane permeabilization. The associated concomitant decrease in membrane fluidity together with increased membrane permeabilization induces liaIH. YydF∗ peptides are likely synthesized by a variety of Gram-positive organisms including Enterococcus, Staphylococcus, and Streptococcus spp. as well as members of the human microbiome.

27Bacillus subtilis also has CESRs induced by polyketide and polyene-type antimicrobials. For example, Streptomyces spp. produce linear polyketides (linearmycins) that depolarize the membrane. Linearmycins strongly activate the LnrJK TCS that regulates an ABC transporter, LnrLMN. This ABC transporter also provides resistance against other polyenes, including the anti-fungal amphotericin.

28Here we provide a brief overview of the diverse ways in which CESRs help modify and protect the membrane in response to environmental threats (Table 1). This is a rapidly evolving field, and the impact of membrane composition on cell physiology is still mysterious. We have much to learn about the synthesis and roles of minor lipids (sesquarterpenes, ether lipids, lysophospholipids). There is a growing need to reconcile current models of lipid rafts, and the role that isoprenoid lipids and flotillins play in their formation. The activities of the VIPP1/IM30/PspA family of proteins in membrane repair and protection, and in particular the specific role of different oligomeric states, are still debated. Finally, the mechanisms by which diverse CESRs sense membrane perturbations are largely unknown, although considerable progress has been made in the specific cases of the DesK sensor kinase, flux-sensing by peptide detoxification modules, and the lysozyme-mediated induction of the σV protein. The overall picture is of the cell membrane as a complex and adaptable assemblage of many different lipid and protein species that still has many secrets to reveal.

29Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

30The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.