Download Susceptibility of phospholipids of Proteus mirabilis smooth and

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FEMS MicrobiologyLetters 5 (1979) 13-16
© CopyrightFederation of European.MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
SUSCEPTIBILITY O F P H O S P H O L I P I D S O F P R O T E U S M I R A B I L I S
S T R A I N S TO E N D O G E N I C P H O S P H O L I P A S E A A C T I V I T Y
SMOOTH AND R O U G H
SHLOMO ROTTEM, SHIMON ULITZUR *, MIRIAM HASIN and SHMUEL RAZIN
Biomembrane Research Laboratory, Department o f Clinical Microbiology, The Hebrew University-Hadassah Medical School,
Jerusalem and * Department o f Food Engineering and Biotechnology, The Technion, Haifa, Israel
Received 15 August 1978
1. Introduction
Determination of phosphotipid distribution
between the inner and outer leaflets of biological
membranes is based to a large extent on the use of
phospholipases, comparing the sensitivity of phospholipid hydrolysis in intact ceils to that in isolated
membranes [ 1]. Yet, previous studies with Gramnegative bacteria showed that these organisms possess
a potent endogenic phospholipase activity located in
the outer membrane [2-5]. This endogenous activity
may interfere with the use of phospholipase for phospholipid localization studies. Nevertheless, previous
studies with Proteus mirabilis showed that the endogenous phospholipase was triggered only when the
structure of the cell envelope was affected [6]. The
present study shows that in rough mutants of P. mirabilis, mainly in the deep rough R 45 strain, high endogenous phospholipase activity can be demonstrated
even in intact cells. The high activity was expressed
by rapid degradation of the outer membrane phospholipids as well as by hydrolysis of an exogenously
supplied phospholipid. The triggering of the endogenous enzyme activity in intact cells of the deep
rough mutants is discussed in view of possible differences in the organization of components and permeability properties of the smooth and deep rough
strains [7,8].
2. Materials and Methods
The smooth P. mirabilis strain S 1959 and its
rough mutant R 110, R 51 and R 45 were kindly pro-
vided by Dr. K. Kotelko (University of,Lodz, Lodz,
Poland). The organisms were grown at 37°C with
vigorous shaking in 250 ml volumes of a basal salt medium [9] supplemented with 0.5% peptone (Difco),
1.2% NaC1 and 0.5% glucose. To label membrane
phospholipids, 1 HCi of [1-14C] oleic acid (50 mCi/
mmole, The Radiochemical Centre, Amersham, England) was added to the growth medium. The organisms were harvested at the mid-exponential phase of
growth (85-130 Klett units, measured with a KlettSummerson photoelectric colorimeter equipped with
filter No. 54), washed with 50 ml of deionized water
and resuspended in 100 mM Tris • HC1 buffer, pH 7.5.
The washed cells were then disrupted by sonic oscillation in a Raytheon model DF 101 sonic oscillator at
0.9 A for 3 min, and the outer and cytoplasmic membrane fractions were separated by sucrose density gradient centrifugation [ 10].
Phospholipase A activity was measured either by
the release of radioactive fatty acids from endogenous
14C-labeled phospholipids of cells or membrane preparations [6] or by the release of myristic acid from an
exogenously supplied L-a-dimyristoylphosphatidylcholine (L-a-DMPC). The degradation of the endogenous radioactive phospholipids was determined in a
reaction mixture (1 ml) containing [ 1-14C] oleatelabeled cells (10 mg protein) or membrane preparations (1 mg protein) and 2.5 mM CaC12 in 100 mM
Tris • HC1 buffer, pH 7.5. After 2 h of incubation at
37°C, the reaction was stopped by the addition of
EDTA. Lipids were extracted, chromatographed on
silica gel G plates and the radioactivity in the various
lipid fractions was determined [6]. Phospholipase
activity was expressed as the percentage of radioactiv-
14
ity in the free fatty acid fraction as compared with
the total radioactivity in membrane phospholipids.
Degradation of exogenously added phospholipids
(L-a-DMPC) was measured by a bioluminescence
assay of the myristic acid released, using the procedure of Ulitzur and Heller [11]. The reaction mixture
(1.25 ml) contained 0.9 ml of artificial sea water in
0.02 M morpholinopropane sulfonic acid (Sigma, St.
Louis, Mo.), pH 7.2, 50 laM L-a-DMPC, 0.1 ml of
Beneckea harveyi M 17 culture (70 Klett units) and
0.1 ml ofP. mirabilis S or R cultures (about I00
Klett units), in a scintillation vial. The luminescence
was recorded with time at 20°C in a photomultiplier
photometer and expressed as light units, where one
light unit is equivalent to 3 • 107 quanta sec -1 [12].
3. Results and Discussion
As previously described [6], P. mirabilis, like other
Gram-negative bacteria [ 2 - 5 ] , possesses potent phospholipase A activity. The endogenous activity of
intact cells of the smooth S 1959 strain was however
very low, but became pronounced after disruption of
the cells, and was found associated with the outer
membrane fraction (Table 1). Almost all the phospholipids of the outer membrane were degraded after
2 h of incubation at 37°C resulting in the liberation
of free fatty acids with no significant accumulation of
lyso compounds. Phospholipase C activity described
in E. coli envelope preparations [2] was not detected
in the P. mirabilis strains.
Table 2 shows that, whereas phospholipase activities of sonicated preparations ofP. mirabilis S and R
strains were roughly the same, the activities of intact
cells differed markedly. Phospholipase A activity of
intact S 1959 cells was low while the activities of the
R mutants were considerably higher. Most pronounced was the high activity of the deep rough R 45
strain ofP. mirabilis which contains a lipopolysaccharide known to lack most of its polysaccharide chain
[13]. The activity of cells of the rough mutants
washed once with deionized water after harvesting
was about three times higher than the activity of unwashed cells (Table 2). Nevertheless, cell viability was
only marginally affected by the washing as determined by the colony counting technique using nutrient agar (Difco) plates.
If the enzyme located in the outer membrane
would also hydrolyze the phospholipids present in
the cytoplasmic membrane, one would expect that
extensive dilution of the sonicated cell preparations
containing both outer and cytoplasmic membrane
fragments would decrease hydrolysis by decreasing
the chances for contact between the enzyme present
in the outer membrane and the substrate present in
the cytoplasmic membrane. Yet serial two-fold dilutions (up to 1 : 128) of sonicated cells of the P. mirabilis R 45 strains had almost no effect on the percent-
TABLE 2
TABLE 1
Degradation of P. mirabilis phospholipids by endogenous
phospholipase A activity.
The phospholipids of the organisms were labeled during
growth with [1-14C]oleate. The preparations were incubated
for 2 h at 37°C before the lipids were extracted.
Degradation of phospholipids in intact and sonicated cell
preparations ofP. rnirabilis S and R strains.
Membrane phospholipids were labeled during growth with
[1-14C]olei c acid. Cells were harvested at the mid-exponential phase of growth, washed, and sonicated as described in
Materials and Methods.
Strain
Preparation
Radioactivity in lipid fraction
(% of total)
Phospholipid degradation (radioactivity in free
fatty acids, % of total)
Intact cells
Polar
lipids
Intact cells
95.9
Isolated cytoplasmic
88.0
membrane
Isolated outer membrane
6.5
Diglycerides
Free fatty
acids
0.2
0.9
3.7
11.0
0.7
92.5
S 1959
R 110
R 51
R 45
Sonicated cells
Unwashed
Washed
2.3
2.1
4.6
11.1
3.1
6.6
14.5
30.3
41.4
41.7
44.1
44.6
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age of endogenous phospholipids degraded (about
45% of the total) within 2 h of incubation at 37°C.
This finding suggests that the degradation is largely
due to an intramembranous enzyme-substrate interaction rather than a transmembranous hydrolysis.
Thus, the presence of degradation products is mainly
due to hydrolysis of outer membrane phospholipids
by an enzyme present in the outer membrane fragments rather than to hydrolysis of cytoplasmic membrane phospholipids.
The much higher phospholipase activity of intact
R 45 cells may be related to the differences in the
structural organization of the outer membrane components between smooth and deep rough strains [7,
8], or to differences in the outer membrane integrity
and/or permeability properties [7,14]. Based on
results with a non-penetrable, covalent labeling
reagent, Kamio and Nikaido [8] concluded that the
distribution of phospholipids between the outer and
inner leaflets of the outer membrane differs in
smooth and deep rough strains. In the smooth strain
most phospholipid molecules are located in the inner
leaflet and the outer leaflet contains protein and lipopolysaccharide molecules, whereas in the deep rough
strain 20-40% of the phospholipids are present in
the outer leaflet, forming phospholipid bilayer
regions. If the phospholipase, like most outer membrane proteins, is located in the outer leaflet, its activity on endogenous phospholipids may be restricted
due to their absence from the outer leaflet of intact
S 1959 cells. Accordingly, such enzyme-substrate
interactions could take place in the R 45 strain,
which does contain phospholipids in the outer leaflet.
Upon disrupting the cells by sonic oscillation, rearrangement of outer membrane phospholipids might
occur which could result in a pronounced endogenous
activity also in the smooth strain. However, this
hypothesis does not explain the increased activity in
washed cells (Table 2), and it was further challenged
by showing the degradation of exogenous phospholipids using a bioluminescence assay. This system is
based on recent findings of Ulitzur and Hastings [ 15]
and Ulitzur and Heller [ 11 ] that a dim mutant of the
luminous bacterium Beneckea harveyi will respond to
long chain fatty acids, mainly myristic acid, by
increasing its luminescence, enabling rapid determination of picomole amounts of myristic acid. The kinetics of phospholipase activity in P. mirabilis S 1959
and R 45 strains were therefore easy to follow by
measuring luminescence in a reaction mixture containing L-a-DMPC. Fig. 1 shows that the rate of phospholipid hydrolysis by intact and sonicated R 45 ceils
was about the same, whereas intact cells of the S
1959 strain remained inactive unless disrupted by
sonication. Although this result suggests that the high
phospholipase activity of cells of the R 45 strain and
low activity of the S 1959 cells is not due to the nonavailability of substrate, one cannot rule out the possibility that the long polysaccharide chains in the
smooth S 1959 strain restrict the access of the exogenous substrate to the enzyme by steric hindrance.
These differences in activity can also be explained on
the basis of differences in the outer membrane integrity and/or permeability properties between the
smooth and deep rough strains. Phospholipase activity was enhanced in cells of the smooth strains of
Escherichia coli subjected to adverse conditions that
affect the integrity of the outer membrane even when
the changes were not sufficient to prevent growth
[3-5]. Likewise, the presence in bacterial cells of a
75
e.,I,-¢.,-
50
...J
v
¢-,
Oq
25
1959
E
._1
0
4
I
I
8
12
Time (min)
Fig. 1. Initial rates of luminescencedeveloped in reaction
mixtures containing intact (solid lines) or sonicated (broken
lines) cells ofP. mirabilis S 1959 and R 45 strains and L-~DMPC as substrate. The bar represents the luminescence
equivalent to 20 nM of myristic acid.
16
potent inhibitor o f phospholipase that might be
involved in the regulation of phospholipid catabolism
in vivo was also described [16]. Such an inhibitor, if
present in the periplasmic space, may leak out o f the
highly permeable rough strain [14] and enable phospholipid degradation, but should be retained in the
less permeable smooth strain.
Acknowledgements
We are most grateful to Prof. K. Kote~ko for providing us with the R-mutants ofP. mirabilis. This
work was supported by a grant from the Stiftung
Volkswagenwerk.
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