Vol. 172, No. 7JOURNAL OF BACTERIOLOGY, JUlY 1990, p. 4109-41140021-9193/90/074109-06$02.00/0Copyright © 1990, American Society for Microbiology

Endogenous Transmembrane Tunnel Formation Mediated bypX174 Lysis Protein E

ANGELA WITTE,1 GERHARD WANNER,2 UDO BLASI,1 GABRIELE HALFMANN,1MICHAEL SZOSTAK,1 AND WERNER LUBITZ1*

Institute of Microbiology and Genetics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria,'and Institute ofBotany, University of Munich, 8000 Munich 19, Federal Republic of Germany2

Received 29 December 1989/Accepted 30 April 1990

Biochemical and genetic studies have suggested that a transmembrane tunnel structure penetrating the innerand outer membranes is formed during the lytic action of bacteriophage 4X174 protein E. In this study wedirectly visualized the lysis tunnel by using high-magnffication scanning and transmission electron microscopy.

Gene E of bacteriophage 4OX174 codes for a polypeptide of91 amino acids (2), the production of which is necessary andsufficient to cause lysis of Escherichia coli (6, 14, 25). Noenzymatic activity has been associated with the protein itself(11, 19). This observation is consistent with the capacity ofE-related fusion proteins to cause lysis (8, 9, 13, 18). ProteinE is highly hydrophobic (2) and has been localized exclu-sively in the cell envelope (1, 4). Moreover, oligomerizationof protein E monomers has been demonstrated (7) and seemsto be required for lysis (18). The biochemical characteriza-tion of the lysis process revealed that collapse of themembrane potential and release of cytoplasmic componentssuch as ions, ATP, proteins, or nucleic acids occur simulta-neously (22, 23). On the basis of these data and the fact thatprotein E-lysed cells retain their structural integrity (22), wesuggested a novel model for E lysis. The model envisioned aprotein-E-mediated transmembrane tunnel structure pene-trating the envelope complex of E. coli (7, 22). Here, wereport the direct visualization of the lysis tunnel by usinghigh-magnification scanning and transmission electron mi-croscopy.

Expression of gene E and cell lysis were induced bythermal inactivation (temperature shift from 28 to 42°C) ofthe lambda cI857 repressor in E. coli pop2135 [F- endA thihsdR malT (X cI857 pR):malPQ] (kindly provided by 0.Raibaud, Institute Pasteur, Paris, France) harboring plasmidpAW13. Plasmid pAW13 was constructed as follows. First,the PstI-BamHI fragment of plasmid pSB12 (6) comprisinggene E under control of the lambdaPL promoter was insertedinto the PstI-BamHI sites of pACYC177 (10). Second, theobtained plasmid was cleaved with PstI and treated with T4DNA polymerase to destroy the bla gene. The resultingplasmid, pAW13, carries gene E under lambda PL controland confers kanamycin resistance.Samples of E. coli pop2135(pAW13) were removed at

various times after the onset of lysis. This ensured thepresence of cells in all phases of lysis. Cells in the early stageof lysis showed cytoplasmic material being released to theenvironment. The cytoplasmic content escaping from thecells could be detected as electron-opaque material in allscanning or transmission electron micrographs of lysing cells(Fig. 1A and B). Osmium tetroxide and uranyl acetate,which were used for en bloc staining, contributed to the highscattering power of the extruded matter. Lead citrate wasnot needed to contrast the cytoplasmic material. The rod-

* Corresponding author.

shaped morphology of the E. coli cells remained intact (Fig.1A and C), and the protein E-mediated lysis tunnel wasrestricted to a small part of the total cell surface (Fig. 1A, C,and D). On average, the diameter of the holes variedbetween 40 and 80 nm. Thus, 4X174 progeny with a hydro-dynamic diameter of 32 nm (3) can easily pass through thelysis tunnel. Irregular tunnels were also observed (Fig. 1D).It is conceivable that the irregular shape of the holes wasindirectly caused by the release of cytoplasmic materialduring lysis. The irregular shape of the tunnel might havebeen a consequence of the surrounding rigid peptidoglycandue to paracrystalline areas in the murein. Tunnel formationwas accompanied by a fusion of the inner and outer mem-branes (Fig. 1B). The resulting continuous membrane mayexplain earlier observations concerning the very limitedrelease of periplasmic enzymes during E lysis. At the timewhen 90% of the intracellular P-galactosidase was releasedby E-lysed cells, only 5% of the total alkaline phosphataseand 10% of the 3-lactamase were detected in the culturemedium (22).The role of peptidoglycan degradation in the formation of

the E-mediated lysis tunnel is poorly understood (17). Thereis evidence that an active autolytic system is one of thecellular prerequisites for the E-lysis process (16). So far ithas not been elucidated which specific component of thissystem is needed. However, only limited degradation (8 to12%) of the rigid murein structure has been observed afterprotein E-mediated lysis (A. Witte, J.-V. Holtje, and W.Lubitz, unpublished results). Inspection of a number ofscanning electron micrographs of E-lysed cells showed thatthe majority of cell ghosts contain only one E hole (data notshown). This suggests that murein degradation during E lysisoccurs only at the sites of transmembrane tunnel formation.In very rare cases, two holes in one bacterial ghost weredetected (Fig. 1C). Since protein E-mediated lysis of a singlecell takes less than 30 s (15), it seems reasonable to assumethat two tunnel structures in one bacterium can only emergesimultaneously. Moreover, the occurrence of two holesmight indicate that potential E-lysis structures are built up atseveral sites in the cell envelope. However, the first E tunnelstructure formed would cause lysis and thus should preventfurther tunnel formation.What constitutes the E-lysis tunnel? It is imaginable that

the endogenous transmembrane tunnel structure is similar tothe exogenous lysis complex formed by Staphylococcusaureus alpha-toxin (12); i.e., protein E-like S. aureus alpha-toxin may oligomerize to form a ringlike structure which

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FIG. 1. Electron micrographs of E-lysed cells. (A) High-resolution field emission scanning electron micrograph (FESEM) of E. coli afterE-induced lysis. E. coli pop2135(pAW13) cells were fixed for 60 min at room temperature with 2.5% glutardialdehyde in 75 mM sodiumcacodylate-1 mM MgCl2 (pH 7.2) and stored in the cold. Before further processing, cells were rinsed with the same buffer and postfixed for30 min in 1% osmium tetroxide in buffer. For en bloc staining the cells were incubated with 1% uranyl acetate in 20% acetone for 30 min.Dehydration was with a graded series of acetone solutions. After dehydration with 100% acetone, cells were critical point dried with liquidC02, mounted with Tempfix on copper foils, and sputtered with gold-palladium by using a Polaron high-resolution sputterer producing a layerca. 3 to 5 nm thick. The thickness of the metal coating was determined by reembedding samples and examining ultrathin sections with atransmission electron microscope. The grain size of the gold-palladium sputter coating was determined to be about 2 to 3 nm, and as theresolution of the FESEM is about 2 to 3 nm, this results in an end resolution of about 4 to 5 nm under optimal conditions. All scanning electronmicrographs were taken with a Hitachi S-800 field emission scanning electron microscope. (B) Transmission electron micrograph of an

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ultrathin section of E. coli pop2135(pAW13) immediately after E-induced lysis. Fixation, postfixation, and dehydration of cells were asdescribed for panel A. Cells were then infiltrated and embedded in Spurr low-viscosity resin (21), polymerized, cut with a diamond knife ona Reichert-Jung Ultracut E Ultramicrotome, and mounted on uncoated copper grids. The sections were poststained with aqueous lead citrate(3%, pH 13). All transmission electron micrographs were taken with an Elmiscop 101 (Siemens) electron microscope. Cytoplasmic materialis shown being released. The fusion between the inner and outer membranes is indicated by an arrow. (C) High-resolution FESEM of E. colipop2135(pAW13) after E-induced lysis, showing empty bacterial ghosts. The preferential locations of the lysis tunnels near the division zone(open arrow) and the poles (closed arrow) are indicated. (D) High-resolution FESEM of an E-lysed cell showing the transmembrane lysistunnel.

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FIG. 1-Continued.

constitutes the hole. However, at present we have noevidence that the walls of the observed holes are built up byE oligomers. It is also possible that E oligomers initially forma relatively small hole and that the observed holes are theresult of a secondary effect: the limited blowup of the cellenvelope at the sites at which the primary E oligomericcomplex has been formed.

How many E monomers are required to build the E-specific lysis complex? It has been estimated that less than100 E molecules are necessary for lysis (18). This suggestionis in agreement with the observed low expression rate ofgene E (6). Therefore, it seems likely that a relatively smallnumber of E molecules are sufficient for formation of theE-lysis complex.

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U

FIG. 1-Continued.

Protein E-mediated lysis seems to require host proteins. Ahost gene, slyD, absolutely required for lysis, has beenmapped at 72.5 min on the E. coli chromosome (18). Thelytic action of gene E is blocked in a slyD mutant strain.Thus, the slyD gene product may define a cellular target forprotein E in the cell envelope. Furthermore, several lines ofevidence suggest that E lysis somehow requires components

of the cell division machinery. Stationary cells cannot belysed by protein E (5). In the present electron microscopicstudy the hole was predominantly located in the neighbor-hood of the septum or at the poles (Fig. 1C), implyingformation of the lysis complex at the sites at which celldivision takes place (20). It has been reported that heatshock proteins known to interfere with the regulation of cell

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division also interfere with protein E-mediated lysis (24).Under conditions when heat shock genes were expressed at30°C, E. coli showed an enhanced resistance to E-mediatedlysis (24). Apparently, the interrelation between cell divisionand E-mediated lysis needs further elucidation.

This work was supported by a grant from the Austrian Fonds zurFoerderung der wissenschaftlichen Forschung (P6861 Bio).

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