Download The polar lipid composition of walsby`s square bacterium

MICROBIOLOGY
LETTERS
ELSEVIER
FEMS Microbiology
Letters 138 (1996) 135-140
The polar lipid composition of Walsby’s square bacterium
Aharon Oren
*, Sara
Duker I, Sigalit Ritter
Division of Microbial and Molecular Ecology, Institute of Life Sciences. and the Moshe Shilo Center for Marine Biogeochemistry,
The Institute of Life Sciences, The Hebrew Unkersity of Jerusalem, Jerusalem 91904, Israel
Received 26 January
1996; accepted
17 February
1996
Abstract
Square, gas vacuole-containing Archaea of the type first described by Walsby were found to dominate in a saltern
crystallizer pond in Eilat, Israel. To obtain information on the taxonomic position of these yet uncultured bacteria, we
analyzed the polar lipids present in the microbial community in the saltern brine. In addition to phosphatidylglycerol,
phosphatidylglycerophosphate and phosphatidylglycerosulfate we found one glycolipid, chromatographically identical with
the sulfated diglycosyl diether lipid found as the major glycolipid in Huloferax species. As the square bacteria contributed at
least 85% of the total membrane surface in the biota of the sample examined, we concluded, based on polar lipid
composition, that these organisms are unrelated to the genera Halobacterium and Haloarcula, and probably belong to a new
genus.
Keywords: Square bacteria;
Halophilic
Archaea;
Halobacterium; Haloarcula: Sahern; Glycolipid
1. Introduction
Square flat halophilic Archaea were first found by
in a coastal brine pool (the Gavish Sabkha)
in the Sinai peninsula, Egypt [1,2]. These unusually
shaped bacteria are square to rectangularly shaped,
very thin (0.1-0.2 pm) organisms, easily recognizable by the presence of refractile gas vesicles. Their
presence has since been documented in a variety of
hypersaline environments, such as halite-saturated
saltem ponds in Mexico [3] and in Spain [4], and in
Lake Saxkoye, Crimea [5].
Walsby
* Corresponding
author. Tel: + 972 (2) 658 495 1; Fax f972
(2) 6.52 8008; E-mail: [email protected]
’ Sara Duker was tragically killed on 25 February 1996.
037%1097/96/$12.00
0 1996 Federation
PfI SO378- 1097(96)00085-7
of European
Microbiological
No cultures are extant of this interesting organism, for which the names ‘Quadra’ [2] or ‘ Arcuh’
[3] have been suggested. A single report has been
published on the successful cultivation of halophilic
square vacuolated cells in pure culture [4], but no
details were given on its growth requirement and
physiology, and the isolate has since been lost. Thus,
all studies performed on these unusual prokaryotes
are based on material collected from natural accumulations of the square bacteria in hypersaline environments [l-3,5-7].
As no pure cultures are available for study, the
taxonomic position of the square bacterium within
the family Halobacteriaceae is as yet unclear. The
fact that members of the genus Hulourcula do occasionally produce flat square cells in culture is insufficient as proof for a phylogenetic relationship [Sl.
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The polar lipid composition of the cell membrane,
and particularly the kinds of glycolipids present, is
one of the main characteristics in which the genera
of the Halobacteriaceae differ [8,9]. Thus, lipid analysis of natural communities
of halophilic Archaea
can provide information on the types of organisms
present. Analysis of polar lipids extracted from collected biomass has been used in studies of Archaea
in the Dead Sea and in saltern crystallizer ponds
[IO-121.
An attempt was made by Fredrickson
and
coworkers to obtain information on the nature of
Walsby’s square bacterium in the Gavish Sabkha on
the basis of the types of the glycolipids present [ 131.
Using fast atom bombardment
mass spectrometry
they found, in addition to the diether derivatives of
phosphatidylglycerol
(PG), phosphatidylglycerophosphate (PGP), and phosphatidylglycerosulfate
(PGS).
a trisaccharide glycolipid and its sulfated derivative
(TGD-1 and S-TGD-I), identical to the glycolipids
of Halobacterium cutirubrum (salinarum) [9]. However, some doubt may arise on the nature of the
sample examined: the authors state that they analyzed “the extremely halophilic square archaebacterium originally isolated from the Gavish Sabkha”,
but as noted above, this type of square bacterium has
not yet been cultured.
We found a high percentage of percentage of
gas-vacuolated
square bacteria (around 55% of the
bacteria present) in the bacterial community
in a
saltem crystallizer pond in Eilat, Israel. This dominance of square Archaea presented us with the opportunity to obtain information on their taxonomic
position, based on the use of polar lipids as chemotaxonomic markers.
2. Materials
and methods
2. I. Sample collection and microscopical
zation of the bacterial commun&y
characteri-
Brine samples were collected on 6 December
1995 from crystallizer pond no. 302 of the solar
saltem system at Eilat, Israel [ 121.
To enumerate bacteria, 10 ml portions of brine
were centrifuged at room temperature for 15 min at
12 000 X g. After removal of about 9.5 ml of super-
natant, the cell pellet was resuspended in the remaining liquid, the volume of the final suspension was
measured, and its bacterial density was determined
with a Petroff-Hauser counting chamber and a microscope equipped with phase contrast optics.
To obtain information on the number of actively
respiring cells, we examined the formation of intracellular formazan granules upon incubation with 2( p-iodophenyll-3(
p-nitrophenyll-5-phenyl
tetrazolium chloride @NT) [14,15]. 10 ml portions of
brine were incubated for 4-6 h at 35°C with 0.2 ml
of a solution of 1 g 1-l INT and 0.1 ml of 0.1%
glycerol. Cells were then collected by centrifugation
and examined microscopically as above.
2.2. Lipid extraction and characterization
Bacteria were collected from 10 1 brine by centrifugation (45 min. 5000 X g>. Cell pellets of the
saltem brine community or reference cultures (see
below) were suspended in 1 ml H,O, and extracted
with 3.75 ml methanol-chloroform
2:1 (v/v) for 4
h. The extract was collected by centrifugation.
and
the pellet reextracted with 4.75 ml methanol-chloroform-water (2: 1:0.8). Chloroform and water (2.5 ml
each) were added to the combined supematants to
achieve phase separation, and after centrifugation the
chloroform phase was collected, and dried in a vacuum desiccator.
Lipids were redissolved in a small volume of
chloroform, applied to silica gel plates (Sigma, 20 X
20 cm>, and separated by single development with
chloroform-methanol-acetic
acid-water
(85:22.5:
IO:4, v/v>. For two-dimensional
chromatography
a
solvent
system
of chloroform-methanol-water
(65:25:4) was used in the first dimension, and chloroform-methanol-acetic
acid-water (80: 12: 15:4) in
the second dimension.
Lipid spots on the plates were detected by spraying the following reagents [9- 111: (1) 0.5% cu-naphthol in 50% methanol, followed by 5% H,S04 in
ethanol, and heating the plates at 150°C (allowing
selective detection of glycolipids); (2) 0.1% CeSO,
in 2 N H2S04, followed by heating at 150°C (a
general lipid stain, allowing differentiation of glycolipids from other lipids by color); (3) ammonium
molybdate-sulfuric
acid reagent, for the detection of
phospholipids.
A. Oren et al. / FEMS Microbiology Letters 138 (19%) 135-140
2.3. Reference strains and culture conditions
The following reference cultures of halophilic
Archaea were used in the polar lipid studies:
Halobacterium halobium (salinarum)
Rl, and
Halobacterium salinarum strain 5, grown in medium
containing (all concentrations in g 1-l >: NaCl, 250;
KCl, 5; MgCl, .6H,O, 5; NH,Cl, 5; yeast extract,
5; pH 7; Haloferax volcanii ATCC 29605, Haloferax
mediterranei ATCC 35300, and Halorubrum saccharouorum ATCC 29252, grown in: NaCl, 175;
MgCl, +6H,O, 20; K,SO,, 5; CaCl,. 2H,O, 0.1,
yeast extract, 5; pH 7; Haloarcula marismortui
ATCC 43049, and Haloarcula vallismortis ATCC
29715, grown in medium composed of NaCl, 206;
MgSO,. 7H,O, 36; KCl, 0.37; CaCl, .2H,O,
5;
MnCl,, 0.013, and yeast extract, 5; pH 7.
3. Results and discussion
The brine sample examined (density 1.239 g ml-‘,
360 g 1-l total dissolved salts) contained 1.3 X lo7
137
microscopically recognizable bacteria per ml. Flat
square or rectangular cells formed the most abundant
type of cells observed (Fig. 1, left panel). These
cells, 2.5-7.5 pm in diameter, are morphologically
identical to Walsby’s square bacteria [1,3,7]. Microscopic examination of the square cells in the brine
showed the presence of refractile gas vacuoles in
most of the cells; no gas vacuoles are seen in Fig. 1
as the high-speed centrifugation employed in sample
preparation caused their collapse. Cells with a recognizable square or rectangular morphology contributed about 55 f 3% of the total bacterial counts.
This value probably underestimates the true contribution of the square Archaea to the community, as
some of the squares may mistakenly be counted as
rod-shaped cells when seen in profile (compare also
Fig. 2 in Ref. [3]). While the occurrence of cells with
a square morphology has earlier been reported in the
Eilat saltem ponds [ll], their contribution to the
bacterial community observed earlier (up to 20-23%
of the total bacterial numbers) was much less extensive than in the presently examined brine.
Most of the flat square bacteria observed in the
Fig. 1. Micrograph of the bacterial community present in the Eilat saltem crystallizer brine, before (left panel) and after (right panel)
incubation with INT. Cell suspensions were mixed with an equal volume of 1% molten agar in 25% NaCl to keep cells from moving during
photography. Granules in INT-exposed cells (right panel, arrows) consist of INT-formazan;
the small dark granules occasionally seen in
cells not exposed to INT (left panel, arrow) probably consist of poly-P-hydroxybutyrate
[3]. Phase contrast; bar = 10 pm,
138
bent
PigBlent*
00000~0
active respiration system, and has been previously
used for the estimation of the number of viable cells
in natural samples [15]. We reported similarly high
percentages of active cells (85-90%) in brine samples collected from the Eilat samples in an earlier
study [ 141. Inhibitor studies proved that the formation of intracellular formazan granules depended on
an enzymatic process: formazan accumulation
was
abolished in the presence of formaldehyde (0.5 or
I %)
or HgC12 (0.1 or 1 mM). In the presence of
cyanide (0.2 or 1 mM) formazan formation continued uninhibited.
as was reported for marine and
freshwater bacterial communities [ 151.
The occurrence of a dense bacteria1 community
dominated by active square cells of the type described by Walsby in the saltern brine sample presented us with a unique opportunity to obtain information on the possible taxonomic affiliation of the
species, using polar lipids as chemotaxonomic
markers. While the square bacteria contributed at least
55% to the total numbers of Archaea in the brine,
their contribution
to the total lipid content of the
biomass is expected to be much larger due to their
large surface area. We calculated an average surface
area of 3 I .4 + 15.6 pm’ for the square cells (a
calculation based on a thickness of 0.1 pm [2]), and
7.0 * 3.4 km* for the rods observed. Assuming that
the glycolipid content per unit of surface of the
square cells is comparable to that of the rod-shaped
Archaea, we can calculate that the square bacteria
will contribute about 85% of the total glycolipids.
Thin layer chromatography
of an extract of the
ooooooo
PG
PGP
PGS
. . . . . . .
Fig. 2. Thin layer chromatogram of polar lipids extracted from the
Eilat saltem pond biomass (lane 4), as compared to extracts of
reference strains of halophilic Archaea: Halohacreriun~ solinarum
5 (lane 1). Halobacterium
halobium (salinurum)
RI (lane 2).
Halorubrum
saccharor~orum (lane 3). Haloferux rolctrnii (lane 5).
Halqferas
mediterranei
(lane 6). and Hrrloarcula
murismortui
(lane 7). Lipids were stained with o-naphthol-HzSO,
(left panel),
CeSO, -HISO,
(middle panel). and ammonium molybdate strain
for phospholipid detection (right panel). Glycolipids are shown in
black. The tentative identification of the lipid spots. based on
literature data [9-l I] is indicated. S-TeGD = the sulfated tetraglycosyl lipids of H&bacterium
species: TGD-2 = the triglycosyl
lipid of Hu/ocrrcula;
S-DGD-I = the sulfated diglycosyl
lipid
present as the major glycolipid of Haloferar species.
Eilat brine sample were living, active cells. Upon
incubation with INT, 83 f 2% of the square cells
were found to accumulate intracellular red formazan
granules (Fig. I. right panel). INT reduction with
formazan formation indicates the presence of an
Pigments
I
S-DGD-1
-0
Fig. 3. Two-dimensional
thin-layer chromatograms
of polar lipids extracted from the crystallizer brine biomass, alone (left panel),
cochromatographed
with an extract of Huloferax c~olctmii (middle panel). or with an extract of Halorubrum saccharororum
(right panel).
Lipids were visualized with the CeSO, spray reagent. Glycolipids are shown in black. The tentative identification of the lipid spots is
indicated.
A. Oren et al./ FEMS Microbiology L&ten 138 (1996) 135-140
biomass collected from the saltem brine separated
four types of polar lipids: PG, PGP, PGS, and a
single glycolipid, chromatographically identical with
S-DGD-1, the major glycolipid found in all Haloj&-ax species (Figs. 2 and 3). A similar polar lipid
composition was reported earlier in the biomass of
the Eilat saltems at a time in which the contribution
of the square bacteria to the total bacterial number
was much smaller than in the presently analyzed
sample. This finding alone does not justify the conclusion that the square cells may belong to the genus
Haloferax. Haloferax species lack PGS, a lipid found
in abundance in the extract of the saltem community.
Moreover, DGD- 1, characteristically found as a minor glycolipid in extracts of representatives of the
genus Haloferax, was not detected (Fig. 2).
The results clearly show that contrary to earlier
evidence [ 131,the square bacteria are unrelated to the
Halobacterium salinarum group, as no trace of the
triglycosyl diether lipids TGD-1 and S-TGD-1 could
be detected in the lipid extract of the saltem biomass.
Likewise, a possible taxonomic relationship with the
genus Haloarcula, as suggested earlier (see [8]) can
be excluded, as TGD-2, the characteristic triglycosyl
diether lipid of Haloarcula was not found either.
The polar lipid composition of the saltem brine
biomass examined most closely resembled that of the
recently created genus Halorubrum, whose members
(including H. saccharouorum, H. sodomense, H.
lacusprofundi, and H. trypanicum) are characterized
by the presence of a sulfated diglycosyl diether and
phosphatidylglycerosulfate. However, the single glycolipid found in the brine is chromatographically
different from that of H. saccharouorum. Thus, a
positive assignment of the square bacteria to one of
the known genera within the Halobacteriaceae on the
basis of polar lipid composition is still not possible,
and they may belong to an as yet undescribed genus.
The above attempt to obtain information on the
species present on the basis of polar lipid analysis is
based on the assumption that the types and amounts
of glycolipids present do not vary greatly under
different growth conditions. Little evidence has been
presented to date on the occurrence of phenotypic
variations in the types of glycolipid present in
halophilic Archaea, though the relative amount of the
different glycolipids may vary slightly according to
growth conditions.
139
The possibility cannot be ruled out that the typical
morphology of square flat cells with gas vacuoles
may be common to more than one type of organism,
belonging to different genera. In that case, the sample analyzed by Fredrickson et al. [ 131 and found to
contain TGD-1 and S-TGD-1 may have contained a
square bacterium with a taxonomic affiliation different from that of the square cells abundant in the Eilat
saltem brine.
Acknowledgements
We thank the Israel Salt Co., Ltd. for allowing
access to the Eilat saltems, and the staff of the
Interuniversity Institute of Eilat for logistic support.
This work was supported by a grant from the Israel
Science Foundation administered by the Israel
Academy of Sciences and Humanities.
References
111Walsby,
121Parkes,
A.E. (1980) A square bacterium. Nature 283, 69-7 1.
K. and Walsby. A.E. (1981) Ultrastructure
of a
gasvacuolate square bacterium. J. Gen. Microbial. 126, 503506.
W. (1981) Walsby’s square bacterium: fine
131 Stoeckenius,
structure of an orthogonal
procaryote.
J. Bacterial.
148,
352-360.
[41 Torrella, F. (1986) Isolation and adaptive strategies of
haloarculae to extreme hypersaline habitats. In: Abstracts of
the Fourth International Symposium on Microbial Ecology,
p. 59. Slovene Society for Microbiology, Ljubljana.
151 Romanenko, V.I. (1981) Square microcolonies in the surface
water film of the Saxkoye lake. Mikrobiologiya 50. 571-574
(in Russian).
161Kessel, M. and Cohen, Y. (1982) Ultrastructure of square
bacteria from a brine pool in southern Sinai. I. Bacterial.
150, 85 l-860.
[71 Kessel, M., Cohen, Y. and Walsby, A.E. (1985) Structure
and physiology of square-shaped and other halophilic bacteria from the Gavish Sabkha. In: Hypersaline Ecosystems.
The Gavish Sabkha (Friedman, G.M. and Krumbein, W.E.,
Eds.), pp. 267-287. Springer-Verlag,
Berlin.
[81 Grant, W.D. and Larsen, H. (1989) Group III. Extremely
halophilic archaeobacteria.
Order Halobacteriales ord. nov.
In: Bergey’s Manual of Systematic Bacteriology,
Vol. 3
(Staley, J.T., Bryant, M.P., Pfennig, N. and Holt, J.G., Eds.1,
pp. 2216-2233.
Williams and Wilkins, Baltimore, MD.
M., Rodriguez-Valera,
F., Juez. G., Ventosa,
[91 Torreblanca,
A., Kamekura, M. and Kates, M. (19861 Classification
of
non-alkaliphilic
halobacteria based on numerical taxonomy
[IO]
[I l]
[I21
[13]
and polar lipid composition. and description of Ha/ocwc~/~
gen. nov. and Ha1ofercc.r gen. nov. Syst. Appl. Microbial. 8.
89-99.
Oren. A. and Gurevich. P. (1993) Characterization
of the
dominent halophilic archaea in a bacterial bloom in the Dead
Sea. FEMS Microbial. Ecol. 12. 249-2.56.
Oren, A. (1993) Characterization
of the halophilic archaeal
community in saltern crystallizer ponds by means of polar
lipid analysis. Int. J. Salt Lake Rea. 3. 15-29.
Oren, A. 1994. The ecology of the extremely halophilic
archaea. FEMS Microbial. Rev. 13. 115-440.
Fredrickson. H.L.. Rijpstra. W.I.C. Tab. A.C., con der Creel’.
J., LaVos, G.F. and de Leeuw, J.W. (1989) Chemical characterization of benthic microbial assemblages.
In: Microbial
Mats: Physiological Ecology of Benthic Microbial Communities (Cohen, Y. and Rosenberg, E., Eds.), pp. 455-468.
American Society for Microbiology, Washington, DC.
[l4] Oren. A. (1995) The role of glycerol in the nutrition of
halophilic archaeal communities: a study of respiratory electron transport. FEMS Microbial. Ecol. 16, 281-290.
[15] Zimmerman”,
R.. Itturiaga, R. and Becker-Birch. J. (1978)
Simultaneous determination of the total number of aquatic
bacteria and the number thereof involved in respiration.
Appl. Environ. Microbial. 36. 926-935.