Glycine betaine and polar lipid composition in halophilic

FEMS Microbiology Letters 59 (1989) 157-160
Published by Elsevier
157
FEM 03554
Glycine betaine and polar lipid composition in halophilic
archaebacteria in response to growth in different
salt concentrations
B. N i c o l a u s 1, V. L a n z o t t i
1,.,
A. T r i n c o n e 1, M. D e R o s a 2, W . D . G r a n t 3 a n d A. G a m b a c o r t a 1
J Istitutoper la Chimica di Molecule di Interesse Biologico del Consiglio Nazionale delle Ricerche,
2 Istituto di Biochimica deUe Maeromolecole, I Facoha' di Medicina e Chirurgia, Universita' di Napoli, Napoli, Italy,
and 3 Department of Microbiology, Unioersity of Leicester, Leicester, U.K.
Received 7 January 1989
Accepted 9 January 1989
Key words: Halophiles; Archaebacteria, Ether lipids; Glycine betaine; Osmoregulation
1. SUMMARY
Examples of halophilic archaebacteria contain
low levels of between 1 and 20 mM trimethyl
glycine (glycine betaine). In disrupted cell preparations, the glycine betaine is associated with
the membrane fraction and is not detectable in
cell supernatants. Cells of Natronococcus occultus
grown in different salt concentrations show an
increase in cell-associated glycine betaine along
with an increase in the ratio of phosphatidyl
glycerophosphate (PG) to phosphatidyl glycerol
(PG) in the cell membrane,
Correspondence to: W.D. Grant, Department of Microbiology,
University of Leicester, Leicester LE1 7RH, U.K,
* Present address: Facolta' di Agraria, Universita' del Molise,
Campobasso, Italy
Abbreviations: PG = 2,3-di-O-phytanyl-sn-glycero-l-phosphoryl-3'-sn-glycerol, PGP = 2,3-di-O-phytanyl-sn-glycero-1phosphoryl-3 '-sn-glycero-1'-phosphate.
2. I N T R O D U C T I O N
Organisms which grow at high solute concentrations survive very low water activities. To
maintain turgor pressure in highly saline environments, halophilic or halotolerant organisms must
accumulate considerable quantities of solutes
within the cells. Typical organic osmotic protectants include sugars (sucrose, glucose, trehalose), polyols (glycerol, mannitol, glycosylglycerol)
or substituted amino acids (glycine betaine,
glutamine betaine) [1]. In general, the least osmotolerant forms accumulate sugars, those of intermediate tolerance polyols, whereas the most
osmotolerant or halotolerant accumulate betaines
[2,3]. However, the halophilic archaebacteria are
exceptional in this respect, in that instead of accumulating organic osmoprotectants, the internal
water activity of the cells is balanced by the
exclusion of Na + and the accumulation of K + [4].
Analyses of cell-associated K + of halophilic
archaebacteria have indicated concentrations of
up to 5 M [4] and N M R studies have shown that
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
158
most of the K + is free in the cell [5]. The activities
of enzymes and of protein synthesis are also
highest in ionic conditions similar to those shown
to be intracellular by direct measurements [4].
It has been assumed that halophilic archaebacteria are devoid of organic osmoprotectants since
there is no reason to suppose any deficiency in the
capacity of the cells to osmoregulate with K + [6].
However, we have recently shown [7] that polar
lipid extracts of haloalkaliphilic archaebacteria
contain glycine betaine in a complex formation
with phosphatidyl glycerophosphate (PGP). However, in the absence of any quantitative data at
that time we were unable to speculate on any role
played in osmoregulation by this compound in
halophilic archaebacteria.
We report here the detection of glycine betaine
in cells of a range of halophilic archaebacteria,
and show that it is associated with membranes in
those cells that we analysed by cell fractionation.
We report also modulation of phospholipid composition in response to different salt concentration
in the growth medium.
3. M A T E R I A L S A N D M E T H O D S
3.1, Microorganisms and culture conditions
Natronococcus occultus (NCMB 2192), Natronobacterium pharaonis (NCMB 2191) and Natronobacterium sp. SP8 [8] were grown in the liquid
medium described by Tindall et al. [9]. Halobacterium halobium (CCM 2090), Hb. salinarium
(NCMB 784), Hb. trapanicum ( N R C 34021), Hb.
saccharovorum (NCMB 2081), Hb. cutirubrum
(NCMB 763), Hb. halobium ( N C M B 777),
Haloferax volcanii (NCMB 2012), Haloarcula vallismortis (ATCC 29715), and Halococcus morrhuae
(NCMB 787) were grown in the liquid medium
described by Norton and Grant [10]. Nc. occultus
was also grown in the medium of Tindall et al. [9]
but at 10% ( w / v ) and 30% ( w / v ) NaC1 as well as
the usual 20% (w/v).
Cells were harvested in the late exponential
phase of growth by centrifugation, washed with
basal salt solution of the same composition as that
in the medium, and lyophilized.
3.2. Extraction and quantitative determination of
betaine
Lyophilized cells (2 g) of representatives of
main groups of halophilic archaebacteria were
extracted with 8 ml of trichloroacetic acid and
after centrifugation the supernatants were decanted and saved. The pellets were washed once
with 5 ml of 15% trichloroacetic acid and the
supernatants were combined with the original extract. These solutions were extracted three times
with 10 ml of diethyl ether to remove the trichloroacetic acid. The solutions were subjected to an
air stream for 60 min at room temperature to
remove residual ether. After this, the p H of the
solution was adjusted to between 7 and 8 with
N a O H (1 M), and the solution was adjusted to its
original volume with water.
The betaine content was determined using a
colorimetric method [11] and pyrolysis-gas chromatography [12].
3.3. Cell envelope preparation
Cells from Natronobacterium and Natronococcus spp. [9] were suspended in 10 ml of salt
solution containing (gl-~): NaC1 200.0; Na2CO3
18.5; KC1 2.0; MgSO4- 7 H 2 0 1.0, p H 9.8.
Cells from other halophilic archaebacteria were
suspended in 10 ml of salt solution containing
(gl 1): NaC1 200.0; KCI 2.0; MgSO 4 - 7 H 2 0 20.0;
p H 8.0.
Cells were disrupted by ultrasonication at full
power (50 W) for 2 min on ice (Ultrasonic Ltd).
Cell breakage was checked by phase microscopy. Cell envelope pellets were collected by
centrifugation at 15 000 × g for 40 rain and washed
twice in the same salt solution, pooling the supernatants.
3.4. Extraction and purification of lipids
Lyophilized cells (5 g) of Nc. occultus were
extracted continuously by Soxhlet for 12 h, with
CHCI3/MeOH
(1 : 1, v / v ) and then with
M e O H / H 2 0 (1 : 1, v / v ) . The extracts were pooled
and evaporated under vacuum.
Isolation of lipids was performed as previously
described [7]. Compounds were pure by T L C analysis (solvent system C H C 1 3 / M e O H / H 2 0
6 5 : 2 5 : 4 , by vol.) and were weighed to evaluate
their relative percentages.
159
Table 1
Concentrations of glycine-betaine in halophilic archaebacteria
Organism
Betaine content
( m g / g dry weight)
Natronococcus occultus (10% NaC1)
Natronococcus occultus
Natronococcus occultus (30% NaC1)
Natronobacterium pharaonis
SP8
Halobacterium halobium (2090)
Hb. salinarium
Hb. trapanicum
Hb. saccharovorum
Hb. cutirubrum
H. halobium (777)
Haloferax volcanii
Haloarcula vallismortis
Halococcus morrhuae
1.0
1.2
2.3
0.9
0.8
0.5
0.1
1.8
0.2
0.5
1.8
0.4
0.2
2.3
All microorganisms were grown in the standard medium, as
described in MATERIALS AND tClETrlODS, at 20% NaCI (w/v),
unless otherwise stated.
3.5. Methanolysis of lipids
Acid methanolysis of lipids was done in dry
methanolic 2 N HC1. The reaction mixture was
heated at 110 °C in stoppered reaction tube for 16
h. After being cooled, the hydrolysis products
were dried under vacuum and then purified on a
silica gel column eluted with CHC13. The diether
fraction was resolved into 2,3-di-O-phytanyl-snglycerol and 2-O-sesterterpanyl-3-O-phytanyl-snglycerol by high performance liquid chromatography (Waters Associates) in n-hexane/ethyl
acetate (9:1, v / v ) using a Microporasil column
(flow rate 1 m l / m i n ) [13].
functioning as an osmotic protectant (0.5-2 M)
[1]. In the example we tested (Nc. occultus), the
glycine betaine content did increase from approximately 10 mM in cells grown in 10% (w/v)
NaC1 to approximately 20 mM in cells grown in
30% (w/v) NaC1, but still did not approach the
levels seen in eubacteria [1,2].
Analyses of distribution of glycine betaine in
cells of Hb. halobium (CCM 2090) and Natronobacterium sp. SP8 revealed that the glycine betaine
is exclusively associated with cell envelope fractions. No glycine betaine was detected in the
supernatant fractions derived from these cell envelope preparations. These results, taken together
with the earlier study [7], showing that glycine
betaine is complexed with PGP in polar lipid
extracts, strongly suggest that in these archaebacteria glycine betaine is localized in the cell membrane rather than free in the cytoplasm, and as
such does not participate in osmoregulation in any
role so far described.
Glycine betaine forms an ionic complex with
phospholipid only when two charges are available
for complex formation. Thus phosphatidyl glycerol
(PG) does not form a complex, nor does the newly
described cyclic phosphate derivative of PGP [15].
It is of note that in Nc. occultus, the relative ratio
of P G P / P G increased from 2 to 5 when the salt
concentration of the medium increased from 10 to
30% NaC1 (w/v), although the total lipid content
remained constant, Nc. occultus has both diphytanyl (C20, C20 ) and sesterterpanyl-phytanyl
(C25, C20) forms of PG and PGP. Table 2 shows
that only the levels of the C20, C2o form of PG
and the C25, C20 form of PGP are significantly
4. RESULTS A N D DISCUSSION
Table 2
Halophilic archaebacteria presently comprise
nine groups on the basis of polar lipid analyses
and nucleic acid hybridization studies [14]. Examples from all of these groups are represented in
this study. Table I shows the amounts of cell-associated glycine betaine in the examples grown in
20% (w/v) NaC1. Concentrations of glycine betaine range from 0.1 mg g - 1 dry cells ( < 1 mM) to
2.3 mg g - t dry cells (20 mM), amounts well outside the range normally expected for a compound
Relative proportions (%) of C25, C20 and C2o, C20 forms of PG
and PGP in Nc. occultus grown at different salt concentrations
Salinity of
PG *
growth medium
(% NaC1)
C25,
10
20
30
9
9
8
PGP *
C20 C20,C20 C25,C20 C20,C20
26
16
8
14
26
35
51
49
49
• The total a m o u n t of PG plus PGP = 100.
The yield of total lipids is similar for all salt concentrations.
160
affected by the salt concentration of the medium.
The resulting increase of the C25, C20 form in Nc.
occultus grown in 30% NaC1, supports the hypothesis that the C25 chains may have an effect in
stabilizing the membranes of haloalkaliphilic
archaebacteria [8].
Since p G and PGP comprise the majority of
the polar lipids in Nc. occultus [7], any change in
the ratio of PGP implies a significant change in
the number of negative charges per tool polar
lipid.
The function of glycine betaine in membranes
of halophilic archaebacteria remains to be established. It can be ruled out that the compound has
any obvious osmoregulation solute function. However, it might be involved in some sensory capacity, acting as a trigger for the main osmoregulatory process in much the same way that K + concentration may be the link between growth in
media of high osmolarity and concomitant accumulation of osmoprotectants by eubacteria such
as E. coli [16]. It might be that glycine betaine has
a role in charge shielding in the membranes of
halophilic archaebacteria as phospholipid composition is modulated in response to different salt
concentrations in the growth medium. We are
currently investigating its role.
ACKNOWLEDGMENTS
We thank Mr. G. Pinch for growth of microorganisms and Miss M.C. Manca for her skilled
assistance in some experiments.
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