Blackwell Publishing LtdOxford, UKMMIMolecular Microbiology0950-382X© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd? 2006604894906Original ArticleHelical arrangement of the protein translocaseD. Shiomi, M.
Yoshimoto, M. Homma and I. Kawagishi

Molecular Microbiology (2006) 60(4), 894–906

doi:10.1111/j.1365-2958.2006.05145.x
First published online 21 April 2006

Helical distribution of the bacterial chemoreceptor
via colocalization with the Sec protein translocation
machinery
OnlineOpen: This article is available free online at www.blackwell-synergy.com

Daisuke Shiomi,1† Masayuki Yoshimoto,1Michio Homma1 and Ikuro Kawagishi1,2*
1
Division of Biological Science, Graduate School of
Science, and 2Institute for Advanced Research, Nagoya
University, Chikusa-ku, Nagoya 464-8602, Japan.
Summary
In Escherichia coli, chemoreceptor clustering at a cellpole seems critical for signal amplification and adaptation. However, little is known about the mechanismof localization itself. Here we examined whether the
aspartate chemoreceptor (Tar) is inserted directly into
the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar–GFP,
fluorescent spots first appeared in lateral membraneregions, and later cell poles became predominantly
fluorescent. Unexpectedly, Tar–GFP showed a helicalarrangement in lateral regions, which was more
apparent when a Tar–GFP derivative with two cysteine
residues in the periplasmic domain was cross-linked
to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domainof the double cysteine Tar–GFP mutant was replacedby that of the kinase EnvZ, which does not localize toa pole. Observation of GFP–SecE and a translocationdefective MalE–GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested thatthe general protein translocation machinery (Sec)itself is arranged into a helical array, with which Tar istransiently associated. The Sec coil appeared distinct
from the MreB coil, an actin-like cytoskeleton. Thesefindings will shed new light on the mechanisms
underlying spatial organization of membrane proteinsin E. coli.
Introduction
The proteins involved in many biological systems are apt
Accepted 23 February, 2006. *For correspondence. E-mailFax
i45406a@cc.nagoya-u.ac.jp;
Tel.
(+81) 52 789 2993;(+81) 52 789 3001. †Present address: Microbiology and Molecular
Genetics, University of Texas Medical School, 6431 Fannin Street,
Houston, TX 77030, USA.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

to be spatially organized within a cell or even within asmaller compartment rather than to be freely diffusible
and encountering each other stochastically. Proper spatialorganization of proteins is of vital importance not only ineukaryotes but also in prokaryotes despite their smaller
sizes. Bacterial cellular functions that involve localizationof proteins include virulence, cell division and chemotaxis.
For example, IcsA of Shigella flexneri and ActA of Listeria
monocytogenes, which nucleate actin polymerization andare required for the bacterial movement within a host celland hence virulence, localize to an old pole of the rodshaped bacterial cell (Goldberg et al., 1993; Smith et al.,1995). CpaE and CpaC, which are required for polar pilus
biogenesis in Caulobactor crescentus, localize to one poleand their localization is controlled by PodJ (Viollier et al.,2002). In Escherichia coli, the best characterized bacterial
species, many proteins have been shown to localize (forreviews, see Nanninga, 1998; Lybarger and Maddock,
2001; Shapiro et al., 2002; Margolin, 2003). However, little
is known about mechanisms underlying protein localization. For example, the cell division machinery of E. coli
has to localize to the mid cell position and this localizationis regulated by other proteins (the Min system) (Bi and
Lutkenhaus, 1991; Akerlund et al., 1992), but it remains
to be elucidated whether the localization results directlyfrom interactions with other protein(s), including thosebetween the Min proteins and the division machinery,
peptidoglycan (cell wall) or phospholipids. Experimental
approaches to examine the mechanisms of their localization are limited, because most of these components are
essential for cellular viability and/or morphology.
The chemotactic signalling system of E. coli may beamenable to studying mechanisms underlying spatialorganization of proteins because the lack of any chemotactic signalling component protein does not affect cellmorphology and viability at least under typical laboratory
conditions. Moreover, all of the components (Che proteins
and the chemoreceptors) have been identified and wellcharacterized in terms of genetics, biochemistry andstructural biology (for reviews, see Parkinson, 1993; Stockand Surette, 1996; Djordjevic and Stock, 1998; Armitage,
1999; Falke and Kim, 2000). The chemoreceptors (alsoknown as transducers or methyl-accepting chemotaxis

Helical arrangement of the protein translocase 895
proteins (MCPs)) form clusters with the histidine kinase
CheA and the adaptor CheW at a cell pole and many other
Che proteins target to this polar receptor-kinase cluster(Maddock and Shapiro, 1993; Sourjik and Berg, 2000;
Shiomi et al., 2002; Cantwell et al., 2003; Banno et al.,2004). Polar localization and clustering have been thought
to be critical for signal amplification (Bray et al., 1998;
Shimizu et al., 2000; Ames et al., 2002; Gestwicki andKiessling, 2002; Kim et al., 2002; Homma et al., 2004)
and adaptation (Li and Weis, 2000; Barnakov et al., 2001;
2002; Levit and Stock, 2002; Shiomi et al., 2002; Bannoet al., 2004).
It has been shown that polar localization of thechemoreceptors depends partially on CheA and CheW
(Maddock and Shapiro, 1993; Skidmore et al., 2000;
Shiomi et al., 2005). However, it is not known how thechemoreceptor localizes to a cell pole. To target to a pole,a nascent chemoreceptor protein might be inserted into
the cytoplasmic membrane (i) at or near a cell pole
thereby staying in the vicinity of the insertion point (thedirect membrane insertion model) or (ii) at random positions thereafter migrating or diffusing away from theinsertion point (the indirect membrane insertion model)(Fig. 1A). Both of the mechanisms have been shown tooperate in other systems: (i) IcsA of S. flexneri employs adirect mechanism when targeting directly to an old polein E. coli, which does not have an IcsA homologue(Charles et al., 2001) and (ii) SpoIVFB of Bacillus subtilis
utilizes an indirect mechanism, being randomly inserted
into the cytoplasmic membrane and then diffused to andcaptured in the outer forespore membrane (Rudner et al.,2002).
To elucidate the localization mechanism of thechemoreceptor, it is indispensable to identify the site(s) ofmembrane insertion. It has been reported that the serine
chemoreceptor Tsr of E. coli is inserted into the cytoplasmic membrane via a SecA-dependent process (Gebert
et al., 1988). SecA is an ATPase that associates with the
integral membrane proteins, SecY, SecE and SecG, toconstitute a general protein translocase (for reviews,
see Driessen et al., 1998; Mori and Ito, 2001). Recent
observation of SecY–GFP (green fluorescent protein)suggested that the Sec machinery is evenly distributed
throughout the cytoplasmic membrane in E. coli (Brandon

CA
Indirect

IM OM

Direct

60 s

20

B

10 s

40

60

80

100

HCB436 (AW+)/
Tar-GFPHCB436 (AW+)/TE-GFP
HCB437 (AW-)/
Tar-GFP
Fig. 1. Polar localization of Tar–GFP.
A. Direct and indirect membrane insertion models for polar localization of the chemoreceptors. To target to a cell pole, a nascent chemoreceptor
protein might be inserted into the cytoplasmic membrane (i) at or near a cell pole (a direct model), or (ii) at random positions thereafter migrating
or diffusing away from the insertion point (an indirect model). IM, the cytoplasmic (inner) membrane; OM, the outer membrane.
B. Time-course of polar localization of Tar–GFP. HCB436 (CheAW+) or HCB437 (CheAW–) cells carrying a plasmid encoding Tar–GFP or Taz1–
GFP were observed at indicated time points after the addition of 1 mM arabinose. Numbers above pictures represent harvested time points (min).
C. Time-lapse observation of polar localization of Tar–GFP. HCB436 cells carrying plasmid encoding Tar–GFP were spotted onto a glass slide
covered with 0.5% agarose. Fluorescence was observed with 10 or 60 s intervals.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

896 D. Shiomi, M. Yoshimoto, M. Homma and I. Kawagishi
et al., 2003; Espeli et al., 2003). It has recently beenreported that the Sec translocases of some Gram-positivebacteria show characteristic localization: to a microdomain in Streptococcus pyogenes (Rosch and Caparon,
2004) and to a helical array in B. subtilis (Campo et al.,2004). Thus without any direct evidence it is hard to judge
which model can apply to the chemoreceptor localizationin E. coli: (i) a subset of Sec translocases at a pole might
be devoted to insert the chemoreceptors (and possibly
other polar membrane proteins) into the membrane, or (ii)the chemoreceptors might be inserted into the membrane
at virtually any position throughout a cell.
Here we report that the aspartate chemoreceptor Tar
employs an indirect mechanism for its polar localization:i.e. after induction, Tar–GFP first appeared in lateral membrane regions, clustered in the presence of CheA and
CheW, and then seemed to migrate towards a cell pole.
Unexpectedly, Tar–GFP formed a helical array when it islocated in lateral membrane regions. Further analyses,
including observation of GFP–SecE and the mutant versions of MalE–GFP, as well as indirect immunofluorescent
microscopy on SecG, suggested that this Tar coil reflects
a helical array of the Sec machinery itself, which wasshown to be distinct from that of the actin homologue MreB.
Results
The aspartate chemoreceptor Tar is not directly inserted
into polar membranes
To visualize the localization pathway of a chemoreceptor,
GFP was fused with the C-terminus of the aspartate
chemoreceptor Tar (Homma et al., 2004), and localizationof the resulting fusion (named Tar–GFP) was observed
sequentially after induction. For tight regulation of expression, the tar–gfp gene was placed downstream of thearaBAD promoter (pBAD24-Tar–GFP). Cells expressing
Tar–GFP swarmed only slightly slower than thoseexpressing wild-type Tar (Shiomi et al., 2005), indicatingthat the Tar–GFP retains essential receptor function. A
chimeric protein named Taz1–GFP that does not localize
to a pole was used as a negative control (pBAD24-Taz1–
GFP): Taz1 was constructed by replacing the cytoplasmic
domain of Tar, which is required for interaction with CheA
and CheW and formation of a predicted trimer of the Tardimers, with the cytoplasmic kinase and phosphatasedomains of the osmosensor histidine kinase EnvZ (details
will be described elsewhere). It was previously reported
that the kinase and phosphatase activities of Taz1 (Utsumi
et al., 1989), which was not fused with GFP, are modulated by the Tar-specific ligand aspartate (Utsumi et al.,
1989; Yang et al., 1993). We observed that Taz1–GFP is
distributed almost evenly throughout the cytoplasmic
membrane (Figs 1B and 2F). HCB436 (CheAW+) cells
carrying the plasmid encoding Tar–GFP or Taz1–GFP

were grown in TG medium. After 3 h, 1 mM arabinose wasadded to express the GFP fusion proteins (time 0). Cells
were harvested at indicated time points (Fig. 1B). At
20 min after induction, in most cells both Tar–GFP and
Taz1–GFP appeared in lateral cytoplasmic membraneregions, where the fusion proteins formed small clusters,
whereas in the other cells we hardly detected any fluorescence from Tar–GFP. At 60 min, subpopulations of Tar–
GFP formed clusters at apparently random positions in
the cytoplasmic membrane and others localized to cell
poles. At 80 min, Tar–GFP localized almost completely tocell poles, whereas Taz1–GFP was almost evenly distributed in the cytoplasmic membrane, an expected result as
it lacks the highly conserved domain critical for polar localization (D. Shiomi and I. Kawagishi, unpubl. results). It
should be noted that the expression levels of Tar–GFP
was comparable with chromosome-encoded Tar at 60 min
after the addition of 1 mM arabinose. In the absence of
CheA and CheW, Tar–GFP also formed clusters at apparently random positions (see Results at 20 and 40 min) andeventually localized to cell poles to a lesser extent(100 min) than in the presence of CheA and CheW. Under
our experimental conditions, cell-to-cell variations in thelocalization patterns of Tar–GFP at each time point except
for at 20 min were only limited (data not shown). These
results are consistent with an indirect model, in which theproper polar localization of Tar is achieved by random
insertions into the cytoplasmic membrane followed by the
association with CheA and CheW and/or interaction witheach other through the highly conserved domain among
MCPs (HCD) and the diffusion or transport of the resulting
clusters to cell poles.
To further examine this possibility, we observed theprocesses of polar localization of Tar–GFP in a single cell.
An hour after the addition of 1 mM arabinose, cells were
washed and resuspended in TG medium supplemented
with 25 µg ml−1 rifampicin to prevent further transcription.
Cells were further incubated at 30°C for 10 min and thenobserved by fluorescence microscopy with 1, 10 or 60 s
intervals. Time-lapse images of a representative cell are
shown in Fig. 1C. Tar–GFP formed clusters at random
positions of the cytoplasmic membrane. At a cell pole, afluorescent cluster was detected after the addition ofrifampicin. Again this observation is consistent with thenotion that Tar–GFP clusters at lateral membrane regions
and then migrates towards a cell pole. However, we havenever seen a cluster travelling from the lateral site to thepole, but this cannot argue against the net migration to
the pole: it would be rather hard to imagine that a migrating cluster stays in the same focal plane for a long period.
Anyhow, fluorescent spots did not appear to migrate
straightforward to the pole. They might move in a randomwalk fashion and be trapped when they reach the pole. It
should also be noted that this result may not exactly reflect© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

Helical arrangement of the protein translocase 897
native migration patterns of the chemoreceptor from lateral membrane regions to a pole because cells wereattached to a 0.5% agarose layer on a slide glass andtherefore nutrients and oxygen must be limited.
Tar–GFP shows a helical arrangement
Careful observation of Tar–GFP clusters at lateral cytoplasmic membrane regions (Fig. 1B and C) led us to suspect that Tar could be organized into a coil-like array inthe lateral membrane. The two-dimensional images oflateral clusters of Tar–GFP are reminiscent of those of thebacterial actin homologue MreB that forms a helical structure along the long axis of a cell (Jones et al., 2001; Shihet al., 2003). We therefore wanted to know the threeA

1

2

4

5

dimensional arrangement of Tar in lateral cytoplasmic
membrane regions. Optical dissection of single cells(more than 30 cells for each condition) was carried out
and the resulting series of fluorescent images were
processed by using a deconvolution software to remove
out-of-focus fluorescence and to reconstitute a threedimensional fluorescent image. First, we examined Tar–
GFP in the presence of CheA and CheW (i.e. in strain
HCB436) at 60 min after the addition of the inducer arabinose (Fig. 2A–C). All of the cells checked showed coilor ring-like fluorescence patterns along their long axes.
Similar helical fluorescence patterns were detected evenwhen Tar–GFP was expressed in the absence of CheA
and CheW (i.e. in strain HCB437) (Fig. 2D and E). In
contrast, Taz1–GFP did not show any characteristic pat-

3

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C

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G

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F

Fig. 2. Helical arrays of the chemotaxis machinery in a cell. Cells were subjected to optical sectioning and processing as described inExperimental procedures. GFP-tagged proteins were induced by 1 mM arabinose for 60 min. Three-dimensionally reconstructed images fromdifferent angles (1, 2 and 3) are shown. Each image was rotated around the long axis of the cell to display the helical array. Unprocessed images
are also shown (4,5,6).
A–C. Tar–GFP in HCB436 (CheAW+) cells.
D and E. Tar–GFP in HCB437 (CheAW–) cells.
F. Taz1–GFP in an HCB436 (CheAW+) cell.
G. GFP–CheA in an HCB437 cell expressing Tar and CheW. Scale bars indicate 1 µm.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

898 D. Shiomi, M. Yoshimoto, M. Homma and I. Kawagishi
tern in either host strain (Fig. 2F). We previously observedthat GFP–CheA forms clusters at a cell pole and lateralcytoplasmic membrane regions when Tar was overexpressed (D. Shiomi and I. Kawagishi, unpubl. results).
Under the same condition, we obtained reconstituted
three-dimensional images of GFP–CheA and detected
coiled fluorescence patterns (Fig. 2G). These results suggest that Tar and CheA are organized into a helical arrayin lateral membrane regions. The formation of such anarray might involve the HCD-mediated interactions of Tar–
GFP with neighbouring Tar–GFP (between dimers) and/or CheA.
We now hypothesized two possibilities about the natureof this helical arrangement of Tar–GFP: (i) Tar might
migrate along a helical track within lateral regions of thecytoplasmic membrane; or (ii) the protein translocation
machinery that inserts Tar into the cytoplasmic membranemight be arranged into a helical array. Whichever the case,introduction of Cys residues in the periplasmic region ofTar, which is devoid of Cys residue, might result in a better
helical image if disulphide cross-linking leads to higher
oligomers and occurs immediately after the translocation.
Introduction of a Cys residue at an appropriate periplasmic position (e.g. S36C) of the subunit interface within adimer results in a disulphide bond between subunits.
When another Cys residue is introduced at the externalsurface of the dimer (e.g. D142C), the double Cys mutant
would form cross-linked oligomers (Fig. 3A). This has
been demonstrated for Tar-S36C&D142C (Homma et al.,2004). Among the double Cys mutant proteins tested, TarS36C&A118C was most efficiently cross-linked (H. Irieda,
M. Homma, M. Homma and I. Kawagishi, submitted for
publication). Immunoblotting analyses of the GFP fusionof the double Cys mutant showed that it is indeed highly
cross-linked leaving only a very small amount of theuncross-linked monomer even without the addition of any
catalyst for oxidation (Fig. 3B; note that highly cross-linked
products could not get into the gel). In HCB436 cells,
Tar-S36C&A118C–GFP showed clearer coiled structures(Fig. 3C).
We
also
constructedandexamined Taz1S36C&A118C–GFP, which lacks HCD, as a negative control. Unexpectedly, however, it also formed a helical array(Fig. 3D), indicating that HCD is dispensable for the helical array. A possible explanation of this phenomenon
is that Taz1-S36C&A118C–GFP might be cross-linked
immediately after inserted into the cytoplasmic membrane
thereby trapped in the vicinity of the sites of insertion. It
was previously shown that the serine chemoreceptor Tsr
is inserted into the membrane via the general proteintranslocation apparatus (the Sec machinery) (Gebert
et al., 1988). We suspect that cross-linking immediately
after the insertion would prevent the mutant protein fromdiffusing away from the Sec machinery, resulting in better

helical images. If cross-linking was extremely efficient, anewly inserted Cys derivative of Tar–GFP or Taz1–GFP
might be cross-linked with the neighbouring one, which
would clog the Sec machinery. If this is the case, overproduction of these proteins would affect cell growth. As
expected, the A118C derivative of Tar–GFP or Taz1–GFP
retarded cell growth even when the expression level was
adjusted roughly equal to the total amount of chromosome-encoded chemoreceptors, whereas the corresponding wild-type versions did not show any significant
effect (data not shown). Therefore, we speculate that thehelical arrangements of Tar–GFP and Taz1–GFP within acell may reflect that of the Sec machinery itself.
Cellular organization of the Sec machinery
The Sec machinery contains, as essential components,
three integral membrane proteins, SecY, SecE and SecG
(the SecYEG complex) that forms a translocation pore,
and a membrane-peripheral ATPase, SecA. To examine
subcellular localization of the Sec machinery, we constructed GFP–SecE, which was expressed in strain
HCB436 and three-dimensional images were reconstituted (Fig. 4A–C). GFP–SecE was also organized into ahelical array. This is inconsistent with recent publications
reporting that SecY- and SecE–GFP fusion proteins areevenly distributed throughout the cytoplasmic membranein E. coli (Brandon et al., 2003; Espeli et al., 2003). This
discrepancy could be accounted for by levels of the Sec–GFP fusion proteins: their even distribution might result
from the failure of excess Sec–GFP proteins to form a
SecYEG complex with their partner Sec proteins as we
also observed almost even distribution of GFP–SecE
when it was mildly overexpressed (Fig. 4D and E).
We also wanted to observe subcellular localization of
the Sec machinery without GFP fusion and at the wildtype stoichiometry. First, we employed indirect immunofluorescence microscopy (IFM) to see localization of SecG,
which is other component of the Sec machinery. Immunoblotting of wild-type cells (W3110) with anti-SecG antibody
detected a major band of approximately 12 kDa, whichshould correspond to SecG, with low levels of crossreacting bands (Fig. 5A). Wild-type cells were then fixed,
treated with anti-SecG antibody and then with the second
antibody labelled with Alexa Fluor 488. In many cells, withsignificant levels of background, diagonal lines wereobserved (Fig. 5B). These lines are consistent with thehelical arrangement of SecG in a cell.
Second, we took advantage of export-defective mutations of maltose-binding protein (MBP or MalE) to observe
subcellular localization of the Sec machinery at the wildtype expression level. The mutant MalE proteins with
M18R or M19R substitution in the leader peptide
sequence are defective in translocation across the mem-

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

Helical arrangement of the protein translocase 899
Aperiplasm
IM
N
C

N
C

N
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N
C

cytoplasm
Cys-36+2ME

B

Tar-GFP

Cys-118-2ME

TE-GFP

Tar-GFP

S36C&
S36C&
A118C WT A118C WTTE-GFP

S36C&
S36C&
A118C WT A118C WTMonomer

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brane, but not in its initiation step, and their precursors arethought to remain trapped in the Sec machinery (Bassford
and Beckwith, 1979; Bedouelle et al., 1980). It has beenshown that wild-type MalE–GFP is exported to the periplasmic space successfully via the Sec machinery but thatthe protein is not fluorescent presumably due to misfolding(Feilmeier et al., 2000). We therefore reasoned that fluorescence of the M18R or M19R derivative of MalE–GFP
would reflect cytoplasmic localization of the Sec machinery (Fig. 6A). Immunoblotting verified that expression lev-

Fig. 3. Helical arrays of disulphide-cross-linked
Tar–GFP and Taz1–GFP.
A. Schematic illustration of the inter-dimer
cross-linking strategy of Tar–GFP or Taz1–GFP.
Two cysteine residues were introduced into theperiplasmic domain of the Tar– or Taz1–GFP
protein to form higher oligomers with disulphidebonds linking subunits both within and between
dimers. IM, the cytoplasmic (inner) membrane;
N and C, the N- and C-termini; open andshaded circles, the introduced Cys residues(S36C and A118C).
B. In vivo disulphide cross-linking of the Cysreplaced Tar– and Taz1–GFP proteins. HCB436
cells (∆MCP ∆CheR ∆CheB) expressing thewild-type (WT) or mutant (S36C&A118C) versions of the fusion proteins (Tar–GFP and
Taz1–GFP) were incubated with (+) or without(–) 60 µM Cu(II)(o-phenanthroline)3 for 5 min at
30°C and their whole-cell extracts were subjected to non-reducing (−2ME) and reducing(+2ME) SDS-PAGE followed by immunoblottingwith anti-GFP (Molecular Probes).
C and D. Three-dimensionally reconstituted
images of HCB436 cells expressing the
S36C&A118C mutant versions of Tar–GFP (C)and Taz1–GFP (D). For each cell, reconstituted
images form different angles (1–3) and unprocessed images (4–6) are shown. Scale bars indicate 1 µm.

els of the wild-type and mutant versions of MalE–GFP
were comparable with each other and that the mutant
versions were not processed (Fig. 6B). Wild-type MalE–
GFP was not fluorescent (data not shown) whereas themutant versions of MalE–GFP formed clusters in the cytoplasmic membrane in strain HCB436 (see Fig. 6C–E). The
reconstituted three-dimensional images of MalE-M19R–
GFP clearly showed helical arrangements (Fig. 6C–E). In
this experiment, no component of the Sec machinery is
overproduced and therefore the result argues strongly that© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

900 D. Shiomi, M. Yoshimoto, M. Homma and I. Kawagishi
forms a helical filament both in B. subtilis (Jones et al.,2001) and E. coli (Shih et al., 2003). The finding of thehelical array of the Sec machinery raised a possibility
that it may be associated with the MreB coil. To address
this issue, we examined whether Tar–YFP colocalizes
with CFP–MreB (Fig. 7). In all of the cells checked, thecoils of CFP–MreB and Tar–YFP were not perfectly
overlapped with each other. The CFP–MreB coilseemed to have a smaller pitch than the Tar–YFP coil.
We therefore conclude that the Sec coil is distinct from
the MreB coil.

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Fig. 4. Helical arrangement of the GFP–SecE fusion protein. Imagesof more than 30 cells were analysed and representative cells areshown.
A–C. Three-dimensionally reconstituted images of HCB436 cells
expressing GFP–SecE. Cells were harvested 15 min after the addition of 1 mM arabinose and subjected to optical sectioning and processing as described in Experimental procedures. For each cell,reconstituted images form different angles (1–3) and unprocessed
images (4–6) are shown.
D and E. Overexpressed GFP–SecE in HCB436 cells. Cells were
harvested 30 min after the addition of 1 mM arabinose.

the Sec machinery is organized into a helical array. We
tried to examine whether MalE-M19R–CFP colocalizes
with YFP–SecE, but it was unsuccessful probably
because the expression level of the latter fusion was difficult to control (data not shown). Instead, we examined
colocalization of the coils of MalE and Tar (Fig. 6A and F).
CFP and YFP were fused to MalE-M19R and Tar respectively. MalE-M19R–CFP and Tar–YFP colocalized in thelateral membrane regions (Fig. 6F). We therefore conclude that the Sec machinery is organized into a coil-likestructure in a cell.
The Sec coil is distinct from the helical filament of MreB
It has recently been shown that E. coli has cytoskeletal
proteins that are homologues of actin (FtsA and MreB)
and tubulin (FtsZ) (van den Ent et al., 2001). FtsA plays
a key role in cytokinesis whereas MreB is critical for therod shape. More recently, it has been shown that MreB

Discussion
In this study, the pursuit of the mechanisms underlying
polar localization of the chemoreceptor Tar leads us to thefinding that the general protein translocation machinery(i.e. the SecYEG complex) is organized into a helicalarray. Sequential observations of cells expressing Tar–
GFP supported the indirect model for polar localization(Fig. 1A), in which Tar is inserted into lateral membraneregions and then migrates through a lipid bilayer to a cellpole. Further analyses of the Tar– and Taz1–GFP fusions

A
(kDa)
29
20
SecG
7.1

B

Fig. 5. Helical arrangement of SecG.
A. Detection of chromosome-encoded SecG by immunoblotting.
W3110 (wild-type) cells were subjected to immunoblotting with antiSecG antibody. Arrow head indicates bands of SecG. Open triangle
on the top indicates increasing amounts applied (from left to right,
one-, two- and fourfold).
B. Detection of chromosome-encoded SecG by IFM. W3110 (wildtype) cells were subjected to IFM with anti-SecG as the first antibody
and Alexa Fluor 488-labelled goat anti-rabbit IgG antibody. Typicalcells are shown.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

Helical arrangement of the protein translocase 901
A

MalE

periplasm
IM

B

CFP

GFP

MalE-GFP
WT

M18R

M19R

Sec machinery

cytoplasm
MalE* CFP

Tar

YFP
C

1

2

3
F

D

E

1

2

1

2

Tar-YFP
1

MalE-M19R-CFP
2
2'

Merge
3

3
4

5

5'

6

7

8

8'

9

3

Fig. 6. Helical arrangement of the translocation-defective mutant MalE–GFP protein.
A. Schematic illustration of double labelling of Tar and MalE. YFP and CFP/GFP were fused with the C-terminus of Tar and MalE to yield Tar–YFP and MalE–CFP/GFP respectively. Exported MalE–CFP was not fluorescent whereas the signal sequence mutant versions of MalE–CFP(M18R and M19R) were fluorescent probably because they remain clogged in the Sec machinery. MalE–CFP and MalE*–CFP represent wildtype and mutant versions of MalE–CFP respectively.
B. Expression of the mutant MalE–GFP proteins. HCB436 cells carrying a plasmid encoding GFP or the wild-type (WT) or mutant (M18R or
M19R) version of MalE–GFP were grown in the presence (+) or absence (–) of 1 mM arabinose and their whole-cell lysates were subjected to
SDS-PAGE followed by immunoblotting with anti-GFP.
C–E. Three-dimensionally reconstituted images of HCB436 cells expressing MalE-M19R–GFP. For each cell, reconstituted images form different
angles (1–3) are shown. Scale bars indicate 1 µm.
F. Tar–YFP and MalE-M19R–CFP in strain HCB436. Individual images of Tar–YFP (images 1, 4 and 7; artificially coloured green) and MalEM19R–CFP [original (2, 5 and 8) and processed (2′, 5′-and 8′) images; artificially coloured red] were merged (images 3, 6 and 9).

identified helical arrays of fluorescence, which werededuced to reflect a similar array of the Sec machinerythat inserts Tar into the membrane (Gebert et al., 1988).
The helical array of the Sec machinery was confirmed by
IFM observation of chromosome-encoded SecG and theobservation of GFP–SecE and the translocation-defective

mutant versions of MalE–GFP. Previous immunoelectron
microscopic analyses detected 80% of the cytoplasmic
membrane population of Tsr, the serine chemoreceptor,
at cell poles (Maddock and Shapiro, 1993). In light of ourfinding, a minor population of Tsr detected at lateral cytoplasmic membrane regions could represent nascent Tsr© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

902 D. Shiomi, M. Yoshimoto, M. Homma and I. Kawagishi

Tar-YFP

CFP-MreB

MergedFig. 7. Subcellular localization of Tar–YFP with CFP–MreB within thesame cells. Tar–YFP and CFP–MreB were expressed in strain
HCB436. Images of Tar–YFP (left) and CFP–MreB (middle) weremerged.

inserted via the Sec machinery into the cytoplasmic
membrane.
It has recently been reported that the Sec machinery isevenly distributed throughout the cytoplasmic membranein E. coli (Brandon et al., 2003; Espeli et al., 2003). However, this could be due to overexpression of Sec proteinsbecause GFP–SecE was evenly distributed throughout
the cytoplasmic membrane when mildly overexpressed(Fig. 4D and E). To visualize the localization of the Sec
machinery without violating wild-type cellular levels andstoichiometry of the components, we took advantage ofthe mutant versions (M18R and M19R) of MalE–GFP,
which are thought to be stuck in the Sec machinery(Bassford and Beckwith, 1979; Bedouelle et al., 1980).
Wild-type MalE–GFP is transported through the Sec
machinery but is not fluorescent probably because the
GFP part cannot be folded properly in the periplasmic
space (Feilmeier et al., 2000). In contrast, the mutant versions of MalE–GFP were fluorescent, suggesting thatthese proteins reside in the cytoplasm probably by associating with the Sec machinery. Fluorescence patterns of
the M18R (not shown) and M19R versions of MalE–GFP
further supported the notion that the Sec machinery isorganized into a helical array. We failed to detect significant effects of the mutant MalE–GFP protein on theprocessing of other exported proteins and cell growth.
Considering its fluorescence images, we gather that the
mutant MalE–GFP protein does not occupy all of thetranslocases. Recently, GFP fusions to SecA, SecY andthe precursor of a translocation substrate protein of a
Gram-positive bacterium B. subtilis have been reported to
localize with similar helical patterns (Campo et al., 2004),
raising a possibility that such a cellular organization is acommon feature of general protein translocases amongrod-shape bacteria.
The size and the subunit stoichiometry of the Sec
machinery have not been unambiguously determined.
Electron microscopy with negative staining suggested
that the Sec machinery may consist of dimeric SecA andfour SecYEG complexes (Manting et al., 2000). In contrast, cryo-electron microscopy suggested that the translocation pore consists of dimeric SecYEG complex(Breyton et al., 2002). Based on our fluorescence observation, we roughly estimated the average length of the
Sec coil to be ∼10 µm assuming that one coil is continuous from one pole to the other. Given the published value(10.5–12 nm) of the diameter of the SecYEG tetramer(Manting et al., 2000), the maximal number of SecYEG
tetramers that can be packed into a helical path would
then be 800–1000. Another line of evidence estimates thenumber of SecY, SecE or SecG monomers per cell to be∼500 (Matsuyama et al., 1992) and therefore the numberof SecYEG tetramers should be ∼125. Considering thatnegative staining does not reflect the actual size of aprotein and that SecYEG tetramers might not be packed
tightly within a helical path, our rough estimation (800–1000 tetramers per helix) may be in good agreement withthe predicted size of a cellular pool (∼125 tetramers percell).
How can the Sec machinery, a complex of integral
membrane proteins, be organized into a helical array? The
Sec machinery (i) might associate with some rigid helicalsupport, such as cytoskeletal protein(s) or peptidoglycan(cell wall), or (ii) might itself assemble into a coil. It has
been shown that an integral membrane protein subunit of
topoisomerase IV (SetB) interacts with MreB and forms ahelical array (Espeli et al., 2003). Although the Sec coil
was shown to be distinct from the MreB coil in this study
as well as in B. subtilis (Campo et al., 2004), it is still
possible that unknown cytoskeletal protein(s) serve toanchor the Sec machinery to form a helical array. It has
recently been demonstrated that morphology of cells lacking some penicillin-binding proteins, which are involved incell wall elongation, is abnormal (de Pedro et al., 2003)
and some penicillin-binding proteins localize with a spotlike pattern like MreB (den Blaauwen et al., 2003;
Scheffers et al., 2004), raising a possibility that it mightform a helical polymer and that E. coli might have cytoskeletal proteins other than the identified ones. Anothercandidate for a helical support may be peptidoglycan as
it is newly synthesized as a helix in B. subtilis (Daniel and
Errington, 2003). To our knowledge, there is no experimental evidence that the SecYEG complex forms oligomers higher than a tetramer. However, the native complex
within the membrane environment might have a property
distinct from detergent-solubilized preparations. Moreover, E. coli shows lateral heterogeneity of phospholipid
distribution in the cytoplasmic membrane and such heterogeneity is involved in cell division (Fishov and Woldringh,1999). Therefore, it is possible that such a certain kind of
membrane lipids might help the Sec machinery to form a
coil even without a rigid anchoring structure. In B. subtilis,a decrease in phosphatidylglycerol causes delocalization© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

Helical arrangement of the protein translocase 903

IM OM

of SecA (Campo et al., 2004). It would be intriguing to see
how GFP–SecE and MalE-M19R–GFP localize in roundshaped E. coli mutants (rodA or mreB) or mutants in membrane lipid synthesis.
Physiological significance of the helical array of the Sec
machinery is unclear. The helical arrangement of the Sec
machinery along the long axis of a cell might have someadvantage in distributing translocated proteins. If SecYEG
tetramers have to be anchored to some rigid support
rather than randomly distributed and freely diffusing, thena helical array would be one of the best solutions to
spread proteins efficiently throughout the cytoplasmic
membranes as well as the periplasmic space and theouter membrane (Fig. 8). Proteins that localize to certain
parts of the cell, such as a pole and a septum, might be
actively transported or passively diffused away towards
their destinations.
It is not clear whether a chemoreceptor is actively transported within a lipid bilayer to a cell pole or passively
diffused and somehow trapped when it reaches a pole. In
either case, the fact that both the Tar- and Taz1–GFP
fusions colocalized transiently with the Sec coil indicatesthat membrane proteins with different destinations mustbe ‘sorted’ after insertion by the Sec machinery. Suchsorting event(s) remain to be characterized but might provide a clue to understand the mechanisms of membraneprotein localizations as well as the functions of the Sec
machinery.
Experimental procedures
Bacterial strains and plasmids
All strains used in this study are derivative of E. coli K-12.
Strain RP437 is wild-type for chemotaxis (Parkinson and
Houts, 1982). Strains HCB436 (Wolfe and Berg, 1989) and
HCB437 (Wolfe et al., 1987) lack all of the four chemoreceptors. In addition, the former lacks the methylesterase CheB
and the methyltransferase CheR, and the latter lacks all of
the cytoplasmic Che proteins, including the histidine kinase
CheA and the adaptor CheW.
The vector plasmid pBAD24 (Apr) (Guzman et al., 1995)
carries the araBAD promoter, the araC gene, which encodes

Fig. 8. Model for helically arranged Sec proteintranslocases within a cell. The Sec machineryis organized into a helical array and translocates proteins from the cytoplasm to the cytoplasmic membrane and the periplasmic spacethat migrate. Some integral membrane proteins
are inserted into the cytoplasmic membrane via
the Sec machinery and may be sorted after
insertion to migrate towards their destined
regions (e.g. polar or random distribution).
Outer membrane proteins are omitted for clarity. IM, the cytoplasmic (inner) membrane; OM,
the outer membrane.

the positive and negative regulator of the araBAD promoter.
Plasmids pEGFP, pECFP and pEYFP, which encode theenhanced green, cyan and yellow fluorescent protein,respectively, were purchased from Clontech.

Construction of plasmids encoding fluorescentfusion proteins
For construction of plasmids encoding Tar–YFP under thecontrol of the nahG promoter, the AvaI-BsrGI fragment ofpEYFP was subcloned between the AvaI and BsrGI sites ofpTrc-Tar–GFP (Homma et al., 2004). A BamHI site was introduced downstream of the tar–gfp/yfp gene by polymerase
chain reaction (PCR) and the EcoT22I-BamHI fragment was
subcloned into pLC113 (Cmr) (Ames et al., 2002), which iscompatible with a pBR322-derivative plasmid, to yield
pLC113-Tar–GFP/YFP.
For construction of pBAD24-Tar-S36C&A118C–GFP,
the AatII-NdeI fragment of pBAD24-Tar-S36C&A118C (M.
Homma and I. Kawagishi, unpublished) was subcloned into
pBAD24-Tar–GFP (D. Shiomi and I. Kawagishi, unpublished). The NdeI-HindIII fragment of pBAD24-EnvZ–GFP (D.
Shiomi and I. Kawagishi, unpublished) was subcloned into
pBAD24-Tar-S36C&A118C–GFP to yield pBAD24-Taz1S36C&A118C–GFP.
For construction of pBAD24–GFP–SecE or GFP/CFP–
MreB, the secE or mreB gene of E. coli was amplified by PCR
using chromosomal DNA of strain RP437 as a template. The
forward and reverse primers used were designed to introduce
unique BsrGI and EcoRI sites at the 5′ and 3′ ends of thesecE or mreB coding region respectively. The BsrGI-EcoRI
fragments were subcloned into pEGFP or pECFP. The NcoIHindIII fragments from the plasmids were subcloned into
pBAD24 to yield pBAD24–GFP–SecE or pBAD24–GFP/
CFP–MreB.
A PstI site was introduced at the 5′ end of the ecfp coding
region of pECFP by PCR. The PCR product was digested
with PstI and EcoRI and the resulting fragment containing theecfp gene was subcloned into pTrcHisB. The NcoI-XbaI fragment of the resulting plasmid was subcloned into pBAD24 toyield pBAD24-CFP(NX). For construction of MalE–CFP, themalE gene was amplified from pMalE (S. Banno and I.
Kawagishi, unpublished) by PCR to introduce unique EcoRI
and XhoI sites at the 5′ and 3′ ends of the malE gene
respectively. The PCR product was digested with EcoRI and
XhoI and the resulting fragment containing the ecfp gene was© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 894–906

904 D. Shiomi, M. Yoshimoto, M. Homma and I. Kawagishi
subcloned into pBAD24-CFP(NX) to yield pBAD24-MalE–
CFP. The M18R or M19R mutation was introduced by twostep PCR to yield pBAD24-MalE-M18R/M19R–CFP.

Immunoblotting
Immunoblotting was carried out as described previously(Shiomi et al., 2002) using anti-GFP antibody (Molecular
Probes).

Fluorescence microscopy(pH 7.0)] supplemented with 2 mg ml−1 lysozyme. An aliquot
was spotted on a cover slip and allowed to dry. PBST
(140 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4,
0.05% Tween20, 2% BSA) were then spotted onto the sample. After 15 min of incubation, the sample was treated withthe first antibody against SecG (provided by Dr H. Tokuda)for 1 h, washed twice, treated with the second antibody [goatanti-rabbit IgG labelled with Alexa Fluor 488 (Molecular
Probes)], washed twice and observed under the fluorescencemicroscope.

Acknowledgements

Observations of fluorescent proteins were carried out essentially as described previously (Shiomi et al., 2002). For timecourse experiments, cells carrying a plasmid encoding Tar–
GFP or Taz1–GFP were grown in TG medium (1% tryptone,
0.5% NaCl, 0.5% glycerol) at 30°C and after 3 h, 1 mM arabinose was added to each culture. Cells were harvested withappropriate intervals (see Results), spotted onto a glass slide
coated with 0.5% agarose and observed under an inverted
fluorescence microscope (Olympus IX71). The fluorescentimages were processed by using the software Metamorph
5.0r4 (Molecular Devices) and Photoshop ver.7 (Adobe). For
time-lapse experiments, after the addition of 1 mM arabinose,
cells were further incubated at 30°C for 1 h, harvested,washed and resuspended in TG medium. Rifampicin(25 µg ml−1) was added to inhibit transcription and cells were
further incubated for 10 min, harvested and observed withintervals of 1 s, 10 s or 1 min. For GFP–SecE or CFP–MreB,
cells were grown at 30°C and after 3 h, 1 or 10 mM arabinose
was added respectively. Cells were further incubated for 15
or 30 min, respectively, harvested and observed. For double
labelling with MalE-M19R–CFP and Tar–YFP, cells weregrown in TG medium supplemented with 0.5 µM sodium salicylate (to induce Tar–YFP) at 30°C for 3 h. Then, 0.1 mMarabinose was added to induce MalE-M19R–CFP. Cells were
further incubated for 15 min, harvested and observed. For
double labelling with CFP–MreB and Tar–YFP, cells weregrown in TG medium supplemented with 0.5 µM sodium salicylate at 30°C for 3 h. Then, 10 mM arabinose was added toinduce CFP–MreB. Cells were further incubated for 30 min,harvested and observed.
For optical sectioning, a fluorescence microscope with
piezo drive (Zeiss Axiovert) was used to obtain a series of zsections with a fixed spacing of 0.2 µm. Each stack of 20–30
sectioned fluorescence images was deconvolved by using
CELLscan (Scanalytics). Three-dimensional images werereconstituted by using IPLab (Hamamatsu Photonics).
Expressions of fluorescent proteins were carried out under
the conditions described above.

Immnofluorescence microscopy
Immnofluorescence microscopy was carried out according to
the method of Maddock and Shapiro (1993) with modifications. Cells were harvested and resuspended in MLM and
fixed by adding an equal volume of 0.6% folmaldehyde in
MLM and by incubating on ice for 2 h. Cells were thenwashed three times with MLM and resuspended in GTE[50 mM glucose, 20 mM Tris-HCl (pH 8.0), 10 mM EDTA

We thank Dr Koutarou D. Kimura for technical advice on
deconvolution, Dr Toshiharu Yakushi for critically reading themanuscript and invaluable discussion and Dr Hajime Tokuda
for invaluable discussion and anti-SecG antibody. This work
was supported in part by grants-in-aid for scientific researchfrom the Japan Society for the Promotion of Science (to D.S.
and I.K.).

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