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Journal of General Microbiology (1983), 129, 3085-3090.
Printed in Great Britain
3085
The Intracellular Localization of Pseudumonas aeruginosa Lectins
ByJ. GLICK AND N. GARBER*
Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52 100, Israel
(Received 29 March 1983; revised 3 May 1983)
The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-11) was studied using
methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release
of the two lectins occurred when P . aeruginosa was exposed to magnesium-osmotic shock or was
frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred,
whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected.
Formation of spheroplasts from P . aeruginosa by gradual removal of the bacterial envelopes
revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme
treated mureinoplasts resulted in low release of glucose-6-phosphatedehydrogenase and the two
lectins (1O-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the
lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P .
aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two
lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer
membranes and in the periplasmic space.
INTRODUCTION
Bacterial lectins resemble plant lectins in being proteins or glycoproteins exhibiting specific
binding of simple sugars (Hart, 1980) or oligosaccharides (Draper et al., 1978; Richards et al.,
1979). Some lectins are found in the extracellular growth medium (DasGupta & Sugiyama, 1977;
Draper et al., 1978; Fujita et al., 1975; Richards et al., 1979). Other bacterial lectins are either
exposed on the cell surface (Eshdat et al., 1978) as appendages (Buchanan & Pearce, 1976 ;Cisar
et al., 1981 ; Duguid & Gillies, 1957 ; Salit & Gotschlich, 1977) or are found in the periplasmic
space (Nelson et al., 1981) or in cell extracts (Gilboa-Garber, 1972a; Gilboa-Garber et al., 1977).
Pseudornonas aeruginosa ATCC 33347, grown in nutrient broth containing choline chloride for
3 d with shaking produces two lectins (Gilboa-Garber, 1972a; Gilboa-Garber et al., 1977). One
is a D-galactose-binding lectin (PA-I) (Gilboa-Garber 1972a, b), while the other binds Dmannose, L-fucose, L-galactose and D-fructose (PA-11) (Garber et al., 198 1 ;Gilboa-Garber et al.,
1977). The two lectins are low molecular weight glycoproteins, relatively resistant to heat and
proteolytic enzymes, which require divalent cations for their activity (Gilboa-Garber, 19723;
Gilboa-Garber et al., 1977). They agglutinate papain-treated erythrocytes more strongly than
untreated erythrocytes as well as other blood cells and various unicellular eukaryotes and
prokaryotes (Garber et al., 1981 ; Gilboa-Garber, 1972 b; Gilboa-Garber & Mizrahi, 1979;
Sharabi & Gilboa-Garber, 1980). Our preliminary studies showed that these lectins are not
associated with pili since the P . aeruginosa ATCC 33347 was poor in pili and exhibited maximal
lectin activity under conditions unfavourable for pili production. The present study was
undertaken to further examine the localization of the Pseudomonas lectins.
METHODS
Organism. Pseudomonas aeruginosa ATCC 33347 was grown at 30 "C with shaking for 72 h in 2 1 nutrient broth
(Difco) supplemented daily with 0.2% (w/v) choline chloride (NBC medium). Organisms were harvested by
centrifugation at 13000g for 10 min in a Sorvall RC2B centrifuge at 4 "C. Lysed cell debris was removed from the
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3086
J. GLICK A N D N . G A R B E R
intact cell sediment by gentle whirling and aspiration. The bacteria were washed twice with a 0.15 M-NaC1
solution. The yield of intact cells was 4 g (wet weight) or 736 mg (dry weight). Viable cell counts were made by
using MacConkey agar plates (Difco).
Release of enzymes and lectins from P . aeruginosa by various treatments. (a) Magnesium-osmotic shock.
Pseudomonas aeruginosa was grown in NBC medium and harvested as above. The packed cells were suspended
(37 mg dry wt ml- I ) in 0.01 M-Tris/HCl buffer (pH 8.4) containing 0.2 M-MgCl, (Cheng et al., 1970)at 22 "C and
stirred for 30 min. The suspension was centrifuged at 13000g and 4 "C for 10 min. The supernatant was separated
and kept at -20°C. The cell pellet was then suspended in distilled water at room temperature at the same
concentration of cells and stirred for 30 min. The bacteria were recovered by centrifugation as above and the
supernatant (osmotic shock water) was kept for various determinations. The treated cells were finally suspended
(3-7mg dry wt ml- l ) in Tris/HCl buffer (pH 7.2) containing 0.15 M-NaCI and were disrupted by treatment in an
ultrasonic disintegrator (MSE, 150 W) for 3 min at 0 "C.
(b)Freezing and thawing treatment. Bacteria were grown in NBC medium, harvested and washed as described
above. The cells were treated according to Furth (1975) with the following modifications: packed cells were
suspended at a concentration of 37 mg dry wt ml-I in 0-2 M-acetate buffer (pH 4.8). Samples (2 ml) were frozen in
an acetone/solid C 0 2 mixture and thawed in a 37 "C water bath. After two cycles of freezing and thawing the
samples were centrifuged at 30000 g for 20 min and the supernatant was stored. The treated cells were suspended
at a concentration of 3.7 mg dry wt ml-1 and disrupted ultrasonically as described above.
(c) Preparation of mureinoplasts and spheroplasts. Mureinoplasts and spheroplasts were prepared according to
Forsberg et al. (1970) with modifications (Glick et al., 1981). The spheroplasts were suspended in 0.01 M-Tris/HCl
buffer (pH 7.6) containing 0.5 M-sucrose and 0.01 M-M~CI,(TSM buffer) to a cell concentration of 36.8 mg dry wt
ml-1 and kept at 0 "C.
Lectin assays. The activity of P . ueruginosu lectins PA-I and PA-I1 was measured in U-type microtiter plates
(Nunc). Samples (100 pl) containing lectins were incubated with 100 p10.3 M-D-mannOSe (Sigma) (for inhibition of
PA-I1 activity) or 100 pl0.3 M-wgalactose (Sigma) (for inhibition of PA-I activity) for 1 h at room temperature.
Twofold dilutions in 0.15 M-NaCl of the treated samples were then carried out and 50 pl of 5 % (v/v) papain-treated
human erythrocytes suspension (Gilboa-Garber, 1972b) was added to each well. The suspensions were mixed by
gentle shaking and then allowed to stand at room temperature until the non-agglutinated erythrocytes settled into a
pellet. A unit of lectin activity is defined as the reciprocal of the dilution end-point (Cumsky & Zusman, 1979).
Enzyme assays. Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) was chosen as a cytoplasmic
marker (Cheng et al., 1971) and determined according to Malamy & Horecker (1964). One unit of G6PD was
defined as the amount of enzyme which reduced 1 pmol NADP h-' at 25 "C. 5'-Nucleotidase (EC 3.1 .3.5) was
chosen as a periplasmic marker (Bhatti et ul., 1976) and determined according to Neu & Heppel (1965) with a
slight modification (Garber & Nachshon, 1980). One unit of 5'-nucleotidase was defined as the amount of enzyme
which liberated 1 pmol inorganic phosphate from 5'-AMP h-' at 37 "C and pH 5.8.
R ESULTS
PseudomonasaeruginosaATCC 33347 produced PA-I and PA-I1 lectins with maximal activity
(per bacterial weight unit) after 72h incubation in NBC at 30 "C with shaking (Fig. 1). The
addition of choline chloride increased the lectin activity and the number of viable cells (Fig. 1).
The intracellular level of PA-I1 was generally lower than that of PA-I (Fig. 1, Tables 1,2). The
activity of each lectin was determined in the presence of the sugar which inhibits the activity of
the other lectin. The addition of both D-galactose and D-mannose abolished the entire
haemagglutinating activity of the cell extract. Washing the bacteria with 0.2 M-Mg2+followed
by a water osmotic shock or treating them by freezing and thawing induced release of
considerable amounts of the periplasmic marker 5'-nucleotidase, while there was no release of
the cytoplasmic marker G6PD and almost no release of lectin activity into the supernatants
(Table 1). Gradual conversion of the intact cells to mureinoplasts and spheroplasts led to a
considerable increase in their surface lectin activities, which was also detected by their ability to
agglutinate papain-treated human erythrocytes (Table 2). It has been shown that procedures
suitable for spheroplast formation exponentially growing Escherichia coli cells failed when used
for stationary phase cells, probably due to higher stability to destabilizers, such as Tris and
EDTA. Since EDTA, which is routinely used for inducing spheroplast formation in E . coli,
caused lysis of P . aeruginosa, a modification of the Forsberg et al. (1970) method was developed
which was used to form spheroplasts from the stationary phase cells. The modification included :
decreasing the cell concentration (in NaCl and sucrose washings, and in the lysozyme treatment
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3087
Lectins of Pseudomonas aeruginosa
Table 1. Release of lectins and enzymes from P . aeruginosa by M g 2 + washing, osmotic shock,
and freezing and thawing
Pseudomonas aeruginosa grown at 30 "C in NBC medium for 12 h was harvested and washed twice in
0.15 M-NaC1. The cells were treated either by osmotic shock or by freezing and thawing as described in
Methods. Cell samples were resuspended in 0.15 M-NaCl in 0.01 M-Tris/HCl buffer, pH 7.2 (3.7 mg dry
wt ml- *), ultrasonically disrupted, centrifuged and the supernatant fluids were used as the untreated
cell extracts. Lectin and enzyme activities were measured in Mg2+ wash, water shock and freeze-thaw
supernatant fluids as well as in the cell extracts of the treated and untreated cells. The untreated cell
extracts were used to estimate the residual cell-bound lectin and enzyme activities. Lectin and enzyme
activities were measured as described in Methods and calculated per 100 mg dry weight. The data
represent an average of five experiments.
Enzyme activity
Lectin activity
-A
r
5'-Nucleotidase
1
2
3
4
5
6
Treatment
0.2 M-MgCl, wash of cells
Water shock fluid of cells
washed by 0-2 ~ - M g c l ,
Cell extract of cells washed by
0.2 M-M~CI,and treated by
water shock
Freezing and thawing
supernatant
Cell extract of cells after
freezing and thawing
Untreated cell extract
G6PD
% Units
3
%
r
PA-I
%
Units
0.7
1.2
0
0
Units
586
0.3
27.9
47.9
0
0
554
29.6
50-8
7-6
100
21.5
36.9
0
36.7
63-0
100
58.2
7.6
7.6
\
PA-I1
Units
%
0
0
0.3
5
0.0
181423
99.3
14777
99.9
0
5565
3.0
43
0-3
100
100
177043
182608
96.9
100
14739
14782
99.7
100
Table 2. Pseudomonas aeruginosa lectin activity on the cell surface during their conversion to
mureinoplasts and spheroplasts
Pseudomonas aeruginosa was grown at 30 "C in NBC medium for 72 h as described in Table 1. Mureinoplasts and spheroplasts were prepared and their haemagglutinating activity against papain-treated
human erythrocytes was examined. Each value represents an average of five experiments.
Lectin activity
(units per 100 mg dry weight)
Cell preparation
Intact cells
Mureinoplasts
Lysozyme-treated mureinoplasts
Spheroplasts
Cell extract
PA-I
%
PA-I1
%
815
3 585
3978
21 521
312826
0.3
1.1
1.7
6.9
00
0
0
0
1448
16 666
0
0
0
8.7
00
of the mureinoplasts), increasing the lysozyme concentration and adding an osmotic shock
treatment by suspending the lysozyme-treated mureinoplasts in a low ionic strength solution
(Glick et al., 1981). Under these conditions we obtained a 90-100% yield of spheroplasts.
However, the amount of the lectins present on the surface of the mureinoplasts and of the
spheroplasts was low compared with that found in the cytoplasm of the same cells (Table 2).
Osmotic shock treatment of the lysozyme-treated mureinoplastsresulted in low release of G6PD
and the two lectins (10-1 3 %) and considerable activity (38.4%) of 5'-nucleotidase.
DISCUSSION
Pseudomonas aeruginosa lectins were found and purified from extracts of sonicated stationary
phase cells (Gilboa-Garber, 1972a, b; Gilboa-Garber et al., 1977). Their exact biological
function and localization were not fully revealed. Recently a close relationship between the level
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3088
J . GLICK AND N. G A R B E R
10"
1OO
'
I
3
4
*
109
0
108
107
---24
48
Time (h)
72
96
168
Fig. 1. Pseudomonas aeruginosa ATCC 33347 was grown overnight in nutrient broth at 30 "C with
shaking. Samples (10 ml) from this culture were transferred to 1 1 nutrient broth in 2 1 flasks with or
without the addition of 0.2%(final concentration) choline chloride (C). Samples were removed at 0,4,
8, 12, 24, 48,96 and 168 h and serial 10-fold dilutions were made. A 0-1 ml sample from 3-4 dilutions
was streaked over MacConkey agar plates in duplicate. After 24 h at 37 "C the colonies were counted
and the viable cell number was calculated in nutrient broth (0)and NBC (a).PA-I (HI) and PA-I1 (0)
levels were determined in the cell extracts of samples after disruption by sonication and centrifugation
as described in Methods.
of the intracellular lectins and the ability of the bacteria to secrete protease, haemolysin and
pyocyanin was described (Gilboa-Garber, 1982, 1983). The aim of the present study was to
systematically examine the localization of these lectins. We have used P . aeruginosa cells which
were grown in NBC medium for 72 h to obtain maximal production of the two lectins (Fig. 1).
The fact that transfer of the P . aeruginosa cells from 0.2 M-Mg2+(which stabilized the outer
membrane of stationary phase bacteria; Witholt et al., 1976) to distilled water caused
considerable 5'-nucleotidase release (Table 1) together with only low lectin activity indicates
that these lectins are located more internally. In order to confirm the cytoplasmic localization of
the two lectins attempts were made to convert P . aeruginosa cells to spheroplasts by conventional
techniques (Cheng et al., 1971; Forsberg et al., 1970; Weiss & Fraser, 1973). Only 5-20% of the
cells were converted to spheroplasts by these methods. Changes in P . aeruginosa wall
composition on ageing were reported (Collins, 1964) and these might contribute to the difficulty
of converting stationary phase cells to spheroplasts.
Gradual removal of P . aeruginosa envelopes enabled us to demonstrate the presence of some
activity of both lectins on the surface of intact cells, mureinoplasts and spheroplasts. This was
shown by the ability of these cell preparations to agglutinate papain-treated human erythrocytes
(Table 2), and to induce cap formation in human peripheral blood lymphocytes (Glick et al.,
1981). With the gradual removal of the cell envelopes, increasing lectin activity was revealed
(Table 2).
These results suggest that the two lectins (PA-I and PA-11) which are produced by P .
aeruginosa, are located principally in the cell cytoplasm and in small amounts on the
cytoplasmic and outer membranes and in the periplasmic space. The low activity of the lectins
located on the surface of the spheroplasts could be due either to originally surface-bound
glycoproteins or to cytoplasmically located lectins which were released by the modification of
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Latins of Pseudomonas aeruginosa
3089
the cell envelope and subsequently bound to the outer surface. The cytoplasmic location of these
bacterial lectins seems to differ from other known bacterial lectins but it is similar to that
described for the slime mould Dictyusteliurn purpureurn (Springer et al., 1980).
The authors wish to thank Miss E. Guggenheimer for the revision and Mrs B. Lederhendler for typing the
manuscript. This study is part of the Ph.D thesis of J. Glick, submitted to Bar-Ilan University, Ramat-Gan, Israel.
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