FEMS Microbiolo~ Letters 67 (19qO) 39-44
Published by Elsevier
39
FEMSLE 03859
Characterization of adhesion zones in E. coli cells
M. L e d u c t a n d C. Frehel 2
: Unild ass~o~e du CNRS, Biochimie Moli~ulaire ¢t cellulaire Universftd paris-sud, Orsay. and ? UnR~ de Micrctscopie Electronique,
Diparlemem de Biologie Moldculaire. Instftut Posteur, paris, France
Received 26 July 1989
Revision received6 September 1989
Accepted 8 September 1989
Key words: Escherichia coil; Adhesion zone; Outer membrane protein
1. S U M M A R Y
After plasmolysis oi' Escherichia coil cells, the
adhesion zones were characterized using the cyto~
chemical PTA and SP procedures which stain
peptidoglycan and lipopolysaccharides (LPS) respectively. A VIA-stained layer was detected at
the adhesion sites. This layer was visualized irrespective of the electron microscopy procedure
used. Also, using SP staining an outer membrane
in which LPS molecules were asymmetrically dis+
tributed, was observed.
2. I N T R O D U C T I O N
Plasmolysis of Escherichia coil cells by exposure to hypertonic sucrose solution induced a
retraction of the cytoplasmic membrane from the
peptidoglycan-outer membrane layer and a subsequent enlargement of the periplasmic space. Such
an enlargment was discontinuous forming periCo~eespondence to: M. Leduc, Unit6 Associ~edu CNRS. Bio-
ehlmie Mol&-ulaire et cellulalre, Universit~ Pmis-sud. 91405
Orsay. France.
plasmic bays delimited by bridges (attachment
zones) first described by Bayer, [1]. These bridges
were localized all along the periplasm [2]. Their
structure could result either from a direct interaction between outer membrane and inner membrane [2] or from an interaction between the three
cell envelope-layers [3].
Recently, we demonstrated that in E. coil cells,
peptidoglycan was the main component responsible for the staining of the periplasmic space by the
PTA-procedure and that this macromolecule
formed a muldiayered structure unevenly distributed all around the cell [4,5]. Also, the outer
membrane LPS molecules can be specifically
stained by the Thiery procedure (SP-staining) [6].
With such stainings it was therefore possible to
further characterize Bayer's junctions in plasmolyzed E. coil cells. In this study the conventional
glutaraldebyde-osmium fixation was compared to
a fixation with dithiobis (succinimidyl propionyl)
(DSP), a cleavage ernss-linking agent [7].
Also, to reduce possible alterations due to electron microscopy procedure, plasmolyzed cells were
processed according to the recently developed
eryosabstitution method [8] prior to PTA-staining
of thin sections.
0378-1097/90/$03.50 © 1990 Fed©ralion of European MicrobiologicalSocieties
4o
3. MATERIAL A N D METHODS
3.2. Plasmolysis
Cells were plasmolyzed according to Cook et aL
3.1. Strain and growth medium
E. coil AB 2497 (Thr, Lcu, pro A, his, Arg E,
lac, gal, ara, xyl, mtl, tiff, tsa, str R, thy) was grown
in 50 ml rich medium at 37 o C (antibiotics medium,
Difco). Cells were harvested in mid exponential
phase (O.D. = 0.4 at 600 nm) by centrifugation
and washed once with phosphate buffer 50 raM,
pH 7.2.
[9].
3.3. Fixation and embedding
After 3 rain of plasmolysis cells were fixed
either with DSP in dimethyl sulfoxide (DMSO) (in
the ratio 2 : 1 , DSP/protein) for -~0 ,an at room
temperature or with 2.5~ ( v / v ) 81utaraldehyde in
0.1 M, pH 6.8, cacodylate buffer for 16 h at 4 ° C .
All samples were washed for 4 h in the cacodylate
Fig- 1. Thin sectionsof p l ~ l y z e d ~.lls stoned eitherwhb PTA pnx~lure (la, ib~ 1¢) or withSP.stainlag(ld), In I a. ld, cellswere
fixed with DSI~,in lb, th~ were fixedwith 8lutaxaldehydc~ia Ic, cells we~ ¢¢yo~t~titutedin DS~ascetonc, In ta, Ib, lc ( ~ )
showsthe PTA stainedlayer; in Id (e,) zhowsthe silver~ranula¢ion,Bar - 0,W3/4m,
41
buffer concentrated in 2% ( w / v ) agar and postfixed successively with 1~ ( w / v ) osmium tetroMde in the same cacodylate buffer and for 1 h
with lfe ( w / v ) uranyl acetate in veronal buffer. In
some cases the fixation procedure was modified
by replacing the glutaraldehyde in cacodylate fixation step by glutaraldehyde in DMSO. or by omitting post fixation with osmium tetro~de. After
dehydration with acetone, bacteria were embedded in Epon. In any case, no modifications of
plasmolyzed bacteria ultrastructure were observed.
The cryosubstitution procedure was as described by Hobot et al. [8] with substitution in 2.5
glutaraldehyde or DSP (80 ttg/ml) and embeddlng in Epoo.
3.4. Cytochemical stainings
Thin sections gathered on plastic rings were
submitted either to the PTA-staining of Rambourg [10] or to the SP staining of Thiery [11] as
described earlier [4-6].
4. RESULTS
When the PTA-staining was applied to intact
E. coli cells, peptidoglycan appeared as an electron-dense layer spanning the periplasmic space
from the outer to inner membranes [4,5]. After
plasmolysis, a PTA-stained layer was still observed all along the outer membrane without any
interruption in adhesion sites. This stained layer
was detected whether cells had been fixed with
gintaraldehyde or DSP followed by embedding in
Epon (Fig. la, b), or processed by cryosubstitution (Fig. lc). In the case of glutaraldehyde fixation the stained layer in the adhesion zones loosely
spread out between the cytoplasmic and outer
membranes (Fig. la); in DSP-fixed cells the
peptidoglycan layer in the adhesion zones appeared as a well-defined layer tightly enclosed by
the outer and inner membranes (Fig. lb). Such a
well-defined structure between the outer and cyto-
Fig. 2, Thin sections of plasmolysedcells stained with lead citrate (a) DSP-fixedcells; (b) cryosubstltuted cells; ( ~ ) shows adhesion
zones. Bat ~ 0.05/Jm.
42
plasmic membranes was also observed in the case
of DSP-cryosubstimted cells (Fig. lc). It is worth
noting that this structure was present but not
stained when thin scetions were treated with lead
citrate (Fig. 2a, b).
To detect possible changes in LPS distribution
during plasmolysis the outer meanbrane was
specifically stained by the SP-procedure. A single
granular line was detected along the outer leaflet
of the outer membrane, both along the bays and
the attachment sites (Fig. ld). This means that
plasmolysis induced no modifications of LPS distribution, lit remained asymmetrically distributed
as already shown in exponentially growing cells
[6].
the synthesis of several components. It might then
be possible to determine whether the protein
molecules present in these bridges are in juxtaposition, despite the presence of peptidoglycan, thus
permitting molecule exportation.
Since adhesion zones were observed irrespective
of the electron microscopic procedure used it can
be concluded that these zones form a morphological entity. Recently, with another electron microscopic technique Bayer et al. [16] could also
visualize these zones. The existence of these cell
entities is reinforced by recent results showing that
they are dependent on cell physiology since their
number increases after MS2 phage infection [17]
and decreases after membrane-derived-oligosaccharide deficiency [18].
5. DISCUSSION
ACKNOWLEDGEMENTS
In the present work the presence of peptidoglycan at the adhesion sites was specifically
demonstrated for the first time. The peptidoglycan
layer was visualized irrespective of the electron
microscope procedure used. During plasmolysis
there was no peptidoglycan degradation, If this
had b ~ n the case, a change in LPS distribution,
from asymmetrical to symmetrical, would have
been observed [6].
The peptidoglycan layer was loosely bound to
the outer and inner membranes in glutaraldehyde-fixed cells (Fig. Ib) while it was tightly bound
to the membranes with DSP-fixed cells (Fig. la).
The constant and short length of DSP could be
responsible for a better role ill fixation than
glntaraldehyde which might be under a polymerized state [12]. Bayer mentioned the importance of
the fixation procedure to visualize plasmolysis in
E. coil cells [2]. We chose DSP in this ohrastructural and cytochemieal study because this crosslinking agent was also used in biochemical studies
of the protein surrounding the E, eoli cell envelope peptidoglycen [13,14]. The use of such a
erosslinking agent could help 1o establish a direct
relationship between these adhesion zones and the
E. coil envelope fractions isolated on isopyenic
gradient, called the O M L fraction [15], This fraction was shown to contain peptidoglycen, outer
and inner membranes and to be able to catalyze
We are indebted to Mrs. R. Daty and 1o Mr.
J.-C. Brnichou for their precious technical assistance. This work was supported by grants from
the Centre National de la Recherche Scientifique
( U A 1131) and from the Fondation pour la Recherche M~licale.
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