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. Author manuscript; available in PMC: 2015 Dec 2.
Published in final edited form as: Structure. 2014 Nov 20;22(12):1875–1882. doi: 10.1016/j.str.2014.09.017

Controlled Bacterial Lysis for Electron Tomography of Native Cell Membranes

Xiaofeng Fu 1, Benjamin Himes 1, Danxia Ke 1, William J Rice 2, Jiying Ning 1, Peijun Zhang 1,3,*
PMCID: PMC4255137  NIHMSID: NIHMS638564  PMID: 25456413

SUMMARY

Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryo-FIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage φX174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and show that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrate the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes.

Keywords: CryoET, cryoEM, FIB, SEM, Membrane, Phage φX174, E gene, Lysis, Bacteria, Chemotaxis, ATP synthase

INTRODUCTION

Currently, cryo-electron tomography (cryoET) is the only method that offers three-dimensional (3D) structures of pleomorphic subjects, such as cells, organelles, and macromolecular assemblies, in a close-to-native state and at nanometer resolution. It has provided a wealth of information on the cellular ultrastructure of small bacterial cells (Kurner et al., 2005; Zhang et al., 2007; Khursigara et al., 2008; Briegel et al., 2009) and very thin peripheral regions of eukaryotic cells (Medalia et al., 2002; Koning et al., 2008; Carlson et al., 2010), where useful information can be recovered in cellular tomograms. With the advent of new generation direct electron detectors (Bammes et al., 2012; Campbell et al., 2012; Bai et al., 2013; Li et al., 2013) and phase plates (Fukuda et al., 2009; Murata et al., 2010; Barton et al., 2011; Guerrero-Ferreira and Wright, 2014), the application of cryoET to study small prokaryotic cells will potentially produce results of significant impact in the field of prokaryotic cell biology (Li and Jensen, 2009; Milne and Subramaniam, 2009). The main limitation for obtaining high quality cellular tomograms is the specimen thickness which is typically close to 1 μm for most bacterial cells. To address this issue, methods have been developed to physically reduce the thickness of frozen-hydrated specimens, including vitreous sectioning (Zhang et al., 2004; Al-Amoudi et al., 2005) and, more recently, cryo-focused-ion-beam (cryoFIB) milling (Marko et al., 2007; Hayles et al., 2010; Wang et al., 2012). Others have used genetic approaches, to create mini-cells (Briegel et al., 2012; Liu et al., 2012), or biochemical approaches, to make leaner cells by varying the cell culture conditions (Trueba and Woldringh, 1980). While these approaches facilitate 3D visualization of intracellular structural details, cryoET analysis of cell membrane structures and important membrane protein complexes within intact, native cell membrane remains difficult.

Conventional cell membrane preparation methods, such as ultracentrifugation of bacterial cell lysates by lysozyme and EDTA treatment, usually yield heterogeneous membrane fragments (Poole, 1993). Phage lysis offers an attractive alternative approach for producing intact native cell membranes in growing cells; in this case, intact ghost cells, which have much of the cytoplasm removed, are generated. Bacterial phages uses two strategies to achieve lysis of the bacterial host cells in a timely manner: double-stranded DNA (dsDNA) phages, such as lamda phage, employ a complex binary system with a holin and a muralytic enzyme, endolysin, for lysis; small single-stranded DNA and single-stranded RNA phages, on the other hand, produce host lysis by creating a single lytic protein without muralytic activity (Young, 1992; Wang et al., 2000). A remarkable example of this is the small DNA phage φX174, in which a single gene, E, which encodes a 91 amino acids integral membrane protein, mediates host cell lysis at a concentration of 100–300 molecules per cell (Young and Young, 1982; Maratea et al., 1985). Furthermore, overexpression of the E gene product is sufficient to cause rapid cell lysis (Young and Young, 1982). Owing to the simplicity and unique properties of the E gene, here, we employ the E lysis system to produce intact bacterial cell membranes for high resolution cryoET structural analysis. We demonstrate that ghost membranes can be obtained via spot lesion upon E gene induction. CryoET analysis of E gene-induced cell ghosts showed highly-ordered chemotaxis membrane receptor signaling complexes, with up to 25 Å resolution in a single cellular tomogram. The methods described here will be of great interest to those studying the biophysical and biochemical properties of cell membranes and membrane proteins in their native environment.

RESULTS

Expression of φX174 E gene results in rapid lysis of E. coli cells

Expression of phage φX174 gene E was previously shown to be necessary and sufficient for the lysis phenomenon exhibited by phage-infected cells (Young and Young, 1982). To examine the structural effect of E gene on host cells, we used a tightly controlled plasmid expression system to produce E gene product in E. coli cells. Under a tacP promoter and a lacIQ repressor (Roof et al., 1997), E gene expression was triggered by addition of IPTG, at two different time points, OD=0.2 or OD=0.6, during the log phase of cell growth. In both cases, the optical density of the cell culture started to decrease within 10 minutes of IPTG addition, suggesting a very rapid activation of cell lysis by E gene product (Fig. 1A). The lysis process was nearly complete at about 30 minutes. This is consistent with previously reported results (Bernhardt et al., 2001a; Bernhardt et al., 2002) and, thus, supports a model wherein E-mediated lysis occurs during cell division by inhibiting the peptidoglycan synthesis enzyme MraY (Bernhardt et al., 2000).

Figure 1.

Figure 1

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Phage φX174 E gene induces rapid bacterial cell lysis. (A) Growth and lysis curves of E. coli cultures carrying E gene expression plasmid. The optical density (OD) at 600 nm was measured in control cells (open circles) or after induction of the E gene product at an OD600 of 0.2 (gray, designated time 0 on the bottom axis) or 0.65 (black, designated time 0 on the top axis). (B) The percentage of lysed cells observed in E. coli samples at the indicated time after E gene induction. The number of lysed cells was counted from low magnification TEM images (~300 cells in each sample).

The efficiency of E-mediated lysis was further quantified by examining the morphology of individual bacterial cells, using a transmission electron microscope (TEM). Cultured E. coli cells were collected and frozen under high-pressure at the indicated time points after IPTG induction, followed by freeze-substitution, resin embedding, and sectioning. TEM imaging revealed individual cells undergoing lysis, as evidenced by their less dense cytoplasm compared to intact cells (Fig. 2A–C). To quantify the lysis process, the fraction of cells undergoing lysis was determined at several time points after IPTG induction from EM micrographs. As illustrated in Fig. 1B, E. coli cells begin losing cytoplasm very quickly upon E gene induction, as early as 5 minutes post-induction. Quantitative cell morphology analysis indicates that the onset of lysis was actually earlier than that measured by OD. This is likely because the majority of cells were still growing at the early OD measurements. At 25 minutes, more than 80% of cells were affected, and at 60 minutes, near 95% of the cells had undergone lysis. Thus, compared to the complex binary endolysin/holin lysis system (Young, 1992), E-mediated bacterial lysis is remarkably simple, effective, and efficient.

Figure 2.

Figure 2

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Electron microscopic characterization of E gene-induced cell lysis. (A–F) TEM images of thinly sectioned E. coli cells, recorded at low (A–C) or high (D–F) magnifications. The cells were subjected to high-pressure freezing at 0 min (A&D), 25 min (B&E), or 60 min (C&F) after IPTG induction, followed by freeze-substitution and resin embedding. White arrows, arrowheads, and double arrowheads indicate unlysed, partially lysed, and completely lysed cells, respectively. (G&H) TEM images capture the cells in the lysis process. Black arrows indicate the lesion sites in cell membranes, resulting in the release of cytoplasm. (I) Stereo view of a 3D volume from E. coli cell samples at 30 min after E gene induction. Scale bars, 2 μm in A–C, 200 nm in D–H.

E-mediated lysis produces whole cell ghosts through spot lesion

To further characterize the structural changes during E-mediated cell lysis, cells at different lysis stages were imaged by TEM. As shown in Fig. 2, before E-gene induction, all cells displayed a dense cytoplasm, and many were actively dividing (Fig. 2A&D). At 25 minutes after induction, the majority of cells were either partially (arrowhead) or completely (double arrowhead) lysed, and only a small fraction of cells remained intact (arrow) (Fig. 2B&E). After 60 minutes, nearly all the bacterial cells had lost cytoplasm. In contrast to other cell lysis methods, which produce only membrane fragments (Poole, 1993), E gene-mediated lysis maintained and preserved the cell membranes and cell shape (Fig. 2E&F). More interestingly, upon close inspection of those cells captured instantly at the early lysis stage (Fig. 2G&H), we found localized lesion spots from which cells seemed to be losing their cellular content: the cell membrane appeared to be punctured with the cytoplasm ejected through the compromised membrane. We further characterized the 3D morphology of lysed cells using ion-abrasion scanning electron microscopy. Consistent with our previous observation, a majority of E. coli cell ghosts remained as rod shaped membrane shells, with a few occasional spheroplasts (Fig. 2I).

Membrane proteins and associated protein complexes are retained

To determine whether membrane proteins and protein complexes remain associated with the cell membrane after E-mediated lysis, we examined the lysed cells for the presence of the chemotaxis membrane receptor signaling complex, which is composed of transmembrane receptors, a cytoplasmic histidine protein kinase, CheA, and a small soluble co-factor, CheW. Cell ghosts were pelleted after varying periods of E-gene induction and analyzed for the presence of membrane receptors and CheA. As shown in Fig. 3A, throughout the lysis process, up to near completion (30 min), the chemotaxis membrane receptor and CheA remained associated with the ghost membranes, with little detected in the cell supernatant, even when very little cytoplasm was left (Fig. 3B). This was also true for F0F1 ATP synthase when tested using antibodies against soluble F1 epsilon subunit (Fig. 3A, lower panel). Thus, the cell ghosts produced by E-lysis are good candidates for biochemical, biophysical, and structural analysis of membrane protein complexes.

Figure 3.

Figure 3

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Analysis of chemotaxis membrane receptor signaling complex in E-lysed cells. (A) Western blot analysis of bacterial chemoreceptors and the associated soluble histidine kinase, CheA, in total cell (T), supernatant (S), and pellet (P) fractions at the indicated post E gene induction time. Both chemotaxis membrane receptors (Tsr) and soluble CheAl/s (long and short forms), as well as the epsilon subunit of F0F1ATP synthase remain in the pellet fractions. (B) A TEM projection image of a sample 30 min after E gene induction shows intact cell membranes. Arrowhead and double arrowhead indicate a partially lysed and a completely lysed cell, respectively. Scale bar, 50nm.

CryoEM and cryoET analysis of ghost cell membranes

Because E-mediated lysis produces cell ghosts with intact native membranes, such a system could have great utility for structural, biophysical, biochemical, and functional studies of membrane proteins. We thus assessed its usage for structural analysis of cell membranes and membrane-associated proteins by cryoEM and cryoET. E. coli cells at an early stage of E-gene expression (12 minutes) were directly plunge frozen from cell culture and imaged by cryoEM. Fig. 4A shows the cryoEM image of a lysed cell (light density, black arrow) and an intact unlysed cell (dark density, orange arrow) captured in the same field. At an early stage such as this, about 20% of the cells have begun to undergo the lysis process, exhibiting reduced cell thicknesses (Fig. 4B, red), and more cells become thinner 30 minutes post E-gene induction (Fig. 4B, blue). This is consistent with the previously proposed model for E gene-mediated lysis, in which the E gene product acts on the bacterial cell wall by inhibiting MraY, leading to cell lysis during cell division (Bernhardt et al., 2001a). In fact, the process of cells spouting their cytoplasmic content, at the onset of lysis, was evident in our cryoEM images (Fig. 4). For example, in Fig. 4C, a pool of ribosome molecules that was presumably just released from the bacterial cell is found surrounding the ghost cell, suggesting that these molecules were released at the moment of plunge-freezing. Such ribosomal pools were frequently observed around cell ghosts at the early stages after E gene induction. The membrane structures were well preserved, with the inner cell membrane, peptidoglycan, and outer membrane clearly seen (Fig. 4C and Fig. 5B) and comparable to those observed in vitreous sections (Zhang et al., 2004) and cryo-FIB milled cell lamella (Wang et al., 2012). Furthermore, the onset of lysis was instantaneously captured by rapid freezing and cryoEM (Fig. 4D); the cellular content was observed to be discharging through small lesion holes, about 50 nm in size (Fig. 4D, black bracket), where the inner membrane and peptidoglycan appear disintegrated. The data indicate that E-lysis occurs through small individual lesion spots where cytoplasm is ejected, leaving the major portion of the cell membrane intact.

Figure 4.

Figure 4

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CryoEM analysis of E gene-induced E. coli cell lysis. (A) A low magnification projection image of E coli cells shows an unlysed cell (Orange arrow) and a partially lysed cell with a higher electron transparency (black arrow). (B) Distribution of the thickness of the cells at 15 (red) and 30 (blue) minutes post E-gene induction. The orange and black arrows indicate the unlysed and lysed cell populations, respectively. (C) A high magnification view of the partially lysed E coli cell shown in A. Ribosome molecules were instantaneously released from the lysing cell at the moment of freezing, as seen by the pooling around the cell. Inner membrane, peptidoglycan, and outer membrane are indicated by chevron arrow, arrow, and double arrow, respectively. (D) A cryoEM projection image captures an E. coli cell at the onset of lysis. The arrowhead points to the lesion spot where cytoplasm, containing DNA and protein molecules, is being ejected. The size of the lesion is indicated by a black bracket. Scale bars, 2 μm in A and 200 nm in B&C.

Figure 5.

Figure 5

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CryoET analysis of E. coli cell membranes generated by E gene induced cell lysis. (A) A lysis pore (black arrowhead) across the two cell membranes is captured by rapid freezing and visualized in 3D tomographic slices. The spouting cytoplasm is indicated by dashed profile. See also Movie S1. Inner membrane, peptidoglycan and outer membrane are indicated by the chevron arrow, arrow, and double arrow, respectively, in each panel. (B) The cytoplasmic membrane (chevron arrow) retracts from the cell wall due to release of the pressure inside the cell. DNA strands and ribosome molecules are clearly seen in the surroundings of the partially lysed cell. See also Movie S2. (C) Flagella motors (white arrowheads) are anchored across the two membranes in a partially lysed cell. See also Movie S3. (D) High resolution tomographic reconstruction of chemotaxis receptor signaling complexes within the cell membrane of partially lysed cell. See also Movie S4. The ordered receptor signaling array diffracts to 25 Å resolution, as indicated by the circles in the Fourier transform (upper inset) calculated from a 15 nm thick tomographic slice from the boxed area. Sub-tomogram averaging yielded a 3D density map of the trimeric chemotaxis signaling unit containing 3 CheA dimers (“A”) and 6 receptor trimers of dimers (“R”) (lower inset). See also Figure S1 and S2. Scale bars, 100 nm.

We also carried 3D structural analysis of E-lysed cell ghosts by cryoET. The spot lysis observation was confirmed in 3D tomograms (Fig. 5A, black arrowhead, Movie S1), with pore sizes of about 50–100 nm. This is the first direct observation of lysis holes spanning two membranes in a native hydrated environment. Attempts to visualize the membrane lesions by conventional ultrathin-section EM have been unsuccessful, in part because of the structural deformations associated with the multiple fixation, dehydration, and staining steps (Kellenberger et al., 1992). CryoET allows chemical-free preservation of whole cells within milliseconds and provided better than 5 nm resolution. Interestingly, in the 3D tomograms, the inner and outer membranes, at the lesion spot, appear to be connected at the rim, sealing the periplasmic space at the pore (Fig. 5A, Movie S1), reminiscent of the nuclear pore in eukaryotic cells. The cellular content moving through the pore is clearly visible in Fig. 5A (dashed profile). Since the cell had just begun to leak its content, the thickness of the cell is only partially reduced to ~250 nm (Fig. S1A), compared to the cells that are further along in the lysis process, at 100–150 nm thickness (Fig. 5B–D, Fig. S1B&C). Presumably due to the release of pressure within lysed cells, the rod-shape ghosts collapse into two layers of cell envelopes upon plunge freezing and the inner membrane sometimes retracts from the cell wall (Fig. 5B, Movie S2) but was often kept in place in regions where flagella motors were anchored across the two membranes (Fig. 5C, Movie S3). The fine structure of the peptidoglycan was clearly resolved in the lysed ghosts (Fig. 5B&C, white arrow), similar to those seen in vitreous sections (Zhang et al., 2004). DNA strands and protein complexes released from the cell were clearly visible adjacent to the cell (Fig. 5B&D).

Remarkably, the transmembrane chemotactic receptor signaling complexes were maintained in a highly-ordered crystalline arrangement upon E-mediated lysis (Fig. 5D, Movie S4), with a resolution of at least 25 Å, as seen in Fourier transform of a single receptor cluster in the tomogram (Fig. 5D, top inset). To our knowledge, this is the best resolution demonstrated to date from a single cellular tomogram without subvolume averaging. Sub-tomogram averaging of ~400 trimeric chemotaxis signaling units yielded a 3D density map comprising a trimer of CheA dimer/receptor complexes (Fig. 5D, bottom inset, Fig. S2A), consistent with the previous sub-tomogram averaged structures from other bacterial species (Briegel et al., 2012; Liu et al., 2012). The data from a single native receptor cluster suggest that the chemotaxis arrays in E. coli cells are highly-ordered, in contrast to the chemotaxis arrays observed in other cell types (Khursigara et al., 2008). While previous studies were carried out with thick specimens, such as whole cells (~0.6–1 μm thick) with limited resolution, the cell ghosts used in this study were mainly composed of two layers of cell envelop (~150 nm thick) and yielded much more detailed information at a higher resolution. Therefore, among the many different methods applied to generate samples for cryoEM and cryoET, such as engineered mini-cell (Briegel et al., 2012; Liu et al., 2012), vitreous sectioning (Zhang et al., 2004), or whole cell preparations (Zhang et al., 2007), the E-lysis procedure provides the highest quality native receptor clusters for 3D structural analysis.

DISCUSSION

The mechanism of E-mediated lysis is still not fully understood, although the current model of MraY inhibition is consistent with our results (Roof et al., 1997; Bernhardt et al., 2000; Bernhardt et al., 2001a; Mendel et al., 2006; Zheng et al., 2008). Further biochemical and structural studies are needed to understand the molecular mechanism of E-gene product action on cell membrane integrity and lysis (Zheng et al., 2009). Nevertheless, with millisecond cryo-fixation, which allows instantaneous capturing of biological processes under native conditions, the lethal holes caused by the E gene product of bacteriophages and the resulting discharge of bacterial cytoplasm have now been directly observed in vivo by cryoEM and cryoET. Unexpectedly, the size of the membrane lesion holes caused by the E gene product is only 50–100 nm (nano-sized), much smaller than that produced by holin S105 protein, which exceeds 300 nm to μm (micro-sized) (Dewey et al., 2010), and smaller than the large transmembrane tunnel structures observed in lysed bacterial cells (Witte et al., 1990a; Witte et al., 1990b; Lubitz et al., 1999). Distinct from the holing-endolysin lysis system with λ phage (Dewey et al., 2010), the nano-sized lesion holes transverse both bacterial membranes, creating a small channel from which the cytoplasm is ejected. In addition, unlike the binary holing-endolysin lysis system (Wang et al., 2000), in which spheroplasts were produced with extensive damage of the cell wall through murein degradation by endolysin (Dewey et al., 2010), the E. coli cell ghosts produced by E-mediated lysis maintained the rod shape with very limited cell wall damage. Further, membrane proteins and protein complexes are retained within the ghost membranes with minimal disruption.

Several methods for bacterial cell membrane preparation have been described, including chemical and mechanical disruption or disintegration of cell wall, as well as freezing and thawing of bacterial cells (Leduc et al., 1982; Poole, 1993). Compared to these commonly used methods, such as treatment with penicillin or lysozyme-EDTA, which generally produce spheroplasts and membrane vesicles (Kaback, 1972), the advantages of membrane preparation by E-lysis are several fold: 1) It is a simple system, since expression of a single 91 amino acid E protein causes cell lysis at a concentration of 100–300 molecules per cell (Maratea et al., 1985); 2) It is quick; overexpression of the E gene product is sufficient to cause rapid cell lysis in less than 10 minutes; 3) Lysed cells release cytoplasm through a nano-sized lesion while a majority of the cell membrane remains intact; 4) Both inner and outer membranes are retained (Witte and Lubitz, 1989), along with membrane proteins and associated protein complexes; 5) E-gene lysed cells retain their rod-shape, but have lost intracellular content through focal lesion. When subject to freezing, the cell envelop collapses, yielding very thin cell ghosts with just two layers of cell envelop, compared to the round spheroplasts with a 0.5–1 μm thickness. The method we described here, using controllable E-mediated lysis to rapidly produce intact membrane envelop, provides superior signal-to-noise ratios for structural study of native cell envelop and membrane protein complexes, as demonstrated by cryoET of bacterial chemotaxis signaling arrays and F0F1ATP synthase. We anticipate that such an intact native membrane system, generated by E- mediated cell lysis, will not only constitute a useful tool for cryoEM and cryoET structural studies of membrane receptor complexes, but also for biophysical and biochemical characterization of membranes and membrane protein complexes in a native environment, as well as for protein antibiotic (Bernhardt et al., 2001b) and vaccine development against bacterial pathogens (Szostak et al., 1996; Szostak et al., 1997).

EXPERIMENTAL PROCEDURES

Bacterial strains, plasmids and cell culture

Wild-type E. coli K-12 strain RP437 cells (Parkinson and Houts, 1982) carrying plasmid pRY100 (a kind gift from Dr. Ry Young, Texas A&M University) (Roof et al., 1997) were grown in TB broth (1% Tryptone and 0.4% NaCl, pH 7.0), supplemented with 100 μg/ml ampicillin. The plasmid pRY100 has the phage φX174 lysis E gene under a tacP promoter and a lacIQ repressor control. RP437 cells carrying pRY100 were grown to an OD600 of 0.2 or 0.6 and E gene expression was induced by 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). The progress of cell lysis was monitored at OD600 from the beginning of culture until 60 minutes after addition of IPTG. The lysis profiles were obtained by measuring OD600 at different time points.

Protein analysis with Western Blot

The same volume of E. coli cells was withdrawn from non-induced cultures and from E gene-induced cultures at 20, or 30 min post IPTG addition. 6 μl samples were mixed with 4 × LDS loading buffer (Invitrogen, NY) supplemented with 10 mM DTT for SDS-PAGE analysis (T). The remaining sample was pelleted at 20,000 × g with an Eppendorf centrifuge 5417R for 15 min and supernatants (S) and pellets (P) were mixed with 4 × LDS loading buffer for gel analysis. Total, supernatant, and pellet samples, were loaded on 4–12% SDS-PAGE. Proteins were electrophoretically blotted to nitrocellulose membranes. Chemotaxis receptors and CheA proteins were detected by probing with receptor-specific and CheA specific polyclonal antibodies. The F1 component of F0F1 ATP synthase was detected by probing with polyclonal antibodies that recognize the epsilon subunit. Bands were developed and revealed using BCIP/NBT liquid substrate system (Sigma B1911) with conjugated anti-rabbit antibodies.

Electron microscopy of high pressure freezing, freeze-substituted samples

Bacterial cell culture samples (30 ml) were collected at 0, 5, 15, 20, 25, or 60 min after IPTG addition and were pelleted at 2,500 g for 15 minutes in a Sorvall (RC 3C Plus) swing rotor. The cell pellets were put on ice and transferred to Leica EMPACT 2 membrane carriers (1.5 mm × 0.2 mm) for high pressure freezing in EMPACT 2 (Leica Microsystems, Vienna, Austria), at an average pressure of 2000 bars (Studer et al., 2001). The frozen cells were then transferred to a freeze-substitution machine, Leica EM AFS (Leica Microsystems, Vienna, Austria), for a 5-day solvent substitution. Briefly, frozen samples were warmed from −196 to −90 °C, over a 3-day period, in a precooled (−90 °C) 1% OsO4 and 0.1% uranyl acetate mixture dissolved in acetone. Samples were then gradually warmed to room temperature over 18 h and subsequently rinsed in acetone for further resin infiltration and embedding in Epon. Ultrathin sections (65 nm) were cut with a Reichart Ultracut and laid on 300 mesh carbon-coated EM grids. The thin sections were post-stained with 2% uranyl acetate in methanol for 10 min, followed by Reynold’s lead citrate for 7 min (Reynolds, 1963), and were examined with a Tecnai F20 electron microscope (FEI Corp., OR.) equipped with a 4k × 4k camera (Gatan Inc., Warrendale, PA). Projection images were recorded at a magnification of 2,500 × and 19,000 ×.

FIB/SEM

Resin blocks were attached to SEM stubs using double sided carbon sticky tape then painted with colloidal silver (EMS, Hatfield PA). The blocks were made conductive by coating with Au/Pd in a Denton sputter coater (25 mA current for five minutes at 50–60 mTorr) then inserted into a FEI Helios Nanolab 650 (FEI Corp, OR.) for FIB milling. Areas were identified by viewing with the e-beam at 20 keV then coated with 1 um Pt for protection. A trench was dug in front of the area to be milled and side trenches were dug adjacent to this area. The FEI Slice and View software was used to collect slices using a slice width of 20 nm and a horizontal field width of 10 μm (4.88 nm/pixel). Sections were imaged at a −38 degree viewing angle at an e-beam voltage of 2 keV and 100 pA current. Following collection, images were aligned using IMOD software (Kremer et al., 1996).

Cryo-electron microscopy and cryo-electron tomography

3–5 μl of un-induced and E-gene induced (12 minutes) E. coli cell samples, at an OD600 of 0.5 were withdrawn directly from cultures and placed on R2/1 Quantifoil grids (Quantifoil Micro Tools GmbH, Jena, Germany). 2 μl of 15 nm gold beads were applied to the EM grids to serve as fiducial markers for tomographic alignment. The grids were manually blotted and plunge-frozen in liquid ethane maintained at approximately −180°C using a home-made manual plunger. For projection images, grids containing plunge-frozen cells were loaded onto a Gatan model 626 cryo-holder maintained at temperatures below −180°C and imaged using a Tecnai F20 transmission electron microscope equipped with a field emission gun (FEI Corp., OR) and a Gatan 4K × 4K CCD camera (Gatan, Inc., PA). Low dose (20 e−/Å2) projection images were recorded at a magnification of 31,000 × with under focus values ranging from 2 to 5 μm.

For 3D cryoET, frozen-hydrated EM grids were placed in cartridges and loaded into the cryo-transfer system of a Polara G3 microscope (FEI Corp., OR.). The Polara microscope was equipped with a field emission gun operating at 300 kV. A series of low dose projection images of E. coli cells were recorded at a tilt angle range from −70° to 70°, at a nominal magnification of 27,500 ×, with under focus values between 5–8 μm. The total dose used for each tilt series was typically ~60–80 e−/Å2. Tilt series were aligned using 15 nm gold bead fiducials and refined to standard deviations below 0.8 pixels. A weighted back-projection algorithm, as implemented in the IMOD reconstruction package (Kremer et al., 1996), was used to convert the information present in the series of tilted projection images into 3D density maps (tomograms). The 3D tomographic volumes were visualized in the environment of the program Amira (TGS Inc., CA). Potential areas of interest were denoised using 3D nonlinear anisotropic diffusion edge enhancing program implemented in IMOD, with 15 iterations and κ of 6.4, allowing clearer visualization.

Sub-tomogram averaging

Two of the chemotaxis receptor patches were selected from cryo-tomograms for subtomogram alignment and averaging. Projection series were CTF corrected using TomoCTF (Fernandez et al., 2006), aligned in IMOD (Kremer et al., 1996). Tomograms were then reconstructed using SIRT as implemented in the IMOD reconstruction package. Subvolumes were extracted using a reference free approach, and orientations were determined using the alignment by classification strategy as implemented in Protomo/i3 software (Winkler, 2007). Data from ~400 subvolumes classified as trimer of CheA dimer/receptor complex were aligned iteratively and averaged. The density was low-pass filtered to the frequency at 0.5 FSC (~33 Å), and clearly shows retention of the soluble cytoplasmic protein CheA, as visualized in Chimera (Pettersen et al., 2004).

Supplementary Material

1

Figure S1, related to Figure 5.

Tomography slices along two orthogonal directions to illustrate the thicknesses of the lysed cells. Panel A is related to Fig. 5A, B is related to Fig. 5B&D, and C is related to Fig. 5C. Scale bars, 100 nm.

Figure S2, related to Figure 5D.

Sub-tomogram averaging of the native bacterial chemotaxis signaling complex in E-lysed cells. (A) Fourier shell correlation (FSC) plot of the density map containing a trimer of dimeric chemotaxis signaling unit. (B–D) Orthogonal views of sub-tomogram averaged density map of the chemotaxis signaling unit. Color indicates the height of the complex, from red (CheA) to green (chemoreceptors).

2. Movie S1, related to Figure 5A.

Movie of tomographic slices from a reconstructed 3D volume of an E. coli cell at the onset of E-gene induced cell lysis, displaying a lesion spot where cytoplasm leaks out.

Download video file (8.4MB, mpeg)
3. Movie S2, related to Figure 5B.

Movie of tomographic slices from a reconstructed 3D volume of cell membranes where flagella was found anchored into two membranes.

Download video file (4MB, mpeg)
4. Movie S3, related to Figure 5C.

Movie of tomographic slices from a reconstructed 3D volume of E. coli cell membranes.

Download video file (6.5MB, mpeg)
5. Movie S4, related to Figure 5D.

Movie of tomographic slices from a reconstructed 3D volume of bacterial chemotaxis arrays within the cell membrane.

Download video file (5.9MB, mpeg)

Acknowledgments

The authors thank Dr. Ry Young (Texas A&M) for the φX174 E gene plasmid, Dr. Robert Nakamoto (University of Virginia) for the F0F1 ATP synthase antibodies and Dr. Teresa Brosenitsch for critical reading of the manuscript. This work was supported by the National Institutes of Health GM085043 and RR024424 to P.Z. The FIB/SEM data collected at NYSBC was made possible by a grant from NYSTAR in a facility constructed with support from Research Facilities Improvement Program Grant number C06 RR017528-01 from the National Center for Research Resources, National Institutes of Health. The dual beam scanning electron microscope was purchased with funds from NIH grant S10 RR029300.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1, related to Figure 5.

Tomography slices along two orthogonal directions to illustrate the thicknesses of the lysed cells. Panel A is related to Fig. 5A, B is related to Fig. 5B&D, and C is related to Fig. 5C. Scale bars, 100 nm.

Figure S2, related to Figure 5D.

Sub-tomogram averaging of the native bacterial chemotaxis signaling complex in E-lysed cells. (A) Fourier shell correlation (FSC) plot of the density map containing a trimer of dimeric chemotaxis signaling unit. (B–D) Orthogonal views of sub-tomogram averaged density map of the chemotaxis signaling unit. Color indicates the height of the complex, from red (CheA) to green (chemoreceptors).

NIHMS638564-supplement-1.pdf (1.6MB, pdf)
2. Movie S1, related to Figure 5A.

Movie of tomographic slices from a reconstructed 3D volume of an E. coli cell at the onset of E-gene induced cell lysis, displaying a lesion spot where cytoplasm leaks out.

Download video file (8.4MB, mpeg)
3. Movie S2, related to Figure 5B.

Movie of tomographic slices from a reconstructed 3D volume of cell membranes where flagella was found anchored into two membranes.

Download video file (4MB, mpeg)
4. Movie S3, related to Figure 5C.

Movie of tomographic slices from a reconstructed 3D volume of E. coli cell membranes.

Download video file (6.5MB, mpeg)
5. Movie S4, related to Figure 5D.

Movie of tomographic slices from a reconstructed 3D volume of bacterial chemotaxis arrays within the cell membrane.

Download video file (5.9MB, mpeg)

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