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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. Societies. All rights reserved 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. 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