Type VI secretion requires a dynamic
contractile phage tail-like structure
M. Basler1*, M. Pilhofer2,3*, G. P. Henderson2, G. J. Jensen2,3& J. J. Mekalanos1
related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of
Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally
homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the
type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell
electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or
contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support
a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate
proteins out of effector cells and into adjacent target cells.
Secretion systems allowbacteria totransport macromolecules such as
proteins out of effector cells or into either target host cells during
pathogenesis or target bacterial cells during competition in various
ecological settings. The type 6 secretion systems (T6SS) are encoded
byaclusterof 15–20genesthatispresent inatleast onecopyinabout
25% of all sequenced Gram-negative bacteria. Although linked to
virulence during host infection, recent studies showed that T6SS of
Pseudomonas, Burkholderia and Vibrio species can kill bacterial as
thought to kill target cells through translocation of toxic effector
proteins in a cell–cell contact-dependent process1–3,8. Little is known,
however, about how T6SS transport toxic proteins through their own
cell membranes or across target cell membranes.
T6SS components are structural homologues of components present
in contractile phage tails. For example, secreted VgrG proteins are
Another highly conserved T6SS gene product is predicted to be a
homologue of gp25, a major component of the T4 phage tail base-
plate10,12,13. Two T6SS gene products of V. cholerae, VipA and VipB,
form tubular structures that can be depolymerized by another T6SS
visually resemble T4 contracted tail sheath and were the first to
propose that a sheath-like structure might power T6SS translocation
by a phage tail-like contraction mechanism. Here we show that
T6SS-dependent secretion of Hcp and killing of Escherichia coli by
V. cholerae correlates with the activity of a dynamic intracellular
structure that indeed appears structurally and functionally related
to contractile phage tail sheath.
Fluorescence microscopic imaging of the T6SS
constructed a carboxy (C)-terminal fusion of VipA protein with super-
folder green fluorescent protein (sfGFP)17. As shown in Supplementary
Fig. 1a, VipA–sfGFP complements a chromosomal in-frame deletion
of vipA for Hcp secretion when the fusion protein is expressed from
microscopy revealed that the VipA–sfGFP fusion is associated with
and length of the cell. The number of visible structures in a single cell
Critically, these structures were not visible in vipB mutant cells (Sup-
of VipA with VipB10. Because VipA is not secreted and resides within
cellular fractions (Supplementary Fig. 3) and the expression level of
VipA–sfGFP was comparable to VipA under the conditions used to
visualize these sheath structures (Supplementary Fig. 4), we conclude
that the fluorescent structures were within the cytosol of imaged cells.
Time-lapse imaging revealed these putative sheath structures to be
different subcellular locations and then contracted and disassembled
also within tens of seconds. Most of the extended sheath structures
visible in cells stretched from one lateral side of the cell to the other,
perpendicular to the membrane, and thus had lengths approximately
equal to the width of the cell (about 0.75–1mm). As shown in Fig. 1a,
these sheaths assembled at speeds of approximately 20–30smm21.
Contraction was very fast, occurring in approximately 5ms or less
(unresolvable at frame rates of approximately 200 frames per second;
see Fig. 1b, c, Supplementary Fig. 5 and Supplementary Video 3).
Sheaths contracted to about 50% of their extended length (Sup-
plementary Fig. 6), and then disassembled over the next 30–60 s
(Fig. 1a). The disassembly of the contracted sheath is most probably
a ClpV-dependent event because ClpV is known to disassemble
do not disassemble VipA–sfGFP-labelled contracted sheaths (Sup-
plementary Fig. 2d and Supplementary Video 8). A similar number
of VipA–sfGFP-labelled sheaths were seen in strains 2740-80 and
V52 and at various levels of VipA–sfGFP expression (Supplementary
1Departmentof Microbiology and Immunobiology, Harvard MedicalSchool, 200 Longwood Avenue,Boston, Massachusetts02115, USA.2Division of Biology,California Institute of Technology,1200 East
California Boulevard, Pasadena, California 91125, USA.3Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.
*These authors contributed equally to this work.
1 8 2 | N A T U R E | V O L 4 8 3 | 8 M A R C H 2 0 1 2
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Bacterial strains. V. cholerae 2740-80 is a non-toxinogenic El Tor strain isolated
in 1980 from a patient in Florida, USA25. A streptomycin resistant, lacZ2deriv-
ative of 2740-80 was used as the wild-type parental strain. E. coli DH10b and
Sm10 lpir were used for cloning and conjugation, respectively. Gentamicin-
resistant E. coli MG1655 strain was used in bacterial killing assays. V. cholerae
V52 and its deletion variants were described previously7. Antibiotic concen-
trations used were streptomycin (100mgml21), gentamicin (15mgml21) and
carbenicillin (100mgml21). Luria–Bertani (LB) broth was used for all growth
conditions. Liquid cultures were grown aerobically at 37uC.
VCA0109, the corresponding surrounding DNA was amplified by overlap exten-
sion PCR and cloned into pWM91 (ref. 35) for subsequent sacB-mediated allelic
the replacement the entireopen readingframe,with the exceptionof first andlast
seven codons. Gene deletion was confirmed by PCR with primers outside of the
had been fused with either sfGFP or mCherry2 genes (both separated by a DNA
linker encoding 33Ala 33Gly), were cloned into plasmid pBAD24 (ref. 27). All
cloning products were sequence-verified.
Bacterial killing assay. V. cholerae 2740-80 strains and E. coli MG1655 strain
were incubated for 14–18h at 37uC in LB, then washed in fresh LB and diluted
V. cholerae and E. coli. V. cholerae was mixed with E. coli in a 10:1 ratio and 10ml
of the mixture was spotted on a dry LB agar plate. After 2 and 4h, bacterial spots
was serially diluted in LB and 5ml of the suspensions was spotted on selective
plates (gentamicin for E. coli, streptomycin 100mgml21V. cholerae). Colonies
were detected after incubation for approximately 16h at 30uC.
Cell fractionation and immunoblot analysis. Cellsfromovernight cultures were
washed with fresh LB and diluted 1:100 in 1.5ml of fresh LB (supplemented with
appropriate antibiotics and arabinose to indicated concentrations), cultivated for
acidfor1h onice. Precipitatedproteinswere collected bycentrifugationfor15min
at 21,000g, washedwith100%acetone and re-suspended in60ml SDS–PAGE load-
ing buffer. Twenty microlitres was loaded on an SDS–PAGE for western blot ana-
lysis. Cell and supernatant protein samples were boiled for 5min, separated by 10–
containing Tween 0.05% (TBST), incubated withprimary peptide antibody for 2h,
washed with TBST, incubated for 1h with horseradish peroxidase labelled anti-
rabbit antibody (Jackson Lab) and washed with TBST; peroxidase was detected by
SuperSignal West Pico Chemiluminescent Substrate (Pierce).
and then shaken at 37uC for 2.5–3.0h to reach D60051.0–1.5. Cells were cooled
on ice, centrifuged for 10min at 7000g and lysed in 12ml lysis buffer (150mM
NaCl, 50mM Tris, pH 7.4, lysozyme 200mgml21, DNase I 50mgml21, 1mM
phenylmethylsulphonyl fluoride, 0.53 CelLytic B (Sigma), 1% Triton X-100).
Cell lysis was complete after incubation for 5–10 min at 37uC. After cell lysis,
samples were cooled on ice and intact cells and cell debris were removed by
centrifugation for 15min at 15,000g. Clearedlysates were subjected to ultraspeed
centrifugation at 150,000g for 1h at 4uC. Pellets were re-suspended in 0.5ml of
Complete Mini (Roche) and stored at 4uC or 220uC for electron microscopy
Preparation of sheath for mass spectrometry analysis. Sheath for mass spec-
trometry analysis was prepared from an flgG in-frame deletion mutant of the
parental 2740-80 strain. Cells were prepared and lysed as described above. To
separate the sheath from soluble proteins, the pellet obtained by ultracentrifuga-
and insoluble material removed by a 2min 15,000g centrifugation step. The
sheath was then collected by sequential ultracentrifugation at 150,000g for 1h.
The sheath pellet was again re-suspended in 12ml TN buffer and subjected to
another ultracentrifugation step. After three successive ultracentrifugations,
samples typically showed only two major bands on a 10–20% SDS–PAGE. The
two detectable bands (20 and 50kDa), and the areas above and below the bands,
were cut out from the gel and analysed by tandem massspectrometry for peptide
identity (Taplin Biological Mass Spectrometry Facility, Harvard).
Peptide-specific antibodies. Antigen-purified rabbit polyclonal antibodies
raised against an Hcp peptide (QSGQPSGQRVHKPF) and VipA peptide
(MSKEGSVAPKERIN) were obtained commercially (GenScript). Specificity of
the antibodies was tested on V. cholerae V52 strains expressing or lacking Hcp
protein, or V. cholerae 2740-80 strains expressing or lacking VipA.
Fluorescence microscopy. Overnight cultures of V. cholerae 2740-80 or V52
strains carrying plasmid pBAD24-VipA–sfGFP or pBAD24-VipA-mCherry2
were diluted 1:100 into fresh LB supplemented with carbenicillin and arabinose
(concentrations 0.01%, 0.003% or as indicated) and cultivated for 2.5–3h to an
phosphate buffered saline (PBS), spotted on a thin pad of 1% agarose in PBS,
covered with a cover slip and immediately imaged at room temperature.
Fluorescence and phase contrast micrographs were captured using a Nikon
TE2000 inverted microscope outfitted with a Nikon Intensilight illuminator, a
Apo DM 100 objective lens (1.4 numerical aperture). The sfGFP images were
taken by using the ET-mCherryfilter set (Chroma49008). Images were captured
using Nikon Elements software. Images were collected every 6 or 10s, using an
exposure time of 100–600ms for fluorescence and about 10–20ms for phase
contrast. Phase contrast imaging was used to refocus automatically between
individual time points. Contrast on images for phase and fluorescence channels
was adjusted identically for compared image sets and merged using ImageJ 1.45
software (http://rsb.info.nih.gov/ij/). Small movement of whole field in time was
corrected by registering individual frames using StackReg plugin for ImageJ
(‘Rigid Body’ transformation). The pixel-size was 60nm.
High-frame-rate fluorescent images were collectedwith a Nikon Ti-E inverted
the Perfect Focus System for continuous maintenance of focus. VipA–sfGFP
fluorescence was excited using a Prior Lumen200Pro metal halide epi-fluor-
escence light source, selected with an ET490/203 filter (Chroma) and collected
with an ET535/30m filter (Chroma). Two different cameras and acquisition
settings were used to collect images. A Hamamatsu ORCA-R2 cooled CCD
camera was used to acquire images every 118ms (exposure time 50ms, with
continuous illumination). A Hamamatsu ORCA-Flash2.8 cooled CMOS camera
was used to acquire images every 20ms (no analogue gain) or 5ms (83 on-chip
analogue gain) under continuous illumination light. Both cameras were con-
trolled with Molecular Devices MetaMorph version 7.7 software. Contrast was
adjusted identically for compared image sets. All image processing and analyses
and 78nm for the ORCA-Flash2.8 camera.
Plunge-freezing. For ECT, V. cholerae 2740-80 wild-type and mutant strains
were grown aerobically at 37uC in LB medium. A 5ml overnight-culture was
diluted 1000-fold and grown to D60051.5–2.2. Copper/rhodium electron micro-
scopy grids (R2/2, Quantifoil) were glow-discharged for 1min. A 203-concen-
trated bovine serum albumin-treated solution of 10nm colloidal gold (Sigma)
was added to the sample (1:4 v/v) immediately before plunge freezing. A 4ml
droplet of the mixture was applied to the electron microscopy grid, then auto-
a Vitrobot (FEI Company)36. The grids were stored in liquid nitrogen.
Negative stain electron microscopy. Samples were incubated on carbon-coated
grids for about 1min. Grids were washed in water and stained by 1% uranyl
formate. The grids were examined in a JEOL 1200EX transmission electron
microscope and images were recorded with an AMT 2k CCD camera.
Electron cryotomography. Tilt series were collected using a Polara 300 kV FEG
transmission electron microscope (FEI Company) equipped with an energy filter
CCD represented 0.95nm (322,500) or 0.63nm (334,000) at the specimen level.
Typically, tilt series were recorded from 260u to 160u with an increment of 1u at
(for whole cells) or 80–100 electronsA˚22(for sheath preparations). Leginon29or
UCSF Tomo30was used for automatic tilt-series acquisition. Three-dimensional
reconstructions were calculated using the IMOD software package31or Raptor32.
Sub-tomogram averaging. IMOD31was used to model the centre of the sheath.
The program addModPts was run to fill in model points every 8nm along the tube
axis. The PEET software package33was used to align and average repeating sub-
35. Metcalf, W. W. et al. Conditionally replicative and conjugative plasmids carrying
lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. Plasmid
35, 1–13 (1996).
36. Iancu, C. V.etal.Electron cryotomographysample preparationusing the Vitrobot.
Nature Protocols 1, 2813–2819 (2006).
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