JOURNAL OF BACTERIOLOGY, Aug. 2008, p. 5672–5680 Vol. 190, No. 160021-9193/08/$08.00�0 doi:10.1128/JB.01919-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Direct Visualization of the Outer Membrane of Mycobacteria andCorynebacteria in Their Native State�†

Benoît Zuber,1‡* Mohamed Chami,2 Christine Houssin,3,4 Jacques Dubochet,1§Gareth Griffiths,5 and Mamadou Daffe6,7*

Laboratory of Ultrastructural Analysis, University of Lausanne, Biophore Building, 1015 Lausanne, Switzerland1;M. E. Muller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 CH Basel,

Switzerland2; Institut de Genetique et Microbiologie, Universite Paris-Sud, F-91405 Orsay, France3;Centre National de la Recherche Scientifique, F-91405 Orsay, France4; EMBL, Postfach 102209,

69117 Heidelberg, Germany5; Universite Paul Sabatier (Toulouse III), Institut de Pharmacologie et deBiologie Structurale (IPBS), 205 Route de Narbonne, 31077 Toulouse cedex 04, France6; and

Centre National de la Recherche Scientifique (CNRS), IPBS, Departement desMecanismes Moleculaires des Infections Mycobacteriennes, 205 Route de

Narbonne, 31077 Toulouse cedex 04, France7

Received 10 December 2007/Accepted 9 June 2008

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucialfor their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemicalcomponents of this envelope, it has proven difficult to combine its components into a comprehensive structuralmodel, primarily because the available ultrastructural data rely on conventional electron microscopy embed-ding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has longbeen postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we haveused cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysisof three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accuratelydescribes the different layers of the mycobacterial cell envelope and challenges current models of the organi-zation of its components. We show a direct visualization of an outer membrane, analogous to that found ingram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolicacids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane.In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteriaare apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model ofthe organization of the lipids in the outer membrane is proposed. The architecture we describe should serveas a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

The suborder of Corynebacterineae is a distinct group ofgram-positive bacteria and comprises mycobacteria and othergenera such as Corynebacterium, Rhodococcus, and Nocardia.The medical importance of the group is enormous; it includesthe causative agents of human diseases such as tuberculosisand leprosy, Mycobacterium tuberculosis and Mycobacteriumleprae, respectively. The structure of the cell envelope of thesebacteria has been the subject of numerous studies because it isalready clear that the powerful biological activities of known

wall components contribute significantly to the disease process.Indeed, lipids isolated from the cell envelope can elicit re-sponses by the host immune system very similar to the re-sponses generated by M. tuberculosis infection (e.g., granulomaformation) (22).

Schematically, the envelope of this bacterial group is com-posed of a typical plasma membrane (PM) surrounded by a cellwall core, which, in turn, is surrounded by an outer layer (OL)called the capsule in the case of pathogenic mycobacterialspecies (Fig. 1). The cell wall core consists of peptidoglycancovalently bound to arabinogalactan, which itself is covalentlybound to mycolic acids (very-long-chain, from C30 to C90, �-al-kyl, �-hydroxy fatty acids) (9). This envelope is unusual in thatit is very rich in lipids, and unlike other gram-positive micro-organisms, Corynebacterineae possess an outer permeabilitybarrier. It has been postulated that this barrier is formed by alipid bilayer analogous to the outer membrane (OM) of gram-negative bacteria (33). The arrangement of the lipids in thishypothetical OM has been long debated (26, 32, 34, 43, 44).Based on the chemical structures of the main cell envelopeconstituents, several models of the cell envelope, in particular,the hypothetical OM bilayer of mycobacteria, were developed(26, 32, 33, 43, 44). According to these models, the innermost

* Corresponding author. Mailing address for Benoît Zuber: MRCLaboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH,United Kingdom. Phone: 44 1223 402209. Fax: 44 1223 402310. E-mail:bzuber@mrc-lmb.cam.ac.uk. Mailing address for Mamadou Daffe:Universite Paul Sabatier (Toulouse III), Institut de Pharmacologie etde Biologie Structurale (IPBS), 205 Route de Narbonne, 31077Toulouse cedex 04, France. Phone: 33 561 175 569. Fax: 33 561 175580. E-mail: mamadou.daffe@ipbs.fr.

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ Present address: MRC Laboratory of Molecular Biology, HillsRoad, Cambridge CB2 0QH, United Kingdom.

§ Present address: DEE, Biophore Building, University of Lau-sanne, 1015 Lausanne, Switzerland.

� Published ahead of print on 20 June 2008.

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leaflet consists mainly of the mycolic acids, which are, at leastin part, covalently linked to the cell wall arabinogalactan. Theoutermost leaflet is proposed to be composed of various gly-colipids, including trehalose monomycolate and trehalose di-mycolate; of phospholipids; and of species-specific lipids suchas glycopeptidolipids (GPL), phthiocerol dimycocerosate, andsulfolipids (8, 26, 33, 44). The presence of pore-forming pro-teins in low numbers in mycobacteria relative to Escherichiacoli may explain both the limited permeability of mycobacterialcell envelopes and the generally rather low susceptibility ofthese bacteria to toxic agents (6, 9, 36).

Nevertheless, not only is the precise organization of the OMbilayer debated, but even its existence can be questioned sincethere are no direct observations of this structure. On the onehand, the bilayer model is supported by data from freeze frac-ture electron microscopy which have clearly shown the occur-rence of a major fracture plane in the outer part of the enve-lope, in addition to the expected plane that typifies the PM (5,7, 33, 35, 43).

On the other hand, no sign of an OM bilayer was seen inultrathin sections of Corynebacterineae (Fig. 1) (9, 11, 43),questioning its existence. Importantly, all electron microscopyanalyses of ultrathin sections were done with specimens fromwhich water had been removed, a prerequisite for electronmicroscopy observation at room temperature. We call thistechnique the conventional preparation method. Even whendehydration has been performed at low temperature by freeze-substitution in order to better preserve biological structures,no OM bilayer was seen in Corynebacterineae (9, 28, 39, 40).This is possibly due to the fact that, during dehydration, water-soluble molecules tend to aggregate and lipid molecules maybe prone to extraction or rearrangement by organic solvents(14).

In an attempt to resolve the question of the existence of anOM bilayer in Corynebacterineae, we addressed the nativestructure of both mycobacteria and the closely related coryne-bacteria, whose cell envelope resembles that of mycobacteriaafter conventional electron microscopy (43). We focused our

study on Mycobacterium smegmatis, Mycobacterium bovis BCG,and Corynebacterium glutamicum by cryo-electron microscopy(cryoEM) of vitreous sections (CEMOVIS). In the CEMOVIStechnique, specimens are vitrified by high-pressure freezing(i.e., cooled to liquid nitrogen temperature without water crys-tallization). The vitreous specimens are then cryosectioned andimaged in a cryo-electron microscope in their fully hydrated,native state. The artifacts of aggregation and lipid extractionare therefore prevented (1). Importantly, this is the only sec-tioning technique for electron microscopy involving freezingwhere the vitreous state can be unambiguously confirmed, byelectron diffraction. With this approach, we provide detailedinsights into the structure of the mycobacterial cell envelope inits native state and a direct visualization of the mycobacterialOM. Furthermore, we demonstrate that mycolates are essen-tial constituents of this structure through the use of a wild-typeand a mycolate-free strain of C. glutamicum (41).

MATERIALS AND METHODS

Culture conditions. M. smegmatis mc2155 (ATCC 700084) and M. smegmatistmptB (49) were cultured in LB (Luria-Bertani broth; Difco, Basel, Switzerland)at 37°C with aeration. M. bovis BCG Pasteur (ATCC 35734) was cultured in0.05% Tween 80, oleic acid-albumin-dextrose-catalase-enriched (oleic acid dex-trose complex) Middlebrook 7H9 broth (Difco) at 37°C with aeration. Mycobac-teria were harvested during the exponential growth phase (optical density at 600nm of 3 for M. smegmatis and of 0.24 for M. bovis BCG). Wild-type C. glutami-cum (ATCC 13032) was cultured on 3% agar–brain heart infusion medium(Difco) at 30°C. C. glutamicum �pks13::km (41) was cultured on agar-brain heartinfusion medium supplemented with 25 �g/liter kanamycin at 30°C. Corynebac-terial colonies were harvested after overnight growth. E. coli B/r carrying pUC19was cultured in 2 � YT in the presence of 100 �g/ml ampicillin (45). Cells wereharvested during the exponential growth phase (optical density at 600 nm of0.96). Streptococcus gordonii Challis was cultured and processed for CEMOVISas previously described (58). Harvesting was done by centrifugation at 3,200 � gfor 5 min.

Vitrification and cryosectioning. For CEMOVIS, M. smegmatis and M. bovisBCG were washed twice in phosphate-buffered saline (PBS) supplemented with20% dextran (20% dextran–PBS) (average molecular mass, 40 kDa; Sigma-Aldrich, Buchs, Switzerland). They were then introduced into copper tubes andvitrified with an EM PACT high-pressure freezer (Leica, Vienna, Austria).Corynebacterial colonies were scraped and resuspended in 20% dextran–PBS. E.coli was centrifuged and resuspended in 20% dextran–PBS. Corynebacteria andE. coli were introduced into membrane carriers (Leica) and vitrified with thesame apparatus. Afterwards, tubes were mounted in the tube holder of anFC6/UC6 cryo-ultramicrotome (Leica) and trimmed to a pyramidal shape aspreviously described (51, 58). Membrane carriers were clamped in the flat spec-imen holder of the cryo-ultramicrotome. Copper was trimmed away with adiamond knife (Diatome, Bienne, Switzerland) on part of the specimen holder,and the specimen was trimmed to a pyramidal shape with the same knife. Forty-to 50-nm feed cryosections were cut with a 35 or 45° diamond knife (Diatome)under standard cutting conditions (58). They were collected on carbon-coated1,000-mesh grids or noncoated lacey carbon grids (Agar Scientific, Essex, UnitedKingdom) and stored in liquid nitrogen or transferred immediately to the mi-croscope.

For staining experiments, M. smegmatis mc2155 was washed three times in PBSand fixed for 2 h in 2.5% (wt/vol) glutaraldehyde in cacodylate buffer at roomtemperature. Cells were washed three times in cacodylate buffer and postfixed in1% (wt/vol) OsO4 for 2 h at room temperature. They were washed three timesin cacodylate buffer and then processed for high-pressure freezing as describedabove.

For cryoEM of whole-mount wild-type C. glutamicum, colonies were scrapedand resuspended in 20 mM Tris, pH 7.5. A 4-�l sample volume was allowed toadsorb to a carbon-coated grid for 1 min, blotted with Whatman no. 4 filter paper(Merck, Zurich, Switzerland), and vitrified by plunging into liquid ethane at�178°C.

CryoEM. For CEMOVIS, grids were transferred to a cryoholder (Gatan,Warrendale, PA) kept below �170°C and inserted into CM100, Tecnai 12, andTecnai F30 cryo-electron microscopes (FEI, Eindhoven, Netherlands) equipped

FIG. 1. Thin-section transmission electron microscopy of chemi-cally fixed and dehydrated Corynebacterineae. (A) M. smegmatismc2155. (B) C. glutamicum (CGL2020). The cell envelope of M. smeg-matis is composed of a PM; a thick, electron-transparent layer (Peri ?;interpreted as the postulated periplasmic space); a thick, internal,electron-dense layer (EDL; considered a complex of peptidoglycanand arabinogalactan); a thin, electron-transparent layer (ETL; as-sumed to be composed of mycolic acids and other lipids); and anelectron-dense OL (a complex protein-carbohydrate matrix with somelipids) (15, 16). The EDL and the mycolic acids of the ETL form thecell wall core. The cell envelope of C. glutamicum looks similar, but itdoes not possess an obvious low-density hypothetical periplasmic spaceand the OL is thicker than in M. smegmatis (43). In both cases, the useof ruthenium red stain resulted in enhanced staining of the OL. Bars,20 nm.

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with a LaB6 cathode, a tungsten cathode, and a field emission gun, respectively.The accelerating voltages were 100, 120, and 200 kV, respectively. Specimenswere irradiated with a low electron dose. Electron diffraction was used to checkwhether water was vitreous or crystalline. Crystalline sections were discarded.Images were recorded with a TemCam-F224HD charge-coupled device camera(Tietz Video and Image Processing Systems, Munich, Germany) at magnifica-tions of �22,500, �33,000, and �53,000. No image processing other than thatdescribed in the figure legends was performed.

Whole-mount plunge-frozen cells were transferred to a Gatan cryoholder andimaged at a magnification of �50,000 in a CM200-FEG (FEI) operated at anaccelerating voltage of 200 kV. Images were recorded on Kodak SO-163 platesand scanned at a pixel size of 0.5 nm. For conventional electron microscopy, C.glutamicum (CGL2020) and M. smegmatis mc2155 were grown, prepared, andimaged as previously described (16, 43).

Quantitative measurements. Pixel size was calibrated by using a two-dimen-sional crystal of catalase (Agar Scientific). At magnifications of �22,500,�33,000, and �53,000, the pixel sizes are 0.63, 0.50, and 0.31 nm, respectively.During cryosectioning, material is compressed along the cutting direction. Nev-ertheless, it has been shown that compression does not affect the dimensionsmeasured perpendicularly to the cutting direction (13). Dimensions were mea-sured accordingly on average density profiles calculated along rectangular selec-tions with the software ImageJ (NIH, Bethesda, MD). The width of selectionrectangles is specified in the figure legends.

The density of OsO4-stained cells was measured with the software EMMENU(Tietz Video and Image Processing Systems) in raw image files acquired at 120kV at a defocus between �1.7 and �2.3 �m. The density of the OM wasnormalized to the density of the background.

RESULTS

Vitreous sections—general considerations. We investigatedthe structure of the cell envelopes of two species of mycobac-teria with CEMOVIS, namely, M. smegmatis, a fast-growingnonpathogenic species, and M. bovis BCG, a slow-growingvaccine strain belonging to the same complex as M. tuberculosisand showing more than 99.9% genome sequence identity withthis pathogen (19). Henceforth, M. smegmatis refers to strainmc2155 unless mentioned differently. A low-magnification mi-crograph of M. smegmatis is shown in Fig. 2. Its quality isrepresentative of the majority of the sections that we observed.It does not contain chatter or crevasses, which are cuttingartifacts that complicate image interpretation because theyproduce irregular distortions (inhomogeneities) (2). Knifemarks (arrows) and compression along the cutting direction

cannot not be prevented but are homogeneous and thereforedo not hinder image interpretation.

Structure of the cell envelopes of M. bovis BCG and M.smegmatis. The cell envelopes of M. smegmatis and M. bovisBCG are structurally similar in CEMOVIS (Fig. 3A to F; Table1). They are composed of a PM, a granular layer (GL), aninner wall zone (IWZ) of low density, a medial wall zone(MWZ) of intermediate density, and an OM of higher density.The bilayer aspect of the OM can be best visualized in micro-graphs recorded with a relatively small defocus value (Fig. 3Aand D) and in the corresponding density profiles (Fig. 3C andF), whereas the organization of the GL, the IWZ, and theMWZ is best seen in micrographs recorded with a larger de-focus value (Fig. 3B and E). The GL is found next to the PM,in the IWZ. The appearance of the mycobacterial GL and IWZis similar to that of the GL and the IWZ of typical gram-positive bacteria (e.g., S. gordonii) visualized by CEMOVIS(58) (Fig. 3G and H; Table 1). The MWZ of mycobacteria istopologically identical to the gram-positive peptidoglycan layer(outer wall zone) but is thinner. On images recorded with arelatively large defocus, the MWZ seems separated from theOM by a low-density gap (Fig. 3F). However, on images re-corded very close to focus, the gap is strongly reduced (Fig. 3C)or absent (data not shown), whereas the leaflets of the OM aredistinct. The gap is thus a phase-contrast artifact that can leadto a slight underestimation of the MWZ thickness.

There is no structure similar to the mycobacterial OM inclassical gram-positive bacteria (e.g., S. gordonii), but the my-cobacterial OM is structurally analogous to the OM of gram-negative bacteria (e.g., E. coli) (Fig. 3I and J). The thickness ofthe mycobacterial OM is similar to the published thickness ofthe gram-negative OM visualized by CEMOVIS (Table 1)(29). Even though an OM in mycobacteria has long been sug-gested and supported by freeze fracture electron microscopy,here we report a direct observation of such a bilayer in nativemycobacteria. Together, these comparisons indicate the fol-lowing three points: (i) the IWZ occupies the position shown tobe a periplasmic space in other gram-positive bacteria (30, 31);(ii) the MWZ is likely formed, at least in part, of peptidogly-can; and (iii) the OM is made of molecules specific to myco-bacteria and not ubiquitous in gram-positive bacteria. Becausethese molecules form a bilayer, they are likely to contain ahydrophobic moiety. OsO4 is considered to label predomi-nantly lipids (56). We thus fixed M. smegmatis cells with glu-taraldehyde, postfixed them with OsO4, and subsequently pro-cessed them for CEMOVIS. In a negative control, cells wereprocessed for CEMOVIS directly after glutaraldehyde fixation.The density of the OM is, on average, 39% higher in OsO4-treated cells than in control cells (P � 0.01, Fig. 4). These datasuggest that the OM is mainly made of lipids. Besides, thebilayer aspect of the OM is lost after OsO4 staining in most ofthe images, an observation that requires further investigation.Mycolic acids, which are the hallmark lipids of Corynebacteri-neae, of which a fraction is covalently bound to peptidoglycanvia arabinogalactan, are likely to be a major constituent of theOM.

In order to test our hypothesis that the OM of mycobac-teria is made at least in part of mycolic acids, we wanted toobserve mutant mycobacteria that would be devoid of my-colic acid. However, this was impossible because mycobac-

FIG. 2. CEMOVIS of a cross-sectioned M. smegmatis mc2155 cell.Arrows, knife marks. Bar, 500 nm.

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teria cannot survive in the absence of this compound (3, 18,41, 42, 53). For the same reason, treating cells with drugsthat inhibit the synthesis of mycolic acids (e.g., isoniazid)proved useless. Indeed, at the highest sublethal isoniazid

concentration, the amount of mycolic acid per cell is onlyreduced by 20% (4). Furthermore, probes such as gold-coupled antibodies could not be used because they are toolarge to diffuse through the cell envelope. And since speci-

FIG. 3. Cell envelope of mycobacteria and gram-positive and gram-negative bacteria by CEMOVIS. (A, B, and C) M. smegmatis mc2155. (D,E, and F) M. bovis BCG. (G and H) S. gordonii. (I and J) E. coli. Images were acquired at 100 kV and defocused by �2.5 �m (A, D, and I), �5�m (B), �3.7 �m (E), and �4.5 �m (G). They were denoised by Gaussian filtering in Adobe Photoshop (radius of 0.6 pixel for panels A and Dand of 1 pixel for panels B, E, G, and I). The density profiles in panels C, F, H, and J were obtained from nondenoised images corresponding topanels A, E, G, and I, respectively. They were averaged over a width of 70 pixels (C) and 50 pixels (F, H, and J). Note that the GL is in IWZ. OWZ,outer wall zone; PG, peptidoglycan layer. Bars, 20 nm (A, B, D, E, G, and I) and 10 nm (C, F, H, and J).

TABLE 1. Dimension of cell envelope structures based on CEMOVIS analysis

Organism

Avg position (nm) SD (no. ofmeasurements)a Avg thickness (nm) SD (no. of measurements)b

GL OM PM GL IWZ MWZ OM Cell envelope

M. smegmatis 8.4 1.2 (8) 35.2 1.9 (8) 7.1 0.6 (8) 3.6 0.9 (8) 16.6 1.2 (8) 7.3 1.9 (8) 7.1 0.6 (8) 42.4 2.3 (8)M. bovis BCG 9.0 1.3 (8) 31.5 1.9 (8) 6.3 0.7 (8) 3.8 0.8 (8) 14.1 1.6 (8) 6.3 1.0 (8) 7.5 0.8 (8) 38.7 2.4 (8)Wild-type C.

glutamicum8.7 0.7 (9) 44.5 7.5 (9) 6.7 0.4 (9) 4.3 0.8 (9) 18.0 1.4 (8) 20.9 8.8 (8) 4.7 0.7 (9) 48.6 6.7 (8)

C. glutamicum�pks13

8.4 1.4 (6) NAc 5.5 0.5 (6) 3.6 0.3 (6) 15.4 2.0 (6) 18.7 3.2 (6) NA 37.2 4.1 (6)

E. coli K-12d NA 27.3 5.8 0.4 (8) NA 11.5 6.4 0.5 (8) 6.9 1.0 (8) 33.7S. gordonii

Challise8.3 0.5 (10) NA 6.3 0.3 (6) 4.1 0.9 (9) 12.9 1.0 (10) 26.4 3.6 (10) NA 45.5 3.4 (10)

a Distance between the center of the PM and the center of the designated structure.b In the case of bilayers, the thickness corresponds to the total width of the structure and not to the peak-to-peak distance.c NA, not applicable.d Adapted from reference 29. Where no standard deviation is given, values were calculated from data in Table 1 or measured on Fig. 6 in reference 29.e Adapted from reference 58. Half the thickness of the PM was subtracted from the thickness of the IWZ and the cell envelope (58), respectively, in order to fit the

measurement methods used in the present paper.

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mens must be kept frozen, immunolabeling of cryosectionsis impossible.

Nevertheless, we studied M. smegmatis tmptB, a mutant de-void of GPL and which forms very large cell aggregates (49).GPL are considered to be an important constituent of the OMouter leaflet in several mycobacterial species (26), and theirdisruption may therefore affect the OM. CEMOVIS revealedthe structure of the contact zone between tmptB cells within anaggregate: all of the layers of the cell envelope are present,except the OM outer leaflet, and cells within the aggregate areinteracting via their OM inner leaflet (see Fig. S1A in thesupplemental material). This assertion is confirmed by imagesshowing that, at the edge of the contact zone, the OM outerleaflet is highly curved and is continuous between the two cellsinvolved in the contact, whereas this is not the case for theinner leaflet (see Fig. S1B in the supplemental material). Con-tacts between wild-type cells also exist, and their structure isidentical to that of contacts seen between tmptB cells (see Fig.S1C in the supplemental material). However, the deletion ofGPL generates an increase in the frequency of such contactsand, accordingly, the surface ratio of the OM outer leafletversus the inner leaflet is reduced, which is in agreement withthe expected localization of GPL in the OM outer leaflet andreinforces the hypothesis that the bilayer that we have ob-served is indeed the long-searched-for mycobacterial OM. Thelipidic nature of the OM is further supported by the fact thatthe OM inner leaflet was never observed in direct contact withthe aqueous environment but is always coated with either theOM outer or inner leaflet of another cell in contact.

Structures of the cell envelopes of wild-type and mycolic-acid-deficient C. glutamicum. Corynebacteria are closely re-lated to mycobacteria, and they possess a similar cell envelope.C. glutamicum has become widely used in the study of mycolic

acids because, as opposed to mycobacteria, C. glutamicum cangrow in the absence of mycolic acids (3, 18, 41, 42, 53). In orderto further test our hypothesis that the OM of Corynebacterineaeis made, in part, of mycolic acids, we studied C. glutamicumATCC 13032 (henceforth referred to as wild-type C. glutami-cum), which possesses mycolic acids, and C. glutamicum�pks13::km (henceforth referred to as C. glutamicum �pks13),a viable mutant strain that is deficient in the production ofthese fatty acids (41).

Our CEMOVIS micrographs show that the cell envelope ofwild-type C. glutamicum is similar to that of mycobacteria; itconsists of an IWZ, a GL, an MWZ, and an OM. Thoughsimilar, the cell envelopes are not identical; the MWZ of C.glutamicum is considerably thicker than the MWZ of mycobac-teria, exhibiting values similar to those of classical gram-posi-tive bacteria in CEMOVIS micrographs (Fig. 5A and C; Table1) (58). This suggests that the amount of peptidoglycan/arabi-nogalactan could be more important in corynebacteria than inmycobacteria. On the other hand, the corynebacterial OM isthinner than the mycobacterial OM and the corynebacterialPM. For this reason, we expect that a higher resolution inmicrographs is needed to visualize its bilayer aspect, which ismore difficult to obtain. In addition, in order to be seen, thetwo dense layers of the OM need to be aligned along theviewing axis through most of the section thickness (see Fig. 2 inreference 59). However, the corynebacterial OM rarely ap-pears smooth but shows many defects, which might thus hinderthe bilayer aspect of the corynebacterial OM (Fig. 5A, aster-isk). As a consequence, this aspect is visible only in localportions of micrographs recorded close to focus (Fig. 5C, ar-row).

In order to determine if these defects are a native feature ofthe corynebacterial OM or a cutting artifact, we examinedwhole-mount plunge-frozen wild-type C. glutamicum cells bycryoEM (13). The cytoplasm appears completely featurelessdue to the large thickness of the sample (data not shown), butboth the PM and the OM can readily be observed. The OMappears smoother than in CEMOVIS micrographs, indicatingthat this structure is particularly prone to cutting artifacts (Fig.5D). Furthermore, after Gaussian filtering of whole-mount cellmicrographs, a bilayer aspect can be distinguished in manyplaces of the OM but its dimensions are close to the resolutionlimit due to specimen thickness (Fig. 5D and E). Altogether,our data suggest that the corynebacterial OM is also a bilayer.

Importantly, the cell envelope of mycolate-free C. glutami-cum �pks13 lacks an OM (Fig. 5F to H). The MWZ thicknessdoes not significantly differ between wild-type C. glutamicumand C. glutamicum �pks13. This indicates that mycolic acidsare not present in the MWZ and that the OM found in wild-type C. glutamicum is formed of mycolic acids and possibly ofother lipids that interact with them. Furthermore, the extra-cellular medium of C. glutamicum �pks13 contains a largeamount of filaments. These are 3.9 0.2 nm thick, which isthinner than the OM found in wild-type C. glutamicum. Thus,they possibly represent lipids that normally interact with my-colic acids to form the outer leaflet of the OM. In the absenceof mycolic acid, they would still be exported but would not belinked to the cell wall anymore and would be released in theouter medium. This interpretation needs to be tested by fur-ther analysis of the released material.

FIG. 4. Staining of M. smegmatis mc2155 with OsO4. (A) Glutaral-dehyde-fixed and OsO4-postfixed cell. (B) Glutaraldehyde-fixed cell.(C) Density profile of the cell envelope in panel A. (D) Density profileof the cell envelope in panel B. Images A and B were acquired at 120kV. They were denoised by Gaussian filtering in Adobe Photoshop(radius of 0.6 pixel). They have the same intensity scale. Images weredefocused by �1.9 �m (A) and �2.1 �m (B) Density profiles wereobtained from nondenoised images. They were averaged over a widthof 35 pixels, and both profiles are shown at the same scale. Theapparent difference in the distance between the PM and the OM inpanels C and D is due to the fact that the measured cell envelopes hada different orientation in relation to the cutting direction. Bars, 20 nm(A and B) and 10 nm (C and D).

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DISCUSSION

Insights into the native structure of mycobacterial cell en-velope and comparison with images of conventional prepara-tions. A variety of electron microscopy techniques have beenused to decipher the unusual architecture of mycobacteria andrelated microorganisms. These include ultrathin sectioning ofboth conventionally processed and freeze-substituted samples,negative staining, and freeze fracture. Although the impressionprevails from all of the methods that the cell envelope islayered, different techniques give different pictures of the lay-ering. Consequently, the assignment of the known chemicalcomponents of the wall to ultrastructurally defined layers is notstraightforward (9).

In ultrathin conventional sections, the cell envelope of my-cobacteria is seen as being composed of an asymmetric PMwith a thin inner leaflet and a thick outer leaflet (10, 47, 48); athick, electron-dense layer (EDL); an electron-transparentlayer (ETL); and an OL of variable density and thickness (Fig.1). Because of the ETL’s transparency and the disappearanceof this layer after removal of lipids by alkaline hydrolysis (12),the ETL is assumed to contain lipids, mainly mycolic acids. Thedensity of the EDL makes it likely to contain the peptidoglycanto which the arabinogalactan is attached at many sites. Inmycobacteria, but not in corynebacteria, the PM is separatedfrom the EDL by a space that may correspond to a periplasm(Fig. 1) (9, 43). Based on electron microscopy data, notablythose from freeze fracture (5, 7, 33, 35, 43), and on the chem-ical structures of the main cell envelope constituents, a modelof the envelope was developed in 1982 by Minnikin (33), fol-lowed by several improvements and modifications (26, 32, 34,43, 44). Nevertheless, discrepancies remained between the im-

ages provided by various ultrastructural techniques and chem-ical knowledge.

Application of CEMOVIS to representatives of pathogenicand slow-growing, as well as saprophytic and rapid-growing,mycobacterial species, i.e., M. bovis BCG and M. smegmatis,respectively, resulted in images that differ from those previ-ously obtained by conventional and freeze-substitution elec-tron microscopy in the following important points. (i) The PMhas its typical bilayer aspect, with a thickness similar to thatobserved in other bacterial cells (Table 1 and Fig. 6) (29–31,58). The inner leaflet has the same density as the outer one, incontrast to the asymmetrical appearance often reported by thinconventional sections of mycobacteria, that was speculated tobe due to the glycoconjugates in the outer leaflet (47, 48). Thisasymmetrical appearance of the membrane seen in conven-tional sections could be a consequence of the dehydrationprocess involved in sample preparation, which could cause thecollapse of the GL against the outer leaflet of the PM. (ii) Acompartment similar to the periplasmic space of both gram-positive and gram-negative bacteria (29–31, 58) is apparent inCEMOVIS images not only in mycobacteria, as it was pre-dicted from conventional electron microscopy (9), but also incorynebacteria. This could provide cells with a space whereenzymatic reactions involved in cell envelope maintenance cantake place. As mentioned, the GL lies in the mycobacterial andcorynebacterial IWZ. This layer has been observed in gram-positive bacteria but not in gram-negative bacteria (58). Thus,its presence in both mycobacteria and corynebacteria is con-sistent with the fact that these bacteria belong to the gram-positive group. Recent cryo-electron tomography-based re-ports of a similar structure in cell wall-free Mycoplasma

FIG. 5. Cell envelope of corynebacteria visualized by CEMOVIS and whole-mount cryoEM. (A to E) Wild-type (WT) C. glutamicum. (F to G)C. glutamicum �pks13. (A, C, F, and G) CEMOVIS. (D) Whole-mount cryoEM. (G) Higher magnification of the boxed area in panel F. ImagesA, C, F, and G were acquired at 100 kV, and image D was acquired at 200 kV. Images were defocused by �3.4 �m (A), �1.1 �m (C), �3 �m(D), and �5 �m (F and G). They were denoised by Gaussian filtering in Adobe Photoshop (radius of 1 pixel for panels A and D, 0.8 pixel for panelC, and 2.4 pixels for panels F and G). The density profiles in panels B and H were obtained from nondenoised images corresponding to panelsA and G, respectively. The density profile in panel E was obtained from Gaussian filtered (radius of 1 pixel) image D. The density profiles wereaveraged over a width of 74 pixels (B), 65 pixels (E), and 70 pixels (H). The abbreviations are the same as those in Fig. 3. Double arrowhead, icecontamination; asterisk, cutting-induced defects in the OM; black arrowhead, PM; black arrow, OM; white arrow, filaments. Bars, 20 nm (A, C,D, and G), 10 nm (B, E, and H), and 100 nm (F).

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pneumoniae suggest that it could be made of membrane-boundproteins (20, 46). (iii) No ETL that is visible in conventionalthin sections and that has been considered to be made up ofcell wall-linked mycolates is seen in either M. bovis or M.smegmatis examined by CEMOVIS. The presence of this layerin conventional preparation of mycolate-free corynebacteria(i.e., Corynebacterium amycolatum [43]) indicates that the layereither represents a technical artifact or contains lipids otherthan mycolates. (iv) Most importantly, both M. bovis and M.smegmatis cells treated by CEMOVIS unambiguously show thepresence of an outer bilayer, a key point of validation of thecommon basic feature of the current cell envelope models.

The density of the OM is strongly increased after fixationwith OsO4, a chemical that stains mainly lipids (56). In cellsdevoid of GPL, which in the OM are considered to be mainlypresent in the outer leaflet (26), the OM inner leaflet has notbeen seen uncoated and in direct contact with the aqueousouter medium. Yet, the reduction in the surface area of theOM outer leaflet is correlated with an increase in the con-tact surface between the OM inner leaflets of two differentcells forming an aggregate. These observations strongly sup-port the hydrophobic and lipidic nature of the leaflets com-posing the OM.

To firmly establish the involvement of mycolates in the OM,we used corynebacteria as a surrogate model for studying es-sential mycobacterial compounds, such as mycolic acids, whichare dispensable in corynebacteria (3, 18, 41, 42, 53). We com-pared the architecture of a wild-type strain and a mycolate-freemutant of C. glutamicum (41). Our data clearly show thatwhereas the wild-type strain had a distinct OM, the pks13knockout mutant is devoid of this structure, making a compel-ling argument that the OM is a real structure that containsmycolic acids.

Challenges to the current models of the mycobacterial OM.The thickness of the OM is 4 to 5 nm in C. glutamicum and 7to 8 nm in mycobacteria. This observation is consistent with thepresence of mycolyl residues in the structure and the shorterchain lengths of corynomycolic acids (32 to 36 carbons) com-pared to those of mycobacteria (70 to 90 carbons). The sepa-ration of the density peaks of the PM bilayer (3.9 0.4 nm inM. bovis BCG) corresponds to the separation measured bycryoEM and X-ray scattering in liposomes made of phosphati-dylcholine with acyl chain lengths of 16 to 18 carbons (25, 52).

This is consistent with the lengths of the main fatty acid con-stituents of the PM (11). On the other hand, the mycobacterialOM is only slightly thicker than the PM (Table 1 and Fig. 6),whereas the main (meromycolic) chain of the mycolic acids ismuch longer (49 to 61 carbons in M. bovis BCG [50] and 35 to58 carbons in M. smegmatis [57]). Similarly, both chains of C.glutamicum mycolic acids are made of 16 to18 carbons (8), likethe main constituents of the PM, but the corynebacterial OMis considerably thinner than the PM. It therefore appears thatto accommodate the limited thickness of the OM, the lipidsfacing the arabinogalactan-bound mycolic acids must be inter-calated between mycolic acid chains, resulting in a zipper-likestructure (Fig. 7). These lipids could be represented by extract-able lipids (i.e., noncovalently bound to arabinogalactan) of aubiquitous nature (e.g., trehalose mycolate, phospholipids)

FIG. 6. Schematic representation, at scale, of the cell envelopes of E. coli, M. smegmatis, C. glutamicum, and S. gordonii as seen with CEMOVIS.The abbreviations are the same as those in Fig. 3. The GL is drawn as bound to the PM. This hypothesis is based on the conclusion of our previouswork with gram-positive bacteria (58). The IWZ is attributed, by analogy to other bacteria, to a periplasmic space. The MWZ represents thepeptidoglycan layer in E. coli and S. gordonii and would correspond to the peptidoglycan-arabinogalactan layer in M. smegmatis and C. glutamicum.The OL of M. smegmatis and C. glutamicum is not depicted (see Discussion).

FIG. 7. Zipper model of the OM of Corynebacterineae. (A) Myco-bacteria. (B) Corynebacteria. Hydrocarbon chains of the lipids aredrawn to scale. Black, mycolic acid; dark blue, phospholipids (16- to18-carbon-long chains); dark gray, peptidoglycan-arabinogalactan;light blue, GPL; light gray, porin; orange, trehalose dimycolate; red,trehalose monomycolate. Mycolic acids and trehalose mycolates arefolded (54, 55). An unfolded mycolic acid is shown in panel A. It is toolarge to be accommodated in the OM. Porins are not drawn to scale.Of note, the porin of M. smegmatis MspA is expected to protrude outof the OM (27). The porin of corynebacteria has been proposed to bemade by a stack of short proteins (6 kDa) (43). GPL are species-specific lipids found in M. smegmatis but not in M. bovis BCG (16). Ithas been suggested that the OM inner leaflet of corynebacteria con-tains a substantial amount of mycolic acids noncovalently bound to thepeptidoglycan-arabinogalactan (43).

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and of species-specific types (e.g., sulfolipid, phthiocerol dimy-cocerosate). Such a structure was originally proposed by Min-nikin (33). The models proposed by Rastogi (44) and Liu et al.(26), in which the extractable lipids form a totally distinctmonolayer rather than intercalating with the nonextractablemycolic acids, seems unlikely because they result in an OMthat would be much thicker than what we see by CEMOVIS.Villeneuve et al. have recently proposed a novel conforma-tional model of mycolic acids where the meromycolyl chain isfolded upon itself to create a compact structure (54, 55). In thiscase, the thickness of a monolayer of mycolic acids correspondsto the length of mycolic acid short arm, which is unfolded (20to 26 carbons). Although this model does not contradict ourCEMOVIS data, it is not sufficient to fully explain them. In-deed, the short arm of M. smegmatis mycolic acids is 22 carbonslong (57), i.e., 20 to 30% longer than phospholipids formingthe PM, whereas the thickness of the PM equals that of theOM in our data. Likewise, both chains of corynebacterial my-colic acids should be unfolded, as suggested by the model ofVilleneuve et al.; as mentioned above, these chains have thesame length as the PM phospholipids (16 to 18 carbons),whereas the OM is thinner than the PM. Thus, even if themycobacterial meromycolyl chain is folded and compact, boththe mycobacterial and corynebacterial arabinogalactan-boundmycolic acids forming the inner leaflet of the OM have to beintercalated to a certain extent with the longest chains of freelipids (e.g., trehalose mycolate) forming the OM outer leaflet(Fig. 7).

Since we submitted this work for publication, a differentlaboratory has published CEMOVIS images and cryo-electrontomograms of M. smegmatis, M. bovis BCG, and C. glutamicum(21). Their results are essentially similar to ours, but the mod-els of the OM of mycobacteria that they propose significantlydiffer from ours in that the mycolic acids are unfolded. As wehave explained above, we think that the unfolded meromycolicchain is too long to fit in the OM. Hoffmann and colleagueshave not proposed any model for the OM of corynebacteria.

Future directions and conclusion. In contrast to picturesfrom conventional (Fig. 1) and freeze-substitution (39, 40)techniques, no OL/capsule was seen at the surface of eithermycobacteria or corynebacteria. This situation may be due tothe fact that the components of such a layer would have thesame density as the cryoprotection medium and would there-fore be undistinguishable from this medium in CEMOVISimages. On the other hand, in mycobacteria, the capsular con-stituents are known to be loosely attached to the cell wall andmost of them are found released in the culture fluids of invitro-grown bacteria (23, 24, 37, 38), potentially explaining whythey are not visible in our images. When mycobacteria growintracellularly, these constituents are confined around the bac-teria by the phagosomal membrane (9). Techniques were de-veloped to maintain these constituents around in vitro-growncells and visualize them by conventional transmission electronmicroscopy (17). Further studies are warranted to address theissue of the structure of the mycobacterial OL and capsule byCEMOVIS.

In conclusion, our study brings a new reference structure ofthe cell envelope of mycobacteria and corynebacteria. Thisshould serve as a framework for building new models of theorganization of chemical components in the cell envelope of

mycobacteria. It also provides a basis for analyzing whether thecell envelope becomes significantly modified when mycobacte-ria are enclosed within phagosomes in macrophages, a ques-tion we are currently addressing.

ACKNOWLEDGMENTS

We thank Jeanne Salje (Cambridge, United Kingdom) for providingthe E. coli B/r strain and Gilles Etienne (Toulouse, France) for helpfuldiscussions and for providing M. smegmatis tmptB. We are grateful toPierre Gounon (Nice, France) for his technical help in conventionalelectron microscopy. We thank Nigel Unwin (Cambridge, UnitedKingdom) and Andreas Engel (Basel, Switzerland) for their support.

B.Z. is supported by an EMBO long-term fellowship.

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