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INTRODUCTION |
The periplasmic space lies between the inner and the outer
membranes of Gram-negative bacteria. A number of processes that are
vital to the growth and viability of the cell occur within this
compartment. Proteins residing in the periplasmic space fulfill important functions in the detection, processing, and uptake of essential nutrient substances. These proteins are exported into the
periplasm mainly via two pathways: the unfolded proteins via the Sec
system (1) and the folded enzymes containing redox cofactor via the
Tat1 (or Mtt) pathway
(2-4).
The periplasm might not be a uniformly homogenous compartment;
fine structures known as Bayer patches/bridges (5) and periseptal and
polar annuli (6-9) have been described. The existence of these
structures under physiological conditions is a subject of contention (10, 11). Nevertheless, these structures were
proposed to provide sites required for the export of outer membrane
components, murein synthesis, secretion of bacteriophages, and cell
divisions (12).
On the other hand, polar bacterial organization was observed with a
variety of bacterial species and concerns a disparate array of cellular
functions (13). In addition to the well known examples of polar
organelles such as flagella, pili, and stalk-like appendages at the
bacterial surface, accumulating evidence shows that periplasmic, inner
membranous, and cytoplasmic proteins may also exhibit polar
localization under certain condition. These proteins participate in
various cellular processes including maltose sensing and uptake (14),
chemotaxis (15), conversion of chorismate to phenylalanine (16, 17),
DNA replication, and cell division (18, 19). Despite the convincing
data that established the polar localization of these proteins, the
relationship between their cellular location and fine periplasmic
structure remains unanswered.
The Tat (also called Mtt) system is a recently discovered protein
export pathway that is capable of translocating folded proteins with
peculiar twin arginine (RR) translocation signal peptides (2, 3). Two
classes of genes encoding functional Tat components have been
identified and studied in Escherichia coli. The
tatC gene encodes an integral membrane protein with six
transmembrane segments, and its depletion leads to mislocation of all
the enzymes analyzed (2, 3, 20). On the other hand, The
tatA, tatB, and tatE genes code for
three proteins. They share sequence homology at their N termini,
including one transmembrane segment and an adjacent amphipathic domain,
whereas their C termini vary both in sequence and in length (2, 3, 21).
The depletion of these genes would affect the translocation of various
enzymes differently. In addition to the enzymes containing redox
cofactors, the Tat system probably also exports proteins that fold too
quickly or too tightly to be handled by the Sec system (2, 3). Green fluorescent protein (GFP) from the jellyfish, Aequorea
victoria, has been widely used as a marker for gene expression and
localization of gene products. The chromophore is generated by the
spontaneous cyclization and oxidation of the sequence
Ser65-(or
Thr65)-Tyr66-Gly67. The protein
fold consists of an 11-stranded
-barrel 42 Å long and 24 Å in
diameter with a central coaxial helix carrying the chromophore (22).
GFP is synthesized without an export signal. When it is fused to
maltose-binding protein carrying a typical Sec-dependent
signal peptide, the hybrid GFP is not fluorescent in a wild type
strain, and it is exported into the periplasm in an improperly folded
conformation (23). Interestingly, the expression of the hybrid GFP in
secA, secY, and secB mutants leads to the recovery of fluorescence. Furthermore, deletion of the Sec signal peptide also results in fluorescent colonies. Therefore, the Sec pathway is capable of exporting only the improperly folded GFP.
To assess the capacity of the Tat system to export folded heterologous
proteins and to study the implication of the periplasm in various
cellular processes, we fused the GFP to the twin arginine signal
peptide of the trimethylamine N-oxide (TMAO) reductase. The
folded GFP was exported successfully into the periplasm via the Tat
pathway. Interestingly, we observed that GFP gathered at the poles in
the periplasm in response to osmotic up-shock. It is a reversible
process, implying an adaptation of the bacterium to the medium. Our
results show that the periplasm is a dynamic heterogenous space and
suggest that bacteria might react to environmental changes by
compartmentalization of the periplasm in correlation with the
localization of membrane proteins.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, and Media--
The E. coli strains used in this study are: MC4100 (F'
lac
U169 araD139 rpsL150 thi flbB5301 deoC7 ptsF25
relA1) (24); B1LK0 (as MC4100,
tatC) (20); MCMTA (as
MC4100, tatB::Kan) (21); CU164
(secY39cs, zhd-33::Tn10)
(25); GC3904 (argS
(pbpA-rodA)::Kan, zbf::Tn10 (a gift from D. Vinella); and
TG1 (
(lac-pro) supE thi hsdD5/F' traD36
proA+B+
lacIq lacZ
M15).
Arabinose-resistant derivatives of the araD mutants were
constructed by selecting purple colonies of these strains spread on
eosin methylene blue plates (26) containing 0.2% arabinose. The
tat mutations in the arabinose-resistant derivatives were confirmed by PCR and anaerobic growth on minimal medium with
TMAO as the energy source. The plasmids pBAD24 and gfpmut2 were
described in Refs. 27 and 28, respectively.
The bacteria were routinely grown in Luria-Bertani (LB) medium, on LB
plates, or in the minimal M63 or M9 media (26). Anaerobic growth was
achieved normally in stoppered bottles or tubes filled to the top or on
plates in GasPak anaerobic jars (BBL Microbiology Systems). As
required, ampicillin (100 µg/ml), rifampicin (150 µg/ml),
chloramphenicol (300 µg/ml), TMAO (1 mg/ml), sodium molybdate (2 µM), or sodium selenite (2 µM) were
added. Precultures were inoculated from a single colony and used at
a 100-fold dilution. To assess the energy dependence of
bacterial adaptation to osmotic up-shock, sodium azide and carbonyl
cyanide m-chlorophenylhydrazone (CCCP) were used at final
concentrations of 2 mM and 10 µM, respectively.
Construction of RR-GFP Fusion--
The
gfp-mut2 gene was amplified by PCR using
GFPNHEIUP (5'-aagaaggagatatacatgctagcaaaggag-3') and GFPDOWN2
(5'-tgaccatgaagcttgcatgcctgc-3') as primers. The reaction was performed
using the Expand High Fidelity PCR system according to the
manufacturer's instruction (Roche Molecular Biochemicals). The
amplified fragment was purified, double-digested by NheI and
HindIII, and cloned into the corresponding sites of the
plasmid p8754 (laboratory plasmid stock), which is a derivative of
pBAD24 and contains the region encoding the RR signal peptide of the
TMAO reductase. The fusion of the RR-gfp gene in the
resulting plasmid was confirmed by DNA sequencing, and the expression
of the fusion was analyzed using media containing either glucose or
arabinose. Expression of RR-GFP was under the tight control of the
PBAD promoter as described by Guzman et
al. (27).
Cellular Fractionation, Electrophoresis, and
Immunodetection--
Periplasm, spheroplasts, membrane, and
cytoplasmic fractions were prepared by lysozyme/EDTA/cold osmoshock and
ultracentrifugation as described previously (29, 30). Proteins were
separated by polyacrylamide gel electrophoresis in the presence
(denaturing) or in the absence of SDS (nondenaturing) on 10%
acrylamide gels (31). The proteins present on both nondenaturing and
denaturing gels were immobilized onto a polyvinylidene difluoride
membrane, and cellular distribution of GFP was analyzed by immunoblot.
Immunoblot was performed using the ECL method according to the
manufacturer's instruction (Amersham Phamacia Biotech).
Microscopy and Fluorescence Spectrometry--
An overnight
culture was diluted 1:100 in LB + ampicillin + glucose medium and grown
at 37 °C until the A600 reached
0.6-0.8. Cells were centrifuged, resuspended in LB + ampicillin with
0.2% arabinose, and grown at 30 °C for 45 min. Rifampicin or
chloramphenicol was added, and the culture was incubated at 30 °C
for the times indicated under "Results." The cells were
examined immediately or after the addition of 0.15 M NaCl
or other treatment, as described under "Experimental Procedures,"
by Zeiss PhoMi III fluorescence microscope equipped with a filter set
for fluorescein isothiocyanate. Images were captured using a CCD camera
(Color Cool View, Photonic Sciences) and Image Pro-Plus software. The
fluorescence levels present in crude extracts and different
cellular fractions were quantified by using a Spex Fluorolog III
fluorimeter equipped with a cooled detector (Jobin Yvon).
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RESULTS |
Construction and Translocation of the RR-GFP Fusion
Protein--
To assess the capacity of the Tat pathway and to monitor
in vivo protein translocation via this system, we fused the
GFP with the RR signal peptide of TMAO reductase. The expression of the fusion protein was under the control of the arabinose-inducible promoter (see "Experimental Procedures"). The synthesis of GFP in a
wild type strain and the tatC derivative was assessed by fluorescence spectroscopy and immunoblot analysis using antisera against GFP. As expected, GFP was synthesized only if the cells were
grown in the presence of arabinose. After cellular fractionation, the
distribution of the GFP was analyzed first by fluorescence spectroscopy. In the wild type strain, more than 65% of total fluorescence was located in the periplasm. In contrast, more than 95%
of total fluorescence was found in the cytoplasm of the tatC mutant. We had thus successfully translocated GFP into the periplasm via the Tat pathway.
We then confirmed the cellular distribution of the GFP by visualization
of GFP on a nondenaturing gel or by immunoblot after electrophoresis. A
single fluorescent band was found in the periplasm of the wild type
strain whereas it was absent from the periplasm of the tatC
mutant (Fig. 1A, lanes
3 and 5). It exhibited the same mobility as the GFP
without a signal peptide synthesized from gfp-mut2 (Fig.
1A, lane 1), suggesting that it corresponds to
the processed mature form of the RR-GFP. Three fluorescent bands,
showing slower mobility than the periplasmic GFP, were observed in the
cytoplasm fractions of both the wild type strain and the
tatC mutant. The band with the fastest mobility exhibited the strongest and the most stable fluorescence (Fig. 1A,
lanes 2 and 4, RR-GFP), whereas the other two
bands lost fluorescence very quickly. Moreover, the slowest migrating
band showed a yellowish-green fluorescence. Analysis of the
nondenaturing gel by immunoblot confirmed the presence of a single band
in the periplasm of the wild type strain (Fig. 1B,
lane 3). In addition, no protein was recognized by the
anti-GFP antisera in the periplasm of the tatC mutant (Fig.
1B, lane 5). Four bands were detected in the
cytoplasmic fractions (Fig. 1B, lanes 2 and
4). One showing strongest signal corresponds to the most
fluorescent form of the cytoplasmic RR-GFP (Fig. 1B,
RR-GFP1). The two slower migrating, fluorescence unstable bands
(RR-GFP2 and RR-GFP3) were clearly detected by the immunoblot. We do
not know whether these two forms represent different conformations or
complexes of the RR-GFP. In addition, we observed another band (RR-GFP*) with a mobility between the periplasmic GFP and the cytoplasmic RR-GFP1 on the nondenaturing gel. This band was not fluorescent and might be a degraded form of RR-GFP. When the same samples were analyzed on a SDS-denaturing gel, a single band of 27 kDa
was observed only in the periplasm of the wild type strain (Fig.
1C). The precursor RR-GFP and probably its degraded
derivatives (RR-GFP*) were present in the cytoplasm of both the wild
type strain and the tatC mutant (Fig. 1C). To
confirm the translocation of GFP via the Tat pathway, we also analyzed
the cellular distribution of GFP in MCMTA
(tatB::Kan) and CU164 (secY39cs).
Folded GFP accumulated in the cytoplasm of the
tatB::Kan mutant, but was exported in the
periplasm of the secY mutant (data not shown). Therefore, we
succeeded for the first time to export heterologous cytoplasmic protein
GFP into the bacterial periplasm via the Tat pathway.

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Fig. 1.
Translocation and processing of RR-GFP.
The cytoplasmic (lanes 2 and 4) and
periplasmic (lanes 3 and 5) fractions prepared
from MC4100 (lanes 2 and 3) and B1LK0
(lanes 4 and 5) were separated on 10%
nondenaturing polyacrylamide gels (A and B) or
SDS-gel (C), and GFP proteins were visualized either after
excitation by a transilluminator (UV 365 nm) (A) or
immunoblot using antisera against GFP (B and C).
GFP indicates native GFP without signal peptide (lane
1) or GFP processed from the fusion precursor (RR-GFP).
GFP* and RR-GFP 1, 2, and 3 represent
a degraded form of GFP and GFP in complex or under different
conformations, respectively. The RR-GFP 1 band in panel B
corresponds to the fluorescent band RR-GFP in panel A.
Unfortunately, we did not succeed in photographing the
fluorescent bands RR-GFP2 and RR-GFP3 in panel A (see
"Results").
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In Vivo Cellular Location of GFP--
It was reported that the
GFP-MinD fusion has a halo appearance when it is evenly distributed
along the cytoplasmic membrane (19). We reasoned that GFP should also
exhibit a halo appearance when it is exported into the periplasm.
Therefore, we examined the in vivo cellular localization of
GFP by fluorescence microscope. To reduce uneven level of GFP in
different individual cells due to the PBAD promoter, a
saturating concentration of arabinose (0.2%) was added in the culture
with cells at logarithm phase. To avoid an overproduction and
saturation of GFP, protein synthesis was inhibited by rifampicin or
chloramphenicol 45 min after the induction by arabinose. When a wild
type strain was directly observed in LB medium, GFP showed a halo
distribution in most cells, but it had occasionally a polar
localization (Fig. 2, A and
E). In contrast, GFP diffused throughout the cells of the
tatC mutant (Fig. 2, C and D).
Notably, the tatC cells form chains, suggesting a deficiency
in a late stage of cell division. Recently it was reported that the
tat mutations exhibit pleiotropic defects in the cell
envelope (32). Interestingly, no GFP was observed at the septum, which
confirmed the cytoplasmic accumulation of GFP in the tatC
mutant. Therefore, export of GFP via the Tat pathway resulted in an
even distribution of the fluorescence in the periplasm of E. coli.

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Fig. 2.
Localization of GFP in wild type
strains and in the tatC mutant. After induction
of the expression of RR-GFP, protein synthesis in the wild type strains
MC4100 (A and B) and TG1 (E and
F) and in the tatC mutant (C and
D) was blocked by the addition of rifampicin (see
"Experimental Procedures"). 45 min later, cells were inspected
under a fluorescence microscope immediately (A,
C, and E) or after the addition of NaCl at a
final concentration of 0.15 M (B, D,
and F).
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The occasional polar localization of GFP was a surprise that raised the
question of what might lead to such polarization. Since the polar
localization was never observed in the tatC mutant, we
concluded that GFP must accumulate either in the membrane or in the
periplasm at the poles of the wild type strain. Several possibilities
might lead to the polar localization of GFP. First, it might be an
artifact or a consequence of over-expression of GFP. Second, the Tat
translocase might have a polar localization, which would concentrate
RR-GFP and GFP at the poles before or immediately after the
translocation, respectively. Finally, periplasmic GFP might relocalize
at the poles for some reasons. To examine these hypotheses, we studied
the kinetics of halo formation and the polar localization of GFP. The
fluorescence appeared very slowly and a sufficient level of
fluorescence was observed at about 45 min after the induction. Because
the PBAD promoter has a very fast induction rate (27), the
slow appearance of GFP suggests a rather slow process of fluorophore
formation under the condition used. Although arabinose was used at
saturating concentration, heterogeneity of GFP level in individual
cells was observed, as described by Hashemzadeh-Bonehi et
al. (33). Therefore, timing of GFP folding and export cannot be
accurately measured and hereinafter all phenomena were described for
the majority of cells. The GFP halo appeared at 40 min after blockage of its synthesis. One hour later almost all cells showed the GFP halo
and about 1% cells showed polarization of GFP. This result clearly
indicates that the halo formation occurs before the polarization of
GFP, excluding a possible polar localization of the translocase, and
that GFP polarization is unlikely to be a consequence of overproduction of the fusion protein. In addition, this result also revealed a long
time lag between the inhibition of RR-GFP synthesis and the appearance
of the GFP halo. It is fully consistent with the export of GFP through
the Tat pathway, which is a slow process (30). We attempted to study
the kinetics of halo formation by following individual cells, but this
was unsuccessful because the GFP halo appeared very slowly and the
fluorescence was quenched after a few excitations.
Polarization of GFP in Response to Osmotic Up-shock--
The above
results indicate that the GFP halo formation results from the
translocation of GFP into the periplasm via the Tat pathway. We then
analyzed the relationship between halo formation and polarization.
Surprisingly, resuspending cells in phosphate saline buffer immediately
caused the disappearance of the halo and recruitment of GFP at both
poles. Further analysis revealed that NaCl or KCl alone could trigger
polarization. In addition, the addition of 0.15 M NaCl in
the culture could completely convert the halo to the polar spots (Fig.
2B), suggesting that the polarization might be a consequence
of cellular response to osmotic shock. Indeed, the addition of 0.15 M potassium or 20% sucrose also triggered the polarization
of GFP, although sucrose was less efficient than the sodium and
potassium ions. This rapid halo to polar spot conversion was also
observed for another wild type strain TG1 (Fig. 2, E and
F). Interestingly, fluorescence was often more dominant at one pole than the other. However, GFP remained uniformly distributed in
the cytoplasm when the tatC mutant was resuspended in the
phosphate saline buffer or was subjected to the osmotic up-shock (Fig.
2, compare panels D and C). Therefore, the
polarization of GFP is most likely the consequence of relocalization of
the periplasmic GFP to the poles.
Polarization of GFP in Spherical Cells--
When cells are subject
to an osmotic up-shock, plasmolysis bays are formed as the cytoplasmic
membrane separates from the other components of the wall. It was
proposed that the plasmolysis bays are restricted at the poles because
the bilayer of the cytoplasmic membrane is essentially an
incompressible two-dimensional liquid. Therefore, the way to cope with
the reduction of cytoplasmic volume while keeping the same surface of
the bilayer would be the invagination of the cytoplasmic membrane at
the two poles (11). It is thus interesting to know if the polarization
of GFP is the simple physics phenomena or represents a gathering of
periplasmic proteins at peculiar sites in response to the osmotic
up-shock. To check these hypotheses, we assessed the GFP localization
in spherical cells.
E. coli owes the rigidity of its rod shape to its
peptidoglycan layer, a single macromolecule surrounding the cytoplasmic membrane. Penicillin-binding proteins catalyze the final steps of
peptidoglycan synthesis. The depletion of rodA-pbpA genes
abolishes the synthesis of the penicillin-binding protein 2 and the
mutant strain, GC3904, thus grows as spherical cells (34). Similarly, lysozyme (muramidase) hydrolyzes the peptidoglycan, resulting in the
formation of spherical cells. The plasmid pRR-GFP was introduced into
GC3094 and the cells were inspected in the same way as described above.
We observed a circle appearance of GFP in most cells of the
exponentially growing mutant GC3940 in LB medium. Occasionally, GFP
also gathered at one or two sites or at one side of the spherical cells
(Fig. 3A). Interestingly, the
convertible localization of GFP was considerably enhanced when the
cells were subjected to an osmotic up-shock (Fig. 3B). It is
likely that after EDTA-lysozyme treatment, the cells of the wild type
strain became spherical and the GFP appeared as a circle (Fig.
3C). The further addition of 0.15 M NaCl or 20%
sucrose resulted in the recruitment of GFP in these cells (Fig. 3,
D and F). Intriguingly, GFP gathered at one to
three mostly irregular sites in the rodA-pbpA mutant,
whereas it was located especially at one or two opposite sites in the lysozyme-treated cells. Such different localization of GFP might be
explained by the fact that the rodA-pbpA mutation affects
cellular elongation, a process related to a clear definition of the
poles, whereas the lysozyme probably hydrolyzes the peptidoglycan at random places. Therefore, these results suggest that the periplasm and
cytoplasmic membrane might not be uniform and that the periplasmic proteins might preferentially gather at the poles or the area corresponding to the poles.

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Fig. 3.
Localization of GFP in spherical cells.
The cultivation condition is the same as described for Fig. 2. Cells of
the rodA-pbpA mutant were inspected immediately
(A) or after the addition of NaCl (panel B). For
the wild type strain MC4100, cells were observed after treatment with
EDTA (2 mM) for 2 min and then with lysozyme (0.2 mg/ml)
for 10 min (C), with the addition of 0.15 M NaCl
(D) or of 20% sucrose (E and F).
Panels A-D and F are fluorescent images, and
panel E is the phase contrast image of panel
F.
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Energy-dependent Reconversion of Polar Spots to a Halo
Appearance--
It is likely that the polarization of GFP is a
consequence of cellular response to an osmotic up-shock. In this case,
the adaptation of bacteria to a given osmolarity should lead to
conversion of polar spots to a halo appearance. Indeed, the
polarization of GFP, resulted from the addition of NaCl in the culture
of the wild type strain MC4100, disappeared progressively and the polar spots completely converted back to halo 15 min after the osmotic up-shock (Fig. 4, A and
B). Importantly, simultaneous addition of 2 mM
sodium azide or 10 µM CCCP with 0.15 M NaCl
completely inhibited the reconversion of the GFP spot to halo (Fig. 4,
C and D). Interestingly, after the induction and
the blockage of the GFP synthesis, if the fluorescent cells were
centrifuged and then resuspended in M63 medium, GFP in all cells was
immediately relocalized from the halo to the two poles and polar
localization remained even after 60 min incubation at room temperature
(Fig. 4E). In contrast, it converted completely to halo 20 min after resuspension of the cells in M63 in the presence of 0.2%
glucose (Fig. 4F). Therefore, the acceleration by glucose
together with the blockage by azide and CCCP strongly suggest that the
conversion of the GFP appearance is an energy-dependent
process. The GFP localization might be thus related to the cellular
adaptation to the osmotic change in its medium.

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Fig. 4.
Energy-dependent reconversion of
GFP polar spots to halo appearance. The cultivation condition is
same as described for Fig. 2. NaCl (0.15 M) was added alone
(A and B) or simultaneously with 2 mM
sodium azide (C) or 10 µM CCCP (D)
in LB medium containing fluorescent halo cells. Cells were examined
immediately (A) or 10 min after the addition of NaCl
(B-D) under a fluorescence microscope. Alternatively, the
fluorescent halo cells were centrifuged and then resuspended in M63
medium (E), supplemented with 0.2% glucose (F),
and examined 60 min or 20 min later, respectively.
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DISCUSSION |
The Tat system is a recently identified bacterial protein export
pathway with the remarkable ability to transport folded proteins and
even enzyme complexes across the cytoplasmic membrane (2, 3).
Precursors exported by the Tat system contain a particular signal
peptide with a conserved RR motif, which is distinct from the
Sec-dependent signal peptides. The twin arginine signal
peptide is capable of rerouting proteins that are naturally exported
via the Sec pathway to the Tat pathway (2). However, export of cytoplasmic protein via the Tat pathway had not been reported. In this
paper, we show the successful translocation of a heterologous cytoplasmic protein via the Tat system in E. coli. Although
active GFP was observed and obtained from the periplasm, whether the GFP is exported in the active form is not obvious. The generation of
chromophore is a post-translational autoxidation process that requires
molecular oxygen. It was reported that anaerobically grown E. coli accumulates nonfluorescent GFP at an intermediate stage in
protein folding and that the generation of fluorescence occurs after
the admission of air both in vivo and in vitro
(35). In this study, GFP was synthesized under aerobic conditions; the redox potential in the cytoplasm is high enough to allow the formation of the fluorophore. We observed that fluorescence was developed in the
cytoplasm of both the wild type strain and the tat mutant starting from about one-half hour after the synthesis of the GFP protein, whereas the appearance of the periplasmic halo requires an additional half-hour. Therefore, the activation of GFP occurs prior
to translocation, and GFP is probably translocated in a folded
conformation via the Tat pathway. This conclusion is supported by the
recent observation that unfolded GFP fused to a
Sec-dependent signal peptide could be exported by the Sec
system but could not be folded correctly in the periplasm (23). In this
study, we observed two slow migrating, fluorescence-unstable bands in
the cytoplasm. They may represent folding intermediates of GFP or GFP
associated with some factor required for the export of GFP. We are
currently analyzing these isoforms of GFP.
Bacterial chemotaxis involves a phospho-relay system brought about by
ligand association with a membrane-bound chemoreceptor. The
chemoreceptors sense, alone or indirectly through a periplasmic ligand-binding protein, the gradient of ligands in the medium. Four
chemoreceptors have been localized at the poles of E. coli cells by immunoelectron microscopy and indirect immunofluorescence light microscopy (15). Maddock and Shapiro (14) thus proposed that the
bacterial cell sequesters different regions of the cell for specialized
functions. Interestingly, the maltose-binding protein (MBP), which is
associated with the chemoreceptor Tar to sense maltose, also exhibits a
polar localization (14). In addition, MBP can diffuse laterally in the
periplasm (36), and the induction of its synthesis by adding maltose in
the medium triggers polar cap formation in E. coli (37).
These observations strongly suggest that bacteria detect the nutrient
substances at the poles. Interestingly, we observed in this study a
reversible relocalization of GFP in the periplasm to the poles in
response to an osmotic up-shock. We thus speculate that in addition to the polarization of cytoplasmic membrane-bound receptors, the polar
area of the periplasm and the outer membrane might be the preferential zone where bacteria sense the change in the environment. Therefore, challenged to an osmotic up-shock, bacteria would gather periplasmic proteins to the poles to interact with the membrane-bound receptor and trigger the adaptation process. However, the polar gathering should not be specific to the ligand-binding proteins because
GFP is a heterologous protein for E. coli. Nevertheless, the
polarization might be an active process because it is achieved very
quickly and is reversible. We will analyze the influence of other
environmental changes on the localization of GFP to determine whether
our model of polar gathering for signal detection and transduction
could be generalized to other stimuli.
The periseptal annulus is described as a pair of concentric rings, each
of which consists of a continuous ring in close apposition with
the inner membrane, murein, and outer membrane (6). These organelles
mark the sites of future cell divisions. Following septation and cell
separation, each daughter cell inherits one of the two periseptal
annuli, which remains a polar annulus at the new pole of the newborn
cell. The periseptal and polar annuli divide the periplasm into three
types of subcompartments: two polar and two midcell compartments and
one compartment at the site of future division. By monitoring
fluorescence recovery after photobleaching at the periseptal and polar
annuli, Foley et al. (38) found that the recovery of
fluorescence was uniformly low over the zones of the periseptal and
polar annuli. They proposed that these regions are biochemically
sequestered from the remainder of the periplasmic space. However, Anba
et al. (7) previously reported that diffusion of the
overproduced phosphate-binding protein in the periplasmic space is not
interrupted by the periseptal annuli of E. coli under
conditions of plasmolysis. In addition, other authors (10, 11) have
challenged the biological implication of periseptal and polar annuli,
suggesting that these fine structures result from the physical
constraints on the membrane imposed by mild plasmolysis and its
localized relief during membrane collapse. In this study, we found that
the halo of GFP could quickly convert to polar spots, suggesting a free
movement of proteins within the periplasm. Therefore, if the periseptal
annuli exist under physiological condition, they should not be
permanent periplasmic compartments. The successful translocation of the
active GFP into the periplasm should thus represent a powerful tool to
study the property of periplasm in various cellular processes.