The Bacterial Cell Envelope...Gram-negative bacterial cell envelope we will use Escherichia coli, an...

The Bacterial Cell Envelope

Thomas J. Silhavy1, Daniel Kahne2, and Suzanne Walker2

1Department of Molecular Biology, Princeton University, Princeton, New Jersey 085442Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138; andDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston,Massachusetts 02115

Correspondence: [email protected]

The bacteria cell envelope is a complex multilayered structure that serves to protect theseorganisms from their unpredictable and often hostile environment. The cell envelopes ofmost bacteria fall into one of two major groups. Gram-negative bacteria are surrounded bya thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containinglipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded bylayers of peptidoglycan many times thicker than is found in the Gram-negatives. Threadingthrough these layers of peptidoglycan are long anionic polymers, called teichoic acids.The composition and organization of these envelope layers and recent insights into themechanisms of cell envelope assembly are discussed.

It has been well known since the late 1830sthat all living organisms are composed of

fundamental units called cells. The cell is a finiteentity with a definite boundary, the plasmamembrane. That means that the essence of theliving state must be contained within the bio-logical membrane; it is a defining feature of allliving things. Everything that exists outside ofthe biological membrane is nonliving. Chemiststend to think of membranes as self assemblingbut biological membranes do not self assemble;they require energy to be established and main-tained. In almost all cells this structure is a phos-pholipid bilayer that surrounds and containsthe cytoplasm. In addition to lipid componentsbiological membranes are composed of proteins;the proteins are what make each membrane

unique. Despite its obvious importance, mem-branes and their associated functions remainedpoorly understood until the 1950s. Before this,many viewed the membrane as a semipermeablebag. This view persisted for a number of years,particularly in the case of bacteria, because itcould not be envisioned how such a “simple”organism could have anything but a simplemembrane (Rothfield 1971).

The bacterial cell envelope, i.e., the mem-brane(s) and other structures that surround andprotect the cytoplasm, however, is anything buta simple membrane. Unlike cells of higher organ-isms, the bacterium is faced with an unpredict-able, dilute and often hostile environment. Tosurvive, bacteria have evolved a sophisticatedand complex cell envelope that protects them,

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but allows selective passage of nutrients fromthe outside and waste products from the inside.The following discussion concerns the organi-zation, composition, and the functions of thevarious layers and compartments that make upthis remarkable cellular structure. It is easilyappreciated that a living system cannot dowhat it does without the ability to establish sep-arate compartments in which components aresegregated. Specialized functions occur withindifferent compartments because the types ofmolecules within the compartment can berestricted. However, membranes do not simplyserve to segregate different types of molecules.They also function as surfaces on which reac-tions can occur. Recent advances in microscopy,which are discussed in other articles on thissubject, have revealed strikingly nonrandomlocalization of envelope components. Here, wewill highlight recent advances in our under-standing of how these extracellular organellesare assembled.

More than 100 years ago Christian Gram(1884) developed a staining procedure thatallowed him to classify nearly all bacteria intotwo large groups, and this eponymous stain isstill in widespread use. One group of bacteriaretain Christian’s stain, Gram-positive, and theother do not, Gram-negative. The basis forthe Gram stain lies in fundamental structuraldifferences in the cell envelope of these twogroups of bacteria. For our discussion of theGram-negative bacterial cell envelope we willuse Escherichia coli, an extensively-studiedorganism that has served as a model for under-standing a number of fundamental biologicalprocesses. In comparing Gram-negative andGram-positive cell envelopes we will use Staph-ylococcus aureus as a reference point but willhighlight specific differences between it andBacillus subtilis. B. subtilis is the major Gram-positive model organism and a substantialknowledge base exists for it, but the cell envelopeof S. aureus has been studied more extensivelybecause of interest in how surface features medi-ate interactions with the environment in thecourse of infection. Care should be taken in gen-eralizing from examples drawn from particularmicroorganisms. For example, E. coli inhabits

the mammalian gut. Accordingly, E. coli andother enteric bacteria must have a cell envelopethat is particularly effective at excluding deter-gents such as bile salts. This need not be a press-ing issue for other Gram-negative bacteria, andtheir envelopes may differ in species- and envi-ronmentally specific ways. Nonetheless, the abil-ity to use the Gram stain to categorize bacteriasuggests that the basic organizational principleswe present are conserved. In addition, manybacteria express an outermost coat, the S-layer,which is composed of a single protein that totallyencases the organism. S-layers and capsules,which are coats composed of polysaccharides,are beyond the scope of this review.

THE GRAM-NEGATIVE CELL ENVELOPE

After more than a decade of controversy, tech-niques of electron microscopy were improved tothe point in which they finally revealed a clearlylayered structure of the Gram-negative cell enve-lope (Fig. 1) (Glauert and Thornley, 1969).There are three principal layers in the envelope;the outer membrane (OM), the peptidoglycancell wall, and the cytoplasmic or inner mem-brane (IM). The two concentric membranelayers delimit an aqueous cellular compartmentthat Peter Mitchell (1961) first termed the peri-plasm. During a similar time frame biochemicalmethods were developed to isolate and charac-terize the distinct set of proteins found in theperiplasm (Heppel, 1967), and to characterizethe composition of both the inner and outermembranes (Miura and Mizushima, 1968;Osborn et al. 1972). Studies since then haveonly reinforced their basic conclusions.

The Outer Membrane

Starting from the outside and proceedinginward the first layer encountered is the OM.The OM is a distinguishing feature of Gram-negative bacteria; Gram-positive bacteria lackthis organelle. Like other biological mem-branes, the OM is a lipid bilayer, but impor-tantly, it is not a phospholipid bilayer. The OMdoes contain phospholipids; they are confinedto the inner leaflet of this membrane. The outer

T.J. Silhavy, D. Kahne, and S. Walker

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leaflet of the OM is composed of glycolipids,principally lipopolysaccharide (LPS) (Kamioand Nikaido 1976). LPS is an infamous mole-cule because it is responsible for the endotoxicshock associated with the septicemia caused byGram-negative organisms (Raetz and Whitfield2002). The human innate immune system issensitized to this molecule because it is a sureindicator of infection.

With few exceptions, the proteins of the OMcan be divided into two classes, lipoproteinsand b-barrel proteins. Lipoproteins containlipid moieties that are attached to an amino-terminal cysteine residue (Sankaran and Wu1994). It is generally thought that these lipidmoieties embed lipoproteins in the inner leafletof the OM. In other words, these proteins arenot thought to be transmembrane proteins.There are about 100 OM lipoproteins in E.coli,and the functions of most of these are notknown (Miyadai et al. 2004; but see below).Nearly all of the integral, transmembrane pro-teins of the outer membrane assume a b-barrelconformation. These proteins are b sheets that

are wrapped into cylinders, and we will refer tothese outer membrane proteins as OMPs. Notsurprisingly, some of these OMPs, such as theporins, OmpF, and OmpC, function to allowthe passive diffusion of small molecules suchas mono- and disaccharides and amino acidsacross the OM. These porins have 16 transmem-brane b strands, they exist as trimers (Cowanet al. 1992), and they are very abundant; togetherthey are present at approximately 250,000copies per cell. Other OMPs, such as LamB(18 transmembrane b strands) (Schirmer et al.1995) or PhoE (16 transmembrane b strainds)(Cowan et al. 1992), exist as trimers as welland they function in the diffusion of specificsmall molecules, maltose or maltodextrins andanions such as phosphate respectively, acrossthe OM. When induced by the presence of mal-tose or phosphate starvation, respectively, theseproteins are very abundant as well. OmpA isanother abundant OMP. It is monomeric, andit is unusual in that it can exist in two differentconformations (Arora et al. 2000). A minorform of the protein, with an unknown number

P-Ring Rod

L-ring

AcrA

OM

PE

IM

CYT

AcrB

100 Å

70 Å

50 Å

Hook

Filament

Rotor

60 Å

120 Å

60 Å

S/M-Ring

TolC

Figure 1. Transenvelope machines in the Gram-negative cell envelope. The AcrA/B proteins together with TolCform an efflux pump that expels harmful molecules such as antibiotics from the cell directly into the media(Koronakis et al. 2000; Eswaran et al. 2004; Murakami et al. 2006). The flagellar basal body hook structureconnects the motor to the flagella (DePamphilis and Adler, 1971). Distances shown provide a reasonableestimate of the size of the cellular compartments shown. PE, periplasm; CYT, cytoplasm.


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of transmembrane strands, can function as aporin, but the major, nonporin form has onlyeight transmembrane strands, and the periplas-mic domain of this form performs a largelystructural role (see later discussion). An addi-tional class of OMPs, which are larger b-barrels(20–24 transmembrane b strands), but arepresent at much lower levels, function as gatedchannels in the high affinity transport of largeligands such as Fe-chelates or vitamins such asvitamin B-12 (for review see Nikaido 2003).

The OM is essential for the survival ofE. coli, but it contains only a few enzymes.For example, there is a phospholipase (PldA)(Snijder et al. 1999), a protease (OmpT)(Vandeputte-Rutten et al. 2001), and an enzymethat modifies LPS (PagP) (Hwang et al. 2002).The active site of all of these enzymes is locatedin the outer leaflet, or it faces the exterior ofthe cell (OmpT). Mutants lacking any of theseenzymes exhibit no striking phenotypes. Theonly known function of the OM is to serve as aprotective barrier, and it is not immediatelyobvious why this organelle is essential. Butwhat a barrier it is. Salmonella, another entericbacterium, can live at the site of bile salt produc-tion in the gall bladder (Sinnott and Teall, 1987),and it is generally true that Gram-negativebacteria are more resistant to antibiotics thanare their Gram-positive cousins. Indeed, someGram-negative bacteria, such as Pseudomonas,are notorious in this regard.

LPS plays a critical role in the barrier func-tion of the OM. It is a glucosamine disaccharidewith six or seven acyl chains, a polysaccharidecore, and an extended polysaccharide chain thatis called the O-antigen (Raetz and Whitfield2002). Traditionally pathogenic E. coli are clas-sified by the antigenic properties of their O-antigen and the major protein (flagellin, termedH) component of the flagella (see later discus-sion). Hence, E. coli O157:H7. LPS moleculesbind each other avidly, especially if cations likeMgþþ are present to neutralize the negativecharge of phosphate groups present on the mol-ecule. The acyl chains are largely saturated, andthis facilitates tight packing. The nonfluid con-tinuum formed by the LPS molecules is a veryeffective barrier for hydrophobic molecules.

This coupled with the fact that the porins limitdiffusion of hydrophilic molecules larger thanabout 700 Daltons, make the OM a very effectiveyet, selective permeability barrier (Nikaido2003).

The Peptidoglycan Cell Wall

Bacteria do not lyse when put into distilled waterbecause they have a rigid exoskeleton. Peptido-glycan is made up of repeating units of thedisaccharide N-acetyl glucosamine-N-actylmuramic acid, which are cross-linked by penta-peptide side chains (Vollmer et al. 2008). Thepeptidoglycan sacculus is one very large poly-mer that can be isolated and viewed in a lightmicroscope. Because of its rigidity, it deter-mines cell shape. The enterics are rod shaped,but cell shapes canvary. Forexample, vibrios andcaulobacters are comma shaped. Recent resultssuggest that the glycan chains run perpendicu-lar to the long axis of a rod shaped cell, i.e.,hoops of glycan chains around the girth of thecell (Gan et al. 2008). Agents such as enzymesor antibiotics that damage the peptidoglycancause cell lysis owing to the turgor pressure ofthe cytoplasm. Lysis can be prevented in mediaof high osmolarity. However, without the pepti-doglycan, cells lose their characteristic shape.The resulting cells are called spheroplasts. WithE. coli, normal methods of spheroplast produc-tion produce nonviable cells, but they can con-tinue metabolism and biosynthesis for hours.However, special methods can be used to pro-duce L forms, which are spherical in shapeand can be propagated on high osmolaritymedia (Joseleau-Petit et al. 2007).

The OM is basically stapled to the underly-ing peptidoglycan by a lipoprotein called Lpp,murein lipoprotein, or Braun’s lipoprotein(Braun, 1975). The lipids attached to the aminoterminus of this small protein (58 amino acids)embed it in the OM. Lpp is the most abundantprotein in E. coli, more than 500,000 moleculesper cell. The e-amino group of the carboxy-terminal lysine residue of one third of thesemolecules is covalently attached to the dia-minopimelate residue in the peptide cross-bridge. In addition, proteins such as OmpA





bind peptidoglycan noncovalently. Nonetheless,mutants that lack Lpp shed OM vesicles (Yemand Wu 1978).

The Periplasm

The OM and IM delimit an aqueous cellularcompartment called the periplasm. The peri-plasm is densely packed with proteins and it ismore viscous than the cytoplasm (Mullineax et al.2006). Cellular compartmentalization allowsGram-negative bacteria to sequester potentiallyharmful degradative enzymes such as RNAseor alkaline phosphatase. Because of this, theperiplasm has been called an evolutionary pre-cursor of the lysosomes of eukaryotic cells (DeDuve and Wattiaux, 1966). Other proteins thatinhabit this compartment include the periplas-mic binding proteins, which function in sugarand amino acid transport and chemotaxis,and chaperone-like molecules that function inenvelope biogenesis (Ehrmann, 2007; see laterdiscussion).

The Inner Membrane

One of the hallmarks of eukaryotic cells is thepresence of intracellular organelles. These organ-elles are defined by limiting membranes, andthese organelles perform a number of essentialcellular processes. The mitochondria produceenergy, the smooth endoplasmic reticulum(ER) synthesize lipids, protein secretion occursin the rough ER, and the cytoplasmic mem-brane contains the receptors that sense theenvironment and the transport systems fornutrients and waste products. Bacteria lackintracellular organelles, and consequently, allof the membrane-associated functions of all ofthe eukaryotic organelles are performed in theIM. Many of the membrane proteins that func-tion in energy production, lipid biosynthesis,protein secretion, and transport are conservedin bacteria, but their cellular location is differ-ent. In bacteria, these proteins are located inthe IM.

The IM is a phospholipid bilayer. In E. colithe principal phospholipids are phosphatidylethanolamine and phosphatidyl glycerol, but

there are lesser amounts of phosphatidyl serineand cardiolipin. Other minor lipids includepolyisoprenoid carriers (C55), which functionin the translocation of activated sugar inter-mediates that are required for envelope biogen-esis (Raetz and Dowhan 1990).

Transenvelope Machines

Certain types of surface appendages such asflagella (DePamphilis and Adler 1971; Macnab2003), which are required for bacteria motility;Type III secretion systems (Kubori et al. 1998),which inject toxins into the cytoplasm of eu-karyotic host cells during the infection process;and efflux pumps (Koronakis et al. 2000;Eswaran et al. 2004; Murakami et al. 2006; Sym-mons et al. 2009), which pump toxic moleculessuch as antibiotics from the cell clear across thecell envelope into the surrounding media andare responsible in part for much of the antibi-otic resistance in pathogenic bacteria, are molec-ular machines that are made up of individualprotein components that span the peptido-glycan and are located in all cellular com-partments. The structures of some of thesemachines are known at sufficient resolution toprovide meaningful insight into the size of thevarious cellular compartments in E. coli. Asshown in Figure 1, these size predictions are inreasonable agreement. However, it should benoted that experimental measurements of thevolume of the periplasm, for example, varywidely (Stock et al. 1977).

Envelope Biogenesis

All of the components of the Gram-negative cellenvelope are synthesized either in the cytoplasmor at the inner surface of the IM. Accordingly, allof these components must be translocated fromthe cytoplasm or flipped across the IM. Peri-plasmic components must be released fromthe IM, peptidoglycan components must bereleased and polymerized, and OM compo-nents must be transported across the aqueous,viscous periplasm and assembled into an asym-metric lipid bilayer. All of this constructiontakes place outside of the cell in a potentially





hostile environment that lacks an obvious energysource. It seems clear that there is no ATPout there for example. In this section we willsummarize what is currently known about theassembly of the major envelope components;proteins, including lipoproteins, LPS, andphospholipids.

All proteins, of course, are synthesized in thecytoplasm. Proteins destined for the periplasmor the OM are made initially in precursorform with a signal sequence at the amino termi-nus. The signal sequence targets them for trans-location from the cytoplasm (Driessen andNouwen 2008). This translocation reaction iscatalyzed by an essential, heterotrimeric IMprotein complex called SecYEG (Van den Berget al. 2004). The signal sequence and this heter-otrimeric membrane protein complex are con-served throughout biology (Rapoport 2007).The essential ATPase SecA, together with theproton motive force, drives this translocationreaction (Zimmer et al. 2008). Periplasmic andOM proteins are generally translocated in post-translational fashion, i.e., synthesis and trans-location are not coupled. Proteins must besecreted in linear fashion from the amino tothe carboxy terminus like spaghetti through ahole; SecYEG cannot handle folded molecules.The cytoplasmic SecB chaperone maintainsthese secreted proteins in unfolded form untilthey can be secreted (Randall and Hardy 2002).During the secretion process the signal sequenceis proteolytically removed by Signal Peptidase I(Paetzel et al. 2002). Other components of theSec translocon, such SecD, SecF, and YajC, per-form important but nonessential function(s)during translocation, perhaps facilitating releaseof secreted proteins into the periplasm. Oncereleased, periplasmic proteins are home, but itseems likely that chaperones function to preventmisfolding and aggregation. For example, theperiplasmic protein MalS, which contains disul-fide bonds, requires the periplasmic disulfideoxidase DsbA for proper folding. In the ab-sence of the DsbA the periplasmic protease/chaperone DegP (HtrA) can substitute (Spiesset al. 1999).

Periplasmic chaperones function to protectOMPs during their transit through the periplasm.

Three such proteins have been well character-ized and shown have general chaperone activity:SurA, which also functions as a peptidyl-prolineisomerase (Behrens and Gross 2001; Bitto andMcKay 2003), Skp (Chen and Henning 1996;Walton et al. 2009), and the aforementionedDegP (Krojer et al. 2008; Shen et al. 2009).Genetic analysis indicates that these three pro-teins function in parallel pathways for OMPassembly; SurA functions in one pathway;DegP/Skp function in the other. Mutants lack-ing either one of these pathways are viable, butcells cannot tolerate loss of both (Rizzitelloet al. 2001). Mutants lacking SurA and Skp, orSurA and DegP are not viable and they showmassive defects in OMP assembly. By definitionthen, these chaperone pathways are redundant.However, this redundancy does not reflect equalroles in OMP assembly. The major OMPs,which account for most of the protein mass ofthe OM, show preference for the SurA pathway(Sklar et al. 2007) as does the minor OMP LptD(Vertommen et al. 2009; see later). At present noOMP that prefers the DegP/Skp pathway hasbeen identified. It may be that many minorOMPs show no pathway preference. The pri-mary role of the DegP/Skp pathway may be torescue OMPs that have fallen off the normalassembly pathway, particularly under stressfulconditions. It is also possible that other peri-plasmic proteins have chaperone function thatis important for the assembly of a subset ofOMPs.

The periplasmic chaperones deliver OMPsto a recently identified assembly site in the OMtermed the Bam complex (Fig. 2). This complexis composed of a large b-barrel protein, BamA(aka YaeT or Omp85), and four lipoproteins,BamBCDE (aka YfgL, NlpB, YfiO, and SmpArespectively) (Wu et al. 2005; Sklar et al. 2007).In addition to the b-barrel domain BamA has alarge amino-terminal periplasmic domain com-posed of five POTRA (polypeptide transportassociated). The structure of a large fraction ofthe BamA periplasmic domain has been deter-mined (Kim et al. 2007). Each of the four visiblePOTRA domains has a nearly identical fold,despite the fact that the amino acid sequenceidentity between them is very low. In E. coli





the first two POTRA domains are not essentialfor the life of the organism. Nevertheless,BamA is highly conserved in Gram-negativebacteria, and in these organisms there are alwaysfive POTRA domains. There are homologs ofBamA in both mitochondria and chloroplasts(Moslavac et al. 2005), which are thought tobe derived from Gram-negative bacteria. Thesehomologs have one, two, or three POTRAdomains, and the proteins function to assembleb-barrel proteins in the OM of these organelles.BamD is the only essential lipoprotein in theBam complex (Malinverni et al. 2006), and itis highly conserved in Gram-negative bacteriaas well. The remaining three lipoproteins arenot essential, and they are conserved to varyingdegrees. We do not yet understand the mecha-nism of b-barrel folding nor do we understandthe functions of the individual proteins in the

Bam complex, but there is evidence suggestingthat the POTRA domains of BamA may tem-plate folding by a process termed b augmenta-tion (Kim et al. 2007).

Lipoproteins are made initially with anamino-terminal signal sequence as well, andthey too are translocated by the Sec machinery.However, the signal sequence is removed by adifferent signal peptidase, signal peptidase II(Paetzel et al. 2002). Signal sequence processingof lipoproteins requires the formation of athioether diglyceride at the cysteine residue,which will become the amino terminus of themature lipoprotein. Once the signal sequenceis removed, an additional fatty acyl chain isadded to the cysteine amino group (Sankaranand Wu 1994). These lipid moieties tether thenewly formed lipoprotein to the outer leafletof the IM.

Phospholipid

Lipoprotein

Lpt pathway Bam pathway LOl pathway

OM

PE

IM

CYT

LptE

D

BamA LPS

β-Barrel E

C B

SurA

SecYEG

AATP

Precursor OM or periplasmic protein

ADP+Pi

ATP

ADP+Pi

ATP

ADP+Pi

ATP

ADP+Pi

A

C

F

B B

G C

D D

E MsbA

A

D B

Figure 2. The cellular machineries required for OM biogenesis. The Lpt pathway, together with MsbA, transportsLPS from its site of synthesis to the cell surface. b-barrel proteins and lipoproteins are made initially in thecytoplasm in precursor form with a signal sequence at the amino terminus. The signal sequence directs theseprecursors to the Sec machinery for translocation from the cytoplasm. Chaperones like SurA deliverbeta-barrel proteins to the Bam machinery for assembly in the OM. For OM lipoproteins, after the signalsequence is removed and lipids are attached to the amino-terminal cysteine residue, the Lol machinerydelivers them to the OM.





Some lipoproteins remain in the IM, andtheir biogenesis is complete after signal sequenceprocessing and lipid addition. However, most ofthe lipoproteins in E. coli are destined for theouter membrane. The Lol system, which trans-ports lipoproteins to the OM has been well char-acterized (Fig. 2) (Narita and Tokuda, 2006).There is an ABC transporter (LolCDE) in theIM that utilizes ATP hydrolysis to extract themolecule from the IM and pass it to a solubleperiplasmic carrier called LolA. LolA deliversthe molecule to the OM assembly site, whichis the lipoprotein LolB. IM lipoproteins have a“Lol avoidance” signal so that they remain inthe IM. The most common Lol avoidance signalis an aspartate residue at position two of themature lipoprotein.

There is a second protein translocation sys-tem in the IM called Tat that translocates foldedproteins (Sargent et al. 2006). E. coli uses theTat system for proteins which have prostheticgroups that must be added in the cytoplasm,and this constitutes a small fraction of thesecreted proteins. Other bacteria, such as ther-mophiles, use the Tat system extensively; pre-sumable because it is easier to fold proteins inthe cytoplasm than it is in the hostile environ-ments they live in. In terms of components,the Tat system is remarkable simple; three com-ponents. TatB and TatC function to target pro-teins for translocation by TatA, but how thissystem recognizes that the substrate is folded,and how it accomplishes the translocationreaction are not yet understood.

Proteins destined for the IM are handled bythe Sec machinery as well. However, in general,these proteins are targeted for cotranslationaltranslocation by Signal recognition particle(SRP) and the SRP receptor FtsY (Bernstein2000). Presumably, posttranslational transloca-tion of these hydrophobic substrates would beinefficient and perhaps dangerous, owing totheir great potential for aggregation. Prokary-otic SRP is much simpler than its eukaryoticcounterpart. It contains only a single protein,Ffh (fifty four homolog) and an RNA, Ffs(four point five S RNA). Both Ffh and Ffs areGTPases, as are their more complex eukaryoticcounterparts.

The transmembrane a-helices, which arecharacteristic of most biological membrane pro-teins in both prokaryotes and eukaryotes, serveas secretion signals. The first trans-membranesegment functions as a signal sequence to ini-tiate translocation of the sequences that followit. These transmembrane sequences tend to belonger and more hydrophobic than typical sig-nal sequences, and this serves as the basis forSRP recognition (Hegde and Bernstein 2006).This signal sequence is not cleaved; it remainsattached serving now as a typical transmem-brane helix. The second transmembrane helixfunctions to stop the translocation reactionand this helix exits the SecYEG translocator lat-erally where it remains in the IM (Van den Berget al. 2004; Driessen and Nouwen 2008). Thethird transmembrane helix functions again asan uncleaved signal sequence. These alternatingstart and stop translocation signals stitch IMproteins into the membrane in stepwise fashion.

Small IM proteins, especially those withsmall periplasmic domains, can be inserted intothe membrane by a second IM translocase calledYidC. YidC family members can be found inmitochondria and chloroplasts. Like their mito-chondrial homologs, YidC plays an importantrole in the assembly of energy-transducingmembrane proteins such as subunit c of ATPase.YidC may also play a role in the SecYEG-dependent insertion of larger IM proteins dur-ing the lateral transfer of the trans-membranea-helices into the lipid bilayer (Xie and Dalbey2008).

LPS, including the core polysaccharide, andthe O-antigen are both synthesized on the innerleaflet of the IM. LPS is flipped to the outerleaflet of the IM by the ABC transporter MsbA.O-antigen is synthesized on a polyisoprenoidcarrier, which then flips it to the outer leaflet.The O-antigen is ligated to the LPS core in theouter leaflet of the IM, a reaction catalyzed byWaaL (Raetz and Whitfield 2002). Note thatthe common laboratory strain E. coli K-12 doesnot make the O-antigen. Accordingly, it istermed “rough,” as opposed to the wild-type“smooth” strain. In the last several years acombination of genetics, biochemistry, and bio-informatics was employed to identify seven





essential proteins that are required to transportLPS from the outer leaflet of the OM to thecell surface (Fig. 2). These proteins have beentermed Lpt (lipopolysaccharide transport); LptA(aka YhbN) (Sperandeo et al. 2007), LptB (akaYhbG) (Sperandeo et al. 2007), LptC (akaYrbK) (Sperandeo et al. 2008), LptD (aka Impor OstA) (Braun and Silhavy 2002; Bos et al.2004), LptE (RlpB) (Wu et al. 2006), LptF (akaYjgP) (Ruiz et al. 2008), and LptG (aka YjgQ)(Ruiz et al. 2008). The large b-barrel protein,LptD and the lipoprotein LptE form a complexin the OM. LptA is made with a cleavable signalsequence and resides in the periplasm. LptFand LptG are IM proteins that likely interactwith the cytoplasmic protein LptB, a predictedATPase, to form an ABC transporter that togetherwith the bitopic IM protein LptC, extracts LPSfrom the IM and passes it to the periplasmic pro-tein LptA for delivery to the OM assembly site,LptD and LptE. An alternative model proposesthat all seven proteins together form a transenve-lope machine that transports LPS directly fromthe IM to the cell surface in analogy with effluxpumps. What is clear is that if any of the sevenproteins are removed, LPS accumulates in theouter leaflet of the IM (Sperandeo et al. 2008,Ruiz et al. 2008).

Like LPS, phospholipids are synthesized inthe inner leaflet of the IM. MsbA can flip these

molecules to the outer leaflet of the IM (Doerr-ler et al. 2004), but it is likely that other mecha-nisms to flip these molecules also exist. Howphospholipids reach the OM is not known.What is known is that phospholipids addedinto the OM reach the IM very quickly (Jonesand Osborn 1977). This is true even for lipidslike cholesterol which are not naturally foundin bacteria. This could suggest sites of IM-OMfusion, or hemi-fusion, that allow intermem-brane phospholipid trafficking by diffusion, ahypothesis made by Manfred Bayer (Bayer 1968)long ago that has remained highly controversial.

THE GRAM-POSITIVE CELL ENVELOPE

The Gram-positive cell envelope differs in sev-eral key ways from its Gram-negative counter-part (Fig. 3). First and foremost, the outermembrane is absent. The outer membrane playsa major role in protecting Gram-negative organ-isms from the environment by excluding toxicmolecules and providing an additional sta-bilizing layer around the cell. Because the outermembrane indirectly helps stabilize the innermembrane, the peptidoglycan mesh surround-ing Gram-negative cells is relatively thin.Gram-positive bacteria often live in harsh envi-ronments just as E. coli does—in fact, some livein the gut along with E. coli—but they lack a

Peptidoglycan

O-antigen

Coresaccharide

Outermembrane

Periplasm

Gram-negativeCytoplasmGram-positive

WTA

LTA

OMP

IMPIMP

LP

LPS

Lipid A

Cellmembrane

CAP

Figure 3. Depiction of Gram-positive and Gram-negative cell envelopes: CAP ¼ covalently attached protein;IMP, integral membrane protein; LP, lipoprotein; LPS, lipopolysaccharide; LTA, lipoteichoic acid; OMP, outermembrane protein; WTA, wall teichoic acid.





protective outer membrane. To withstand theturgor pressure exerted on the plasma mem-brane, Gram-positive microorganisms are sur-rounded by layers of peptidoglycan many timesthicker than is found in E. coli. Threadingthrough these layers of peptidoglycan are longanionic polymers, called teichoic acids, whichare composed largely of glycerol phosphate, glu-cosyl phosphate, or ribitol phosphate repeats.One class of these polymers, the wall teichoicacids, are covalently attached to peptidoglycan;another class, the lipoteichoic acids, are an-chored to the head groups of membrane lipids(Neuhaus 2003). Collectively, these polymerscan account for over 60% of the mass of theGram-positive cell wall, making them majorcontributors to envelope structure and func-tion. In addition to the TAs, the surfaces ofGram-positive microorganisms are decoratedwith a variety of proteins, some of which areanalogous to proteins found in the periplasmof Gram-negative organisms (Dramsi et al.2008). Because there is no outer membrane inGram-positive organisms to contain extracellu-lar proteins, all these proteins feature elementsthat retain them in or near the membrane. Somecontain membrane-spanning helices and someare attached to lipid anchors inserted in themembrane. Others are covalently attached toor associated tightly with peptidoglycan (Scottand Barnett 2006). Still others bind to teichoicacids. Studies on S. aureus have shown that thecomposition of surface-expressed proteins canchange dramatically depending on environ-mental cues or growth conditions, reflectingthe important role of the cell envelope in adapt-ing to the local environment (Pollack andNeuhaus 1994). The major structural elementsof Gram-positive cell walls, excluding capsules,will be described below.

Gram-positive Peptidoglycan

The chemical structure of peptidoglycan inGram-positive organisms is similar to thatin Gram-negatives in that it is composed of adisaccharide-peptide repeat coupled throughglycosidic bonds to form linear glycan strands,which are crosslinked into a meshlike frame-

work through the peptide stems attached tothe disaccharide repeat. The major differencebetween Gram-positive and Gram-negativepeptidoglycan involves the thickness of thelayers surrounding the plasma membrane.Whereas Gram-negative peptidoglycan is onlya few nanometers thick, representing one toa few layers, Gram-positive peptidoglycan is30–100 nm thick and contains many layers.

There are many differences among Gram-positive organisms with respect to the details ofpeptidoglycan structure, but perhaps the mostnotable difference relates to the peptide cross-links between glycan strands (Vollmer 2008;Vollmeret al. 2008). S. aureus contains crosslinksin which the peptides are connected through apentaglycine branch extending from the thirdamino acid of one of the stem peptides. Thispentaglycine branch is assembled by a set ofnonribosomal peptidyl transferases known asFemA, B, and X (Ton-That et al. 1998; Rohrerand Berger-Bachi 2003). Staphylococci can tol-erate, albeit with difficulty, the loss of FemA orB, but not of FemX, which attaches the first gly-cine unit to the stem peptide (Hegde andShrader 2001; Hubscheret al. 2007). Many Gram-positive organisms contain branched stem pep-tides, but B. subtilis does not; the stem peptidesand crosslinks in this organism are identical instructure to those found in E. coli.

Branched stem peptides in S. aureus andother Gram-positive organisms play a variety ofroles. Chief among these roles, they serve asattachment sites for covalently-associated pro-teins (discussed in more detail later). They havealso been implicated in resistance to beta lactamantibiotics (Chambers 2003). Beta lactamsinactivate transpeptidases that catalyze the pep-tide crosslinking step of peptidoglycan synthesisby reacting with the active site nucleophile oftranspeptidases. Transpeptidases that couplebranched stem peptides are mechanisticallysimilar to those that couple unbranched stempeptides; however, their substrate specificity issufficiently different that they only recognizeunbranched stem peptides, and some of themare resistant to beta lactams (Rohrer and Berger-Bachi 2003; Pratt 2008; Sauvage et al. 2008). Forexample, methicillin-resistant S. aureus strains





express a transpeptidase, PBP2A, that couplesonly pentaglycine-branched substrates. Manyother Gram-positive organisms are also thoughtto harbor low affinity PBPs that preferentiallyrecognize and couple branched stem peptides.It is thus speculated that the evolution ofbranched peptides in the peptidoglycan biosyn-thetic pathway may be an adaptation that en-ables escape from beta lactams. As describedin the following section, however, branchedstem peptides also play other important roles.

Surface Proteins

S. aureus colonizes human skin and mucosalsurfaces. Breaches in the epithelium occasion-ally result in invasive S. aureus infections thatare extremely serious. The ability to adhere tohost tissue is a crucial first step in effective col-onization by S. aureus, and a variety of surfacefactors are involved in this process. These fac-tors include teichoic acids, which are discussedin the following section, as well as surface pro-teins that recognize components of host extrac-ellular matrix such as fibronectin, fibrinogen,and elastin (Clarke and Foster 2006).

Some of these surface proteins, called adhe-sins, are attached via noncovalent ionic interac-tions to peptidoglycan or teichoic acids, butmany are attached covalently to stem peptideswithin the peptidoglycan layers (Dramsi et al.2008; Sjoquist et al. 1972; Fischetti et al. 1990).Proteins destined for covalent surface displaycontain an amino-terminal signal sequencethat enables secretion through the cytoplasmicmembrane and a carboxy-terminal pentapep-tide cell wall sorting motif, which is commonlyLPXTG (DeDent 2008). Enzymes called sorta-ses catalyze a transpeptidation reaction betweenthese sorting motifs and the glycine branch ofthe stem peptide of peptidoglycan precursors.The transpeptidation reaction is thought tooccur in two steps: in the first, a nucleophilein the sortase active site attacks the amidebond between the threonine and glycine of thesorting motif to produce a covalent intermedi-ate in which the amino-terminal portion ofthe protein substrate is attached to the enzyme;in the second, the glycine branch of the stem

peptide enters the active site and the nucleo-philic amino terminus of the glycine branchattacks the acyl- enzyme intermediate, regener-ating the enzyme and forming a new TG amidebond that anchors the protein to the peptido-glycan precursor. The protein-modified pepti-doglycan precursor is then incorporated intopeptidoglycan (Dramsi et al. 2008; Marraffiniet al. 2006).

In S. aureus, more than twenty protein sub-strates for the major sortase, sortase A, have beenidentified. In addition to adhesins, these proteinsubstrates include proteins involved in immunesystem evasion, internalization, and phage bind-ing. A minor sortase, sortase B, is responsiblefor surface display of proteins involved in ironacquisition, which is necessary for pathogenesisbecause iron is required for the function of manybacterial enzymes. In other Gram-positive organ-isms, sorting enzymes similar to SrtA and SrtBcovalently couple proteins that comprise pili.

In addition to covalent attachment, Gram-positive organisms have other ways of retainingcell surface proteins. Many proteins involved inpeptidoglycan biosynthesis are anchored to thecytoplasmic membrane by membrane span-ning helices. Some proteins required for cell-wall degradation are associated noncovalentlywith peptidoglycan; others appear to be scaf-folded and/or activated by teichoic acids orother types of cell surface polymers.

Teichoic Acids

Teichoic acids are anionic cell surface polymersfound in a wide range of Gram-positive organ-isms, including S. aureus and B. subtilis. Thereare two major types of teichoic acids: wall tei-choic acids (WTAs), which are coupled to pep-tidoglycan, and lipoteichoic acids (LTAs), whichare anchored to the cell membrane. Wall tei-choic acids are attached via a phosphodiesterlinkage to the C6 hydroxyl of occasional Mur-NAc residues in peptidoglycan. Although thestructural variations are considerable, the mostcommon WTAs are composed of a disaccharidelinkage unit to which is appended a polyribitolphosphate (polyRboP) or polyglycerol phos-phate (polyGroP) chain containing as many





as 60 repeats. WTAs extend perpendicularlythrough the peptidoglycan mesh into what hasbeen characterized as a “fluffy” layer beyond.S. aureus produces polyRboP WTAs; B. subtilisproduces either polyRboP and polyGroP de-pending on the strain. The hydroxyls on theRboP or GroP repeats are tailored with othergroups, typically D-alanyl esters or glycosyl moi-eties, and the nature and extent of the tailoringmodifications significantly affect the propertiesand functions of WTAs (see later) (Neuhaus,2003).

LTAs are similar to WTAs in that they arecomposed of polyGroP polymers that are oftenfunctionalized with D-alanine or a sugar moi-ety; however, they also differ in a number ofways. For example, they contain glycerol-phosphate repeats of opposite chirality to thosefound in WTAs. Furthermore, rather than beingattached to peptidoglycan, they are anchored tomembrane-embedded glycolipids and typicallycontain fewer GroP repeats. Thus, they extendfrom the cell surface into the peptidoglycanlayers rather than through and beyond.Together, the LTAs and WTAs comprise whathas been concisely described as a “continuumof anionic charge” that originates at the Gram-positive cell surface but extends well beyond thepeptidoglycan barrier. The importance of thiscontinuum of negative charge is underscoredby the fact that Gram-positive organisms lack-ing WTAs (either because they do not containthe gene clusters or because they are grownunder phosphate-limiting conditions) produceother types of polyanionic polymers in whichthe negative charges are supplied by carboxylateor sulfate groups. Furthermore, although nei-ther LTAs nor WTAs are essential, deleting thepathways for the biosynthesis of either of thesepolymers produces organisms that have celldivision and morphological defects as well asother, less serious growth defects. Moreover, itis not possible to delete both pathways becausethe genes involved are synthetic lethals (Okuet al. 2009; Morath et al. 2005).

Teichoic acids account for a significant frac-tion of the cell wall mass in producing organ-isms and their functions are many, varied, andspecies-dependent. Because they are anionic

they bind cations and thus play a role in cationhomeostasis (Marquis et al. 1976). Networks ofmetal cations between WTAs also influence therigidity and porosity of the cell wall. The nega-tive charge density on WTAs can be modulatedby tailoring modifications that introduce posi-tive charges along the polymer backbone, andthese modifications can have a profound effecton the interactions of bacteria with other cellsor molecules. For example, in S. aureus, a D-alanine transferase couples D-alanine moietiesto free hydroxyls on the polyribitol phosphatebackbone. S. aureus strains lacking D-alanineesters are more susceptible to antimicrobial cat-ionic peptides and to lytic enzymes produced byhost neutrophils (Collins et al. 2002; Peschelet al.1999; Peschel et al. 2000). They also displayreduced autolysin activity, suggesting a role forfunctionalized WTAs in scaffolding or activat-ing hydrolytic enzymes involved in cell wallsynthesis and degradation.

THE CELL ENVELOPE OFCORYNEBACTERINEAE

The Corynebacterineae are a group of bacteriathat includes the very important pathogensMycobacterium tuberculosis and Mycobacteriumleprae. These bacteria are generally classified ashigh GþC Gram positives, however their cellenvelope has characteristics of both Gram-positive and Gram-negative bacteria. Indeed, agenome-based phylogeny places them in betweenGram positives and Gram negatives (Fu andFu-Liu, 2002).

The cell envelope of the Corynebacterineae isvery complex and this complexity contributessubstantially to their virulence. The peptidogly-can layer that surrounds a standard IM containscovalently attached arabinogalactan and this iscovalently attached to mycolic acids (Minnikin,1982). These mycolic acids have very long alkylside chains (up to C90) that give the bacteria awaxy appearance and account for their resis-tance to acid decolorization during stainingprocedures (acid-fast).

Unlike other Gram-positive bacteria, Cory-nebacterineae have an OM. This OM appears tobe symmetrical unlike the Gram-negative OM.





Mycolic acids appear essential for this OM, buthow they are organized remains unclear (Hoff-mann et al. 2008; Zuber et al. 2008). Mycobac-teria have porin proteins in their OM, but thestructure of the main porin, MspA, from Myco-bacterium smegmatis is quite different from thetypical porin proteins of Gram-negative bacte-ria (Faller et al. 2004). Indeed, mycobacteriahave no obvious homologs of the Bam complexmembers. This might suggest a novel mecha-nism for the assembly of proteins in the OMof Corynebacterineae.

CONCLUSION

The cell envelopes of bacteria are complex,dynamic structures that play a variety of protec-tive and adaptive roles. The major conservedcomponent of all bacterial cell envelopes is pep-tidoglycan, which is essential for stabilizing cellmembranes against high internal osmotic pres-sures. But peptidoglycan alone is not enough toenable bacteria to survive in their different envi-ronments. In addition to peptidoglycan, theouter membrane of Gram-negative bacteriathe, dense array of negatively charged polymersembedded in Gram-positive peptidoglycan andthe complex outer layers of the Corynebacteri-neae play important roles in cell envelope integ-rity. One of the major challenges in the nextdecade will be to define the mechanisms bywhich these complex structures are assembledand regulated in response to changing environ-mental conditions.

ACKNOWLEDGMENTS

We thank Juliana Malinverni for help with fig-ures and Susan DiRenzo for help preparingthe manuscript. The authors were each sup-ported by grants from the National Institute ofGeneral Medical Sciences.

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14, 20102010; doi: 10.1101/cshperspect.a000414 originally published online AprilCold Spring Harb Perspect Biol

Thomas J. Silhavy, Daniel Kahne and Suzanne Walker The Bacterial Cell Envelope

Subject Collection Cell Biology of Bacteria

Electron CryotomographyElitza I. Tocheva, Zhuo Li and Grant J. Jensen

Cyanobacterial Heterocysts

James W. GoldenKrithika Kumar, Rodrigo A. Mella-Herrera and

Protein Subcellular Localization in BacteriaDavid Z. Rudner and Richard Losick Cell Division in Bacteria

Synchronization of Chromosome Dynamics and

Martin Thanbichler

Their Spatial RegulationPoles Apart: Prokaryotic Polar Organelles and

Clare L. Kirkpatrick and Patrick H. ViollierMicroscopyAutomated Quantitative Live Cell Fluorescence

Michael Fero and Kit Pogliano

MorphogenesisMyxobacteria, Polarity, and Multicellular

Dale Kaiser, Mark Robinson and Lee KroosHomologsThe Structure and Function of Bacterial Actin

Joshua W. Shaevitz and Zemer GitaiMembrane-associated DNA Transport Machines

Briana Burton and David DubnauBiofilms

Daniel López, Hera Vlamakis and Roberto KolterThe Bacterial Cell Envelope

WalkerThomas J. Silhavy, Daniel Kahne and Suzanne III Injectisome

Bacterial Nanomachines: The Flagellum and Type

Marc Erhardt, Keiichi Namba and Kelly T. HughesCell Biology of Prokaryotic Organelles

Dorothee Murat, Meghan Byrne and Arash Komeili Live Bacteria CellsSingle-Molecule and Superresolution Imaging in

Julie S. Biteen and W.E. Moerner

SegregationBacterial Chromosome Organization and

Esteban Toro and Lucy Shapiro

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