- Email: [email protected]
Taxonomic Analysis of Extremely Halophilic Archaea Isolated from 56-Years-Old Dead Sea Brine Samples
System. Appl. Microbiol. 23, 376-385 (2000) © Urban & Fischer Verlag _htt..c..p_://w_w_w_.ur_ba_nf_is_ch_er_ .de-'./jo_u_rn_als_/s_am_ _ _ _ _ _ _ _ _ _ _ _
SYSTEM4TIC AND APPLIED MICROBIOLOGY
Taxonomic Analysis of Extremely Halophilic Archaea Isolated from 56-Years-Old Dead Sea Brine Samples DAVID R. ARAHAL!, M. CARMEN GUTIERREZ!, BENJAMIN E. VOLCANI 2 ,::., and ANTONIO VENTOSA 1 1
Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, California, USA
2 Marine
Received August 1, 2000
Summary A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the SJ coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et ai., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work. Key words: Extremely halophilic archaea - Dead Sea - Numerical Taxonomy - DNA relatedness -
Haloarcula - Haloferax - Halobacterium
Introduction The Dead Sea occupies the lowest point on earth's surface, in an arid region of the Syrian-African rift valley. Its salinity is so high that it was historically considered to be sterile. Nevertheless, a large number of halophilic microorganisms have been isolated from this neutral hypersaline terminal lake. Extremely halophilic archaea are quantitatively the main group of prokaryotic microorganisms present in the Dead Sea (OREN, 1988, 1993) and they have been continuously found in the lake since the pioneering studies of B.E. VOLCANI (1936, 1940). Indeed, some of these isolates have provided some novel members of the family Halobacteriaceae, namely Haloferax volcanii (MULLAKHANBHAI and LARSEN, 1975; TORREBLANCA et aI., 1986), Haloarcula marismortui (OREN et aI., 1990), Halorubrum sodomense (MCGENITY et aI., 1995; OREN, 1983) and Halobaculum gomorrense (OREN et aI., 1995). In a previous study we proved that some enrichments obtained from Dead Sea water sam* Deceased on 6 February 1999 0723-2020100/23/03-376 $.15.00/0
pies used by Volcani in 1936 and kept by him during more than 50 years in closed bottles enabled the isolation of 22 extremely halophilic microorganisms that were characterized upon several phenotypic tests and the phylogenetic analysis of the 16S rRNA gene sequences of some representative strains (ARAHAL et aI., 1996). The aim of our study was to determine the taxonomic position of a large number of strains isolated from these enrichments from the Dead Sea and to compare them with the studies carried out by Volcani in the period 1936-1944 as well as with respect to the current classification of the family Halobacteriaceae.
Material and Methods Organisms and growth conditions
The strains used in this study were isolated from fifteen enrichments that were set up by Volcani in the 1930s using a Dead Sea water plus 1.0% (wtlvol) peptone medium. They were inoc-
Taxonomy of Dead Sea Halobacteria ulated with Dead Sea water samples collected in 1936 at surface level on the north basin of the Dead Sea, sampling at different spots near the mouth of Jordan river (VOLCANI 1940, 1944). These enrichments were kept in closed 500-ml bottles under aseptic conditions and stored in the dark in a dry place at 18-20 °C over the entire storage period. The isolation media and the maintenance conditions of the strains have been previously described (ARAHAl et aI., 1996). A total of 158 extremely halophilic isolates were obtained. For comparative purposes the following reference strains were used: Haloarcula argentinensis ATCC 29841 T (T = type strain), H. hispanica ATCC 33960T , H. japonica JCM 7785 T , H. marismortui ATCC 43049 T , H. mukohataei DSM 11483\ "H. sinaiiensis" ATCC 33800, H. vallismortis ATCC 29715 T , Halobacterium salinarum DSM 3754T , Halobaculum gomorrense DSM 9297T , Halococcus morrhuae CCM 53?T, H. saccharolyticus ATCC 4925 7T , Haloferax denitrificans DSM 4425 T , H. gibbonsii ATCC 33959T , H. mediterranei CCM 3361 T, H. volcanii NCIMB 2012 T , Halorubrum distributum VKM B-1733 T , H. lacusprofundi DSM 5036 T , H. saccharovorum NCIMB 2081 T , H. sodomense ATCC 33755 T , H. trapanicum NRC 3402JT, and Natrialba asiatica JCM 9576 T• Phenotypic characterization Tests for 126 characteristics, which included morphological, cultural, physiological, biochemical, nutritional and antibiotic susceptibility features, were carried out (Table 1). The methodology used has been described elsewhere (ARAHAl et aI., 1996; GERHARDT et aI., 1981; MONTERO et aI., 1988; NIETO et aI., 1987; RODRIGUEZ-VALERA et aI., 1980; TORREBlANCA et aI., 1986). The guidelines for the phenotypic characterization included into the minimal standards for description of new taxa in the order Halobacteriales, proposed by the subcommittee on the taxonomy of Halobacteriaceae, were also taken into consideration (OREN et aI., 1997). All tests were performed in media containing 25% (wt/vol) salts, at pH 7.2, and incubated at 37°C, unless otherwise indicated. Numerical analysis A total of 104 phenotypic traits out of the 126 studied were found to be differential features and were used for numerical analysis. The data were coded as 1 or 0 for positive and negative results, respectively; while noncompatible or missing data were coded as 9. For the estimation of the strain similarities the Jaccard coefficient (SJ) (JACCARD, 1908) was chosen and the cluster analysis was carried out using the unweighted pair group method of association (UGMA) (SNEATH and JOHNSON, 1972). Twenty strains were examined in duplicate in order to estimate the test error (SNEATH and JOHNSON, 1972). The cophenetic correlation was also evaluated (SNEATH and SOKAl, 1973). All computations were done using the NTSYS-pc version 1.80 program of EJ. ROHLF (1993) on a PC computer. DNA extraction and purification Cells from twenty-two representative strains selected for this study and sixteen reference strains were cultured at approximately the late exponential phase and then harvested by centrifugation. The cells were washed and suspended in 0.15 M NaCI-O.l M EDTA buffer (pH 8.0) (5 g wet weight in 50 ml of buffer). Lysis was accomplished at 60°C for 10 min adding sodium dodecyl sulfate at a final concentration of 2 % (wt/vol). The DNA was extracted and purified by the method of MARMUR (1961). Its purity was assessed from the A26(jA28o and A2301A26o extinction ratios (JOHNSON, 1994). DNA base composition The guanine-plus-cytosine (G+C) content of the DNA was calculated from the midpoint value of the thermal denaturation
377
profile (MARMUR and DoTY, 1962) obtained with a model UVVis 551S spectrophotometer (Perkin-Elmer Corp., Norwalk, Conn., USA) at 260 nm, programmed for temperature increases of 1.0 °C min-I. The G+C content was determined from the thermal denaturation temperature by using the equation of OWEN and HilL (1979). The G+C content of reference DNA from Escherichia coli NCTC 9001 was taken to be 51 mol% (OWEN and PITCHER, 1985). Preparation of 3H-labelled DNA and DNA-DNA hybridization experiments DNA was radioactively labelled by the multiprime system with a commercial kit (RPN 1601 Y; Amersham International, Amersham, England) , using [1',2',VH] dCTP (Amersham). The average specific activity obtained with this procedure was 8.4 x 10 6 cpm ).Ig-I of DNA. The labelled DNA was denatured before hybridization by heating at 100°C for 5 min and then placed on ice. DNA-DNA similarity was studied by the competition procedure described by JOHNSON (1994) . Competitor DNAs were sonicated (Braun Melsungen, Melsungen, Germany) at 50 W for two 15-s intervals. Membrane filters (HAHY; Millipore Corp., Bedford, Mass., USA) containing reference DNA (ca. 25 I1g cm-2) were placed in 5 ml screw cap vials which contained the labelled, sheared, denatured DNA and the denatured and sheared competitor DNA. The ratio of the concentration of competitor DNA to the concentration of labelled DNA was at least 150:1. The final reaction concentrations were 2 x SSC and 30% formamide, and the final volume was 140 ).II. The hybridization experiments were carried out under optimal conditions, with temperatures ranging between 54 and 58°C, which is within the limits of validity for the filter method (DE LEY and TIJTGAT, 1970). The vials were shaken slightly for 18 h in a water bath (Grant Instruments, Cambridge, England); these procedures were done in triplicate. After hybridization the filters were washed in 2 x SSC at the optimal renaturation temperature. The radioactivity bound to the filters was measured in a liquid scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif., USA), and the percentage of similarity was calculated as described by JOHNSON (1994). At least three independent determinations were carried out for each experiment, and the mean values are reported here.
Results Using the appropriate media and culture conditions, we were able to isolate a total of 158 extremely halophilic strains from the 56 years old enrichments obtained from Dead Sea water samples. All isolates required for optimal growth salt concentrations between 20 to 25% (wt/vol). They produced pink to red pigmented colonies on solid media. According to their phenotypic characteristics (Table 1) they were identified as members of the Halobacteriaceae (GRANT and LARSEN, 1989). All strains were Gram-negative, motile and catalase positive. They reduced nitrates and were able to grow on pyruvate and L-asparagine, but were not able to degrade DNA or Tween 80. They were Voges-Proskauer, Simmons citrate, phenylalanine deaminase and arginine dehydrolase negative. Acid was not produced from lactose or D-mannose and no growth was obtained on lactose, L-alanine, DLarginine, L-aspartic acid, L-phenylalanine, L-glutamine, L-proline, L-serine and L-threonine. All strains were resistant to chloramphenicol, erythromycin and neomycin.
378
D. R. ARAHAL et al.
Table 1. Phenotypic characteristics of the 158 extremely halophilic archaea which group into six phenons. The percentage of strains giving positive is indicated for each test. Characteristics:
Phenon No. strains
Pigmentation: Pink" Red" Gas vacuoles" Growth at % (w/v) salts:
3 5 10 15 25 30
B
C
D
28
7
E 24
F
18
38 62 0
22
29 71 0
100 0 0
17 83 0
0 100 100
6 49 89 97 46
0 6 28 50 100 100
4 4 89 100
0 8 67 100 100
36
0 0 86 100 100 100
92
0 0 44 67 100 44
0 7 79 100 0
0 22 61 100 0
0 0 14 100 7
71
100 100 86 0
79 100 100 100 0
78 100 67 0
19 85 100 99 18
0 56 100 100 28
29 86 100
96
78
14
100 100 86 0 0
100 79 38 0
100 56 11 0
24 35 18 24
83 6 11 0
18 82 0 0
14 0 0 86
96
56 33 0
0 1
0 0
11
4
0 0
79 0
44
64 26 67 0
17 22 0 0
86 18 57 0
100 71 0 0
21
79 97 57 31 99 36 1 0 4 6 63 76 6
83 100
21 100
29 86 14 86 86 100 100 71 0 29 100 86 100 0
75 100 8 54 100 17
11 11
92
0 0 67 89 100
0 14 100
4 100 100 100 100 0 42 100 100 100 100 88 42
A 72
11
78
0
96
9
Growth at pH:
5.5 6.0" 6.5 8.5 9.0
78
Growth at (0C):
20 25 55 60 65
96
Minimal Mg2+ requeriment (%):
0.05" O.l" 0.2 0.5* Anaerobic growth with: Nitrate Arginine Hydrolysis of: Gelatine Starch Casein Phosphatase* Acid production from: D-Arabinose D-Fructose D-Galactose Glycerol D-Glucose Maltose Sucrose" D-Trehalose D-Xylose Indol production ,. Methyl red Nitrite reduction HzS production Urease" Utilization of: Starch D-Arabinose D-Cellobiose D-Fructose" D-Galactose " D-Glucose* Inulin Lactose * Maltose D-Mannose D-Raffinose D-Rhamnose D-Ribose"
11
99 93 97 99 93 100 100 100 100 25 99 40 4
11
11
28 94 100
32 100 86 7 14
11
0 11
11
0 100 89 22 94
86 32 86 7 4
61
86 7 4 100 0 100 82 18 14 14 7 21 0
72
94 100 100 100 89 100 100 83 72
83 78
71
100 0 100 43 86 43 100 100 0
0 4 0
13
0 0
8 21 88 100 4 100 0
11
78
78 0 0 100
0 22 11 11
78
100 100 0 100 89 0 100 78
100 0 89 67 11 0 100
Taxonomy of Dead Sea Halobacteria
379
Table 1. (Continued). Characteristics:
Ph en on No. strains
Sucrose" Salicin" D-Trehalose" D-Xylose* Glycerol" D-Mannitol" myo- Inositol" D-Sorbitol <. Acetate" Butyrate" Citrate" Fumarate L-Glutamate':' Hippurate Malate* Propionate" Succinate* L-Isoleucine* L-Leucine" L-Ornithine " Susceptibility to antibiotics: Aphidicolin (5 pg/ml) Anysomicin (10 pg/ml) Bacitracin (10 U) Novobiocin (30 pg/ml) Rifampin (5 pg/ml) Susceptibility to heavy metals (mM): 1.0 • Ag: 0.5 0.1 0.05" 20.0 • As: 10.0 5.0 1.0 • Cd: 0.5 0.1 0.05 1.0 • Co: 0.5 0.1 5.0 • Cr: 2.5 1.0 2.5 • Cu: 1.0 0.5 0.01 • Hg: 0.005 2.5 • Ni: 1.0 0.5 20.0 • Pb: 10.0 5.0 2.5 0.5 • Zn: 0.1
E
A 72
B 18
C 28
D 7
24
85 100 96 33 42 72 100 0 54 63 0 0 14 0 0 13 0 14 0 92
83 83 100 67 100 39 100 0 94 11 0 0 83 0 0 0 17 17 0 89
0 0 0 0 0 43 14 0 0 0 0 0 0 0 0 0 0 100 0 0
100 0 71 0 29 100 0 0 100 0 0 14 0 0 71 100 100 0 0 0
100 100 100 100 100 75 0 88 100 100 29 96 100 25 92 100 100 0 8 33
22 11 0 22 100 0 100 0 100 11 100 22 100 0 100 67
0 83 93 93 0
0 61 100 89 0
7 71 100 100 21
29 71 100 100 86
0 96 79 100 0
0 100 78 100 11
6 39 56 0 0 39 61 0 49 51 0 49 50 1 74 26 0 0 74 26 83 17 28 72 0 0 43 47 10 60 40
33 67 0 0 0 67 33 0 56 44 0 6 78 17 28 72 0 0 78 22 78 22 67 33 0 0 67 33 0
0 0 25 75 4 79 18 11 68 21 0 14 71 14 14 64 21 68 29 4 86 14 14 61 25 68 29 4 0 82 18
0 86 14 0 29 71 0 14 86 0 0 86 14 0 86 14 0 0 86 14 100 0 86 14 0 57 29 14 0 100 0
0 29 71 0 71 29 0 0 83 17 0 25 75 0 17 71 13 0 79 21 83 17 29 71 0 29 71 0 0 67 33
0 0 0 100 78 22 0 0 0 78 22 0 78 22 0 89 11 0 89 11 100 0 0 56 44 0 0
78 22
F 9
78 100 100 100
78 22 89 11
* Differential characteristics between the phenons. All strains were Gram-negative, motile and yielded positive results to the following tests: growth at 20% (w/v) salts, and at pH 7.0 to 8.0, growth in the range from 30 to 50°C, catalase production, nitrate reduction, and utilization of pyruvate and L-asparagine. All strains were negative to: growth at 0.9% (w/v) total salts, growth at pH 5.0 and 9.5, growth at 15 and 70°C, DNAse, acid production from lactose and D-mannose, Voges-Proskauer, Simmons citrate, phenylalanine deaminase, arginine dehydrolase, hydrolysis of Tween 80, utilization of lactate, L-alanine, DL-arginine, L-aspartic acid, L-phenylalanine, L-glutamine, L-proline, L-serine and Lthreonine and susceptibility to chloramphenicol (30 pg/ml), erythromycin (15 pg/ml) and neomycin (30 pg/ml).
380
D. R. ARAHAL et al.
Reproducibility of phenotypic characters The inclusion of the pairs of randomly chosen duplicate strains in the analysis enabled experimental test error to be calculated. The probability (P) of an erroneous result averaged 4.04%. A small number of tests were responsible for most of the test error. These tests, with a test variance of 0.03, were acid production from maltose and sucrose, Voges-Proskauer, and utilization of D-cellobiose, D-trehalose, fumarate and malate as sole source of carbon and energy.
Numerical analysis The result of the numerical study of the strains grouped by means of the SJ coefficient and UPGMA clustering yielded the dendrogram shown in Fig. 1. The cophenetic correlation was 0.901. The SSM coefficient was also used and the clustering of strains was quite similar to that obtained with the SJ coefficient. In the dendrogram obtained, all the isolates grouped into six phenons at similarity levels ranging from 76 to 85% (Fig 1). Table 1 shows the characteristics which differentiate the six phenons studied from one another.
DNA base composition The G+C contents of the DNAs of the 22 strains chosen as representative isolates of the different phenons ranged from 56.5 to 66.9 mol% (Table 2).
DNA-DNA hybridization The results of the DNA-DNA hybridization experiments are shown in Table 2.
Discussion The 158 isolates of this study were obtained from Dead Sea brine samples kept by Volcani for almost 60 years and, since the isolation media were designed for extremely halophilic microorganisms, all of them were members of the halobacteria (GRANT and LARSEN, 1989). The ability of halo bacteria to survive for such a long period was proved in a previous study in which we isolated 22 halo bacteria from these samples (ARAHAL et aI., 1996). The purpose of this study was to know in detail the taxonomic affiliation of a large number of strains obtained from the 56-years-old Dead Sea brine samples, and to compare them with both, the current classification of halo bacteria, as well as the information available in 1936-1944, when the studies of Volcani were reported (VOLCANI 1936,1940,1943,1944). All 158 isolates were grouped, based on the numerical taxonomy study, into six phenons whose phenotypic features are shown in Table 1. Reference strains were not included in any phenon, although some of them were closely related to the phenons; this could be due to their different ability to use organic compounds and different
response to antibiotics and heavy metals. Phenons A, B and C showed features (Table 1) similar to those of the genus Haloarcula (GRANT and LARSEN, 1989). Phenon A comprised pleomorphic, motile cells, rods are rarely observed, and gas vacuoles are not produced. Strain Ell, which is included in this phenon, was used as a representative isolate in a previous study from the enrichments from Dead Sea brine samples (ARAHAL et aI., 1996) and its 16S rRNA sequence was more closely related to Haloarcula hispanica (with a 98.8% similarity). The phenotypic features of phenon A are indeed similar to those of H. hispanica (JUEZ et ai., 1986), except for growth at 65 DC, acid production from D-mannose and D-xylose, and use of salicin, arginine, asparagine and glutamine. The DNA hybridization study shows that the six representative strains chosen from phenon A constitute a homogeneous genotypic cluster, with DNA relatedness ranging from 82 to 100%. Besides, the results clearly support the assignment of phenon A to the species H. hispanica, showing a 76% DNA relatedness with H. hispanica ATCC 33960T (Table 2). With respect to phenons Band C, they could also be assigned to the species H. hispanica, showing differences in some physiological and biochemical features with respect to ph en on A (Table 1). DNA-DNA hybridization results obtained between representative strains from phenons Band C, and those from phenon A where always above 68% (Table 2). The G+C contents of the thirteen representative strains included into these three phenons varies from 56.5 to 63.5 mol%, a range that also comprise the G+C content of H. hispanica (61.8 mol%) (GUTrERREZ et ai., 1989). The close relationship of strains included in phenons A, Band C to H. hispanica is interesting, considering that this species was originally isolated from salterns located in Alicante (Spain) (JUEZ et ai., 1986), and has not been reported in studies carried out in the Dead Sea (VENTOSA and ARAHAL, 1999). Different geochemical conditions were present at the Dead Sea in 1936 and in recent periods, with an increase of the salinity and a higher Mgz+ concentration. Although H. hispanica was never described to occur in the Dead Sea, another species of this genus, Haloarcula marismortui (OREN et ai., 1990) was originally isolated from this habitat in the 1960's (GINZBURG et ai., 1970). Prior to its formal designation it was known as the "Halobacterium of the Dead Sea" and it resembled Flavobacterium (Halobacterium) marismortui, which was one of the extremely halophilic microorganisms described by VOLCANI (1940) and whose cultures were unfortunately lost. Phenons D and E comprise 7 and 24 isolates, respectively. They show similar phenotypic features. They are pleomorphic cells able to grow from 15 to 30% salts, from pH 6.0 to 8.5 and require 0.5% Mgz+ ions. They produce indole and HzS and are able to use a large number of compounds (Table 1). They clustered close to the Haloferax reference strains, indicating a similarity with species of this genus. Ph en on E - and also phenon D to a lower extent - showed very similar phenotypic features with Haloferax volcanii. H. volcanii NCIMB 2012T, that was used as reference strain, did not clustered within the
Taxonomy of Dead Sea Halohacteria
381
% Similarity
60
70
80
90
100
No. strains
Phenon
72
A
18
B
Haloarcula vallismortis Haloarcula hispanica
28
c
Halobacterium salinarum Halorubrum saccharovorum
7
D
24
E
Haloferax volcanii Haloferax gibbonsii Haloferax denitrijicans Haloferax mediterranei
9
F
Halorubrum lacusprofundi Halococcus morrhuae Halorubrum distributum Halococcus saccharolyticus Haloarcula japonica
Fig. 1. Simplified dendrogram showing the clustering of the strains into six phenons based on the SJ coefficient and UPGMA (unweighted average linkage clustering method) for the 158 extremely halophilic archaea and some reference strains of the Halobacteriaceae.
382
D. R. ARAHAL et al.
Table 2. DNA G+C contents and levels of DNA-DNA contents and levels of DNA-DNA relatednedd for some strains representatives of the different phenons obtained by numerical analysis, and of the extreme halophilic genera. Source of unlabelled DNA'f
G+C content (mol%)
% Homology with 3H-labelled DNA from strain: Ell
E2
329
E8
E1
E12
PhenonA: strain 342 strain 344 strain 349 strain 409 strain Ell strain 619
60.4 63.0 63.5 64.0 56.5 59.9
100 88 100 94 100 82
73 ND 70 ND 80 72
ND ND 100 ND 93 ND
ND
ND
11
11
10 11 10 ND
ND ND 42 ND
ND 48 ND ND 0 ND
PhenonB: strain E2 strain 408 strain 647
61.6 60.4 58.0
60 90 78
100 70 86
90 ND ND
18 ND ND
0 ND ND
0 ND ND
Phenon C: strain 320 strain 329 strain 427 strain 634
60.0 58.7 59.0 63.4
ND 73 ND ND
ND ND ND ND
72 100 100 100
ND 60 ND ND
ND ND ND ND
ND 0 ND ND
Phenon D: strain 638 strain E8
65.1 63.0
ND 31
ND 2
ND 42
98 100
68 61
ND 56
Phenon E: strain 719 strain 808 strain 824 strain E1
62.2 63.5 61.6 63.8
ND ND ND 0
ND 0 0 ND
ND ND ND 55
67 66 65 ND
100 78 100 100
ND ND ND 0
PhenonF: strain E12 strain 706 strain 810 Haloarcula argentinensis ATCC 29841 Haloarcula hispanica ATCC 33960 Haloarcula japonica JCM 7785 Haloarcula marismortui ATCC 43049 Haloarcula mukohatei DSMZ 11483 Haloarcula quadrata DSMZ 11927 "Haloarcula sinaiiensis" ATCC 33800 Haloarcula vallismortis ATCC 29715 Halobacterium salinarum DSMZ 3754 Halobaculum gomorrense DSMZ 9297 Haloferax denitrificans DSMZ 4425 Haloferax gibbonsii ATCC 33959 Haloferax mediterranei CCM 3361 Haloferax volcanii NCIMB 2012 Halorubrum lacusprofundi DSMZ 5036 Halorubrum saccharovorum ATCC 29252 Halorubrum sodomense ATCC 33755 Haloterrigena turkmenica JCM 9743 Natrialba asiatica JCM 9576
66.9 60.0 61.4 62.0' 61.8" 63.3' 62.0 b 65.0 a 60.1d 59.7 b 61.0 b 66.4 b 70.0' 64.2b 61.8 1 59.1b 64.9 h 65.3-65.8 g 64.3 b 66.0 b 59.8 h 60.3-63.1i
6 ND ND ND 76 0 0
ND ND ND 15 ND 57 0 43 50 0 0 53 0 20 0 0 16 0 0 0 ND 12
13 ND ND ND ND 30 0 42 2 1 26 0 0 0 54 14 38 0 0 3 30 2
ND ND ND 45 17 0 0 0 0 ND 0 17
ND ND ND 1 26 37 0 53 44 35 0 8 37 1 15 67 98 0 0 0 33 31
100 70 75 0 47 15 0 49 0 21 0 90 31 0 67 0 0 53 65 16 0 0
11 ND 21 6 0 36 ND 0 0 0 28 0 36 ND 0
0 0 29 3 92 7 25 0 0 21
,. ATCC - American Type Culture Collection; JCM - Japan Collection of Microorganisms; DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; CCM - Czechoslovak Collection of Microorganisms; NCIMB - National Collection of Industrial and Marine Bacteria. Data from: aIHARA et al. (1997), bGUTIERREZ et al. (1989), 'TAKASHINA et al. (1990), dOREN et al. (1999), eOREN et al. (1995), IJUEZ et al. (1986), gMCGENITY and GRANT (1995), hVENTOSA et al. (1999b), iKAMEKURA and DYALLSMITH (1995).
Taxonomy of Dead Sea Halobacteria phenon mainly because of its different ability to use compounds as carbon source. The G+C content of the four representative strains chosen from phenon E ranged from 61.6 to 63.8 mol%, which is slightly lower than the G+C content reported for H. volcanii (64.9 mol%) (GuTIERREZ et ai., 1989), but in the case of the two representatives of phenon D it was 63.0 and 65.1 mol% (Table 2). DNA-DNA hybridization experiments show that the four strains of phenon E constitute an homogeneous genotypic group (with DNA relatedness ranging from 78-100%) not related to any other halo bacteria, except to H. volcanii NCMIB 2012\ with a DNA relatedness of 98%, that clearly indicates the assignment of strains of phenon E to the species H. volcanii. These phenotypic and genotypic results correlate with the phylogenetic study based on the comparison of the 16S rRNA sequences, in which isolate E1, representative strain of phenon E, and strain E8, representative from phenon D, showed a 99.7% similarity with H. volcanii (ARAHAL et ai., 1996). It is interesting to note that the species H. volcanii was isolated from the Dead Sea and described in 1975 (MULLAKHANBHAI and LARSEN, 1975). It was named after B.E. Volcani as a recognition of his early studies in the Dead Sea. Although he did not isolate this species in his studies carried out from 1936 to 1944, we show here that H. volcanii was present in the Dead Sea samples that he used at that time. Independently from the molecular techniques used for the characterization of the isolates, this could be also explained by the different isolation procedures used in our studies. Ph en on F comprises nine red pigmented strains that have gas vacuoles and require 0.1 to 0.2% Mg2+ for growth. They are H 2S, phosphatase, urease and methyl red positive. They are not able to hydrolyze starch or casein. They are not able to produce acids from glucose and other sugars. Their features are similar to these of the genus Halobacterium (GRANT and LARSEN, 1989). The DNA-DNA hybridization data and the phylogenetic analysis based on the 16S rRNA sequence comparison of the strain representative of this group E12 (ARAHAL et aI., 1996) clearly support that this phenon belongs to the species Halobacterium salinarum. Among his extremely halophilic isolates, VOLCANI (1940) identified one as Flavobacterium (Halobacterium) trapanicum. Since the cultures were lost and the information available is very limited not much can be concluded, but we have found similarities between the description of that organism and the strains of phenon F. VOLCANI (1940) described also the presence of Micrococcus morrhuae and Sarcina morrhuae - now believed to be one single species, Halococcus morrhuae (VENTOSA and ARAHAL, 1999), but we failed in isolating members of the genus Halococcus, probably because it is present normally in hypersalinic habitats in very low proportions (MONTERO et ai., 1988) or because it could not survive until our days. Therefore, an important question in our work is, how did the microorganisms isolated by us manage to keep their viability after 56 years of preservation? In the literature there are abundant reports of long term survival
383
events, even up to 650 million years, which is more than 10 7 fold longer than the case described here. A broad review about the topic is that of KENNEDY et al. (1994), while that of GRANT et al. (1998) deals only with halobacteria. Despite the abundance of reports by many different authors about longevity of microorganisms and the care taken in the processing of the samples to avoid external contamination, still many objections arise against the claims of revival of long-term isolates microorganisms. Even assuming that the sterilization methods applied have an absolute effectiveness and only microorganisms indigenous to the samples are recovered, there is still the doubt whether microorganisms entered the sample at any time during the storage period, not reflecting, thus, the age of the sample. This can be specially problematic for the longest records whose sources include fossils, rock salt, soils, ice, etc. (KENNEDY et ai., 1994). So far, attempts to find a molecular evidence for longevity have been unsuccessful (GRANT et ai., 1998). In any case, there is very little resemblance between those ancient samples and the ones used in this study. The first and most obvious difference is, as it was mentioned before, the storage time. Besides, our source consist of laboratory material (enrichments prepared from Dead Sea water samples) which brings two important consequences: first, it is a closed system (500 ml-flasks) whose physical parameters were controlled and free from any external environmental change. Second, since the medium was prepared with sterile Dead Sea water enriched with peptone (1 %) (VOLCANI, 1940) and all flasks were incubated and yielded growth before storage, it cannot be expected that their biodiversity reflects that of the Dead Sea at that time. Even more, the samples used for the inoculation of the flasks were collected close to the mouth of the Jordan River at surface level. At that time the lake was stratified and the salinity of the upper layer was slightly lower than 27% (VENTOSA et aI., 1999a). Nowadays, the Dead Sea has evolved to a holomictic lake with a salinity close to 34%. Therefore, there are enough reasons that explain the differences between the diversity obtained by us and that found in other studies performed at the Dead Sea. Still the questions remain, which are the mechanisms that enable this sustained survival and, what is the possible growth rate during their storage? While in nature growth is usually sporadic or unbalanced (DAWES, 1984), in the laboratory it is an optimized situation, not only for the abundance of nutrients, but also for other factors (temperature, osmotic pressure, etc.). The lack of nutrients, and to a lower extent, other adverse environmental factors, can drastically slow down the growth rate (doubling time of months or even years). The term "starvation survival" has been coined to indicate an absence of growth resulting from an insufficient level of nutrients (MORITA, 1990). It is a physiological state in which the metabolism is suspended or arrested (LYNCH, 1990). In the enrichments of our study nutrients (1 % peptone added to sterile Dead Sea water) are not likely to be a limiting factor at least in an initial stage. Even assuming a depletion of nutrients, and since it is a closed system,
384
D. R. ARAHAL et al.
dead lysed cells can serve to feed other cells, a phenomenon known as cryptic growth (POSTGATE and HUNTER,
1962). Acknowledgments This study was supported by grants from the Ministerio de Educaci6n y Cultura, Spain (grants IFD97-1162 and PB981150); and from the Junta de Andalucia.
References ARAHAL, D.R., DEWHIRST, EE., PASTER, B.J., VOLCANI, B.E., VENTO SA, A. Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences. Appl. Env. Microbiol. 62,3779-3786 (1996). DAWES, E.A. Stress of unbalanced growth and starvation in microorganisms pp. 19-43. In: The revival of injured microbes (M.H.E. ANDREW, A.D. RUSSEL eds.) London, Academic Press 1984. DE LEY, l, TIJTGAT, R. Evaluation of membrane filter methods for DNA-DNA hybridization. Antonie Leeuwenhoek 36, 461-474 (1970). GERHARDT, P., MURRAY, R.G.E., COSTILOW, R.N., NESTER, E.W., WOOD, W.A., KRIEG, N.R., PHILLIPS, G.B. (eds.): Manual of methods for general bacteriology. Washington, D. e., American Society for Microbiology 1981. GINZBURG, M., SACHS, L., GINZBURG, B.Z. Ion metabolism in a Halobacterium. l. Influence of age of culture on intracellular concentrations. J. Gen. Physiol. 55, 187-207 (1970). GRANT, W.D., GEMMELL, R.T., MCGENITY, T.J. Halobacteria: the evidence for longevity. Extremophiles 2, 279-287 (1998) . GRANT, W. D., LARSEN, H.: Extremely halophilic Archaeobacteria. Order Halobacteriales ord. nov., pp. 2216-2233. In: Bergey's manual of systematic bacteriology (J.T. STALEY, M .P. BRYANT, N. PFENNIG, J.G. HOLT eds.), vol. 3. Baltimore, The Williams & Wilkins Co. 1989. GUTIERREZ, M.e., VENTOSA, A., RUIZ-BERRAQUERO, E DNADNA homology studies among strains of Haloferax and other halo bacteria. Curro Microbiol. 18,253-256 (1989). IHARA, K., WATANABE, S., TAMURA, T. Haloarcula argentinensis sp. nov. and Haloarcula mukohataei sp. nov., two new extremely halophilic archaea collected in Argentina. Int. J. Syst. Bacteriol. 47, 73-77 (1997). JACCARD, P. Nouvelles recherches sur la distribution florale. Bull. Soc. Vandoise Sci. Nat. 44, 223-270 (1908) . JOHNSON, J.L.: Similarity analysis of DNAs, pp. 655-681. In: Methods for general and molecular bacteriology (P. GERHARDT, R.G.E. MURRAY, W.A. WOOD, N.R. KRIEG eds.), Washington, D. e., American Society for Microbiology 1994. JUEZ, G., RODRIGUEZ-VALERA, E, VENTOSA, A., KUSHNER, D.J. Haloarcula hispanica spec. nov. and Haloferax gibbonsii spec. nov., two new species of extremely halophilic archaebacteria. Syst. Appl. Microbiol. 8, 75-79 (1986). KAMEKURA, M ., DYALL-SMITH, M .L. Taxonomy of the family Halobacteriaceae and the description of two new genera Halorubrobacterium and Natrialba. J. Gen. Appl. Microbiol. 41,333-350 (1995). KENNEDY, M.l, READER, S.L., SWIERCZYNSKI, L.M. Preservation records of microorganisms: evidence of the tenacity of life. Microbiol. 140,2513-2529 (1994) . LYNCH, J.M. Longevity of bacteria: considerations in environmental release. Curro Microbiol. 20, 387-389 (1990).
MARMUR, J. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. BioI. 3,208-218 (1961). MARMUR, J., DOTY, P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. BioI. 5, 109-118 (1962). MCGENITY, T. l, GRANT, W.D. Transfer of Halobacterium sacccharovorum, Halobacterium sodomense, Halobacterium trapanicum NRC 34021 and Halobacterium lacusprofundi to the genus Halorubrum gen. nov., as Halorubrum saecharovorum comb. nov., Halorubrum sodomense comb. nov., Halorubrum trapanicum comb. nov., and Halobacterium lacusprofundi comb. nov. Syst. Appl. Microbiol. 18,237-243 (1995). MONTERO, e.G., VENTOSA, A., RODRiGUEZ-VALERA, E, RUlzBERRAQUERO, F. Taxonomic study of non-alkaliphilic halococci. J. Gen. Microbiol. 134,725-732 (1988). MORITA, R. Y. The starvation-survival state of microorganisms in nature and its relationships to the bioavailable energy. Experientia 46, 813-817 (1990). MULLAKHANBHAI, M.E, LARSEN, H. Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch. Microbiol. 104,207-214 (1975). NIETO, J.l, VENTOSA, A., RUIZ-BERRAQUERO, E Susceptibility of halobacteria to heavy metals. Appl. Environ. Microbiol. 53, 1199-1202 (1987). OREN, A. Halobacterium sodomense sp. nov., a Dead Sea halobacterium with an extremely high magnesium requeriment. Int. J. Syst. Bacteriol. 33, 381-386 (1983). OREN, A.: The microbial ecology of the Dead Sea, pp. 193-229. In: Advances in microbial ecology (K.e. MARSHALL ed.), vol. 10. New York, Plenum 1988. OREN, A.: Ecology of extremely halophilic microorganisms, pp. 25-53. In: The biology of halophilic bacteria (R.H. VREELAND, L.l. HOCHSTEIN eds.), Boca Raton, CRC Press 1993. OREN, A., GINZBURG, M., GINZBURG, B.Z., HOCHSTEIN, L.l., VOLCANI, B.E. Haloarcula marismortui (Volcani) sp. nov., nom. rev., an extremely halophilic bacterium from the Dead Sea. Int. J. Syst. Bacteriol. 40, 209-210 (1990). OREN, A., VENTOSA, A., GRANT, W.D. Proposed minimal standards for the description of new taxa in the order Halobacteriales. Int. J. Syst. Bacteriol. 47,233-238 (1997). OREN, A., VENTOSA, A., GUTIERREZ, M.e., KAMEKURA, M. Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt). Int. J. Syst. Bacteri01. 49,1149-1155 (1999). OWEN, R.l, HILL, L.R.: The estimation of base compositions, base pairing and genome size of bacterial deoxyribonucleic acids, pp. 217-296. In: Identification methods for microbiologists. 2 nd ed. (EA. SKINNER, D.W. LOVELOCK eds.) London, Academic Press, 1979. OWEN, R.J., PITCHER, D.: Current methods for estimating DNA base composition and levels of DNA-DNA hybridization, pp. 67-93. In: Chemical methods in bacterial systematics (M. GOODFELLOW, E. MINNIKIN eds.), London, Academic Press 1985. POSTGATE, J.R., HUNTER, J.R. The survival of soil bacteria. J. Gen. Microbiol. 29,233-263 (1962). RODRIGUEZ-VALERA, E, RUIZ-BERRAQUERO, E, RAMOS-CORMENZANA, A. Isolation of extremely halophilic bacteria able to grow in defined inorganic media with single carbon sources. J. Gen. Microbiol. 119, 535-538 (1980). ROHLF, E].: NTSYS-pc. Numerical taxonomy and multivariate analysis system, version 1.80. New York, Exeter Software 1993. SNEATH, P.H.A., JOHNSON, R. The influence on numerical taxonomy similarities of errors in microbiological tests. J. Gen. Microbiol. 72, 377-392 (1972).
Taxonomy of Dead Sea Halobacteria SNEATH, P.H.A., SOKAL, R.R.: Numerical taxonomy. The principles and practice of numerical classification. San Francisco, Freeman, W.H. 1973. TAKASHINA, T., HAMAMOTO, T., OTOZAI, K., GRANT, W.D., HORIKOSHI, K. Haloarcula japonica sp. nov., a new triangular halophilic archaebacterium. Syst. App!. Microbio!. 13, 177-181 (1990). TORREBLANcA, M., RODRIGUEZ-VALERA, E, JUEZ, G., VENTO SA, A., KAMEKURA, M., KATES, M. Classification of non-alkaliphilic halo bacteria based on numerical taxonomy and polar lipid composition and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst. App!. Microbio!. 8, 89-99 (1986). VENTOSA, A., ARAHAL, D.R.: Microbial life in the Dead Sea, pp. 357-368. In: Enigmatic microorganisms and life in extreme environments (J. SECKBACH ed.) Netherlands, Kluwer Academic Publishers 1999. VENTO SA, A., ARAHAL, D.R., VOLCANI, B.E.: Studies on the microbiota of the Dead Sea - 50 years later, pp. 139-147. In: Microbiology and biogeochemistry of hypersaline environments (A. OREN ed.) Boca Raton, CRC Press 1999a. VENTO SA, A., GUTIERREZ, M.C., KAMEKURA, M., DYALL-SMITH, M.L. Proposal for the transfer of Halococcus turkmenikus,
385
Halobacterium trapanicum JCM 9743 and strain GSL 11 to Haloterrigena turkmenica gen. nov., comb. nov. Int. ]. Syst. Bacterio!' 49,131-136 (1999b). VOLCANI, B.E. (B. WILKANSKY) Life in the Dead Sea. Nature 138,467 (1936). VOLCANI, B.E. (B. ELAZARI-VOLCANI): Studies on the micro flora of the Dead Sea. Ph.D. thesis. The Hebrew University, Jerusalem 1940. VOLCANI, B.E. (B. ELAZARI-VOLCANI) Bacteria in the bottom sediments of the Dead Sea. Nature 152, 274-275 (1943). VOLCANI, B.E.: The microorganisms of the Dead Sea. In: Volume Consists of Papers Collected to Commemorate the 70th Aniversary of Dr. Chaim Weizmann, pp. 71-85. Daniel Sieff Institute, Rehovoth, Israel 1944.
Corresponding author: A. VENTO SA, Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain Telephone: +34 954556702, Fax: + 34 95462 8162, e-mail: [email protected]
SYSTEM4TIC AND APPLIED MICROBIOLOGY
Taxonomic Analysis of Extremely Halophilic Archaea Isolated from 56-Years-Old Dead Sea Brine Samples DAVID R. ARAHAL!, M. CARMEN GUTIERREZ!, BENJAMIN E. VOLCANI 2 ,::., and ANTONIO VENTOSA 1 1
Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, California, USA
2 Marine
Received August 1, 2000
Summary A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the SJ coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et ai., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work. Key words: Extremely halophilic archaea - Dead Sea - Numerical Taxonomy - DNA relatedness -
Haloarcula - Haloferax - Halobacterium
Introduction The Dead Sea occupies the lowest point on earth's surface, in an arid region of the Syrian-African rift valley. Its salinity is so high that it was historically considered to be sterile. Nevertheless, a large number of halophilic microorganisms have been isolated from this neutral hypersaline terminal lake. Extremely halophilic archaea are quantitatively the main group of prokaryotic microorganisms present in the Dead Sea (OREN, 1988, 1993) and they have been continuously found in the lake since the pioneering studies of B.E. VOLCANI (1936, 1940). Indeed, some of these isolates have provided some novel members of the family Halobacteriaceae, namely Haloferax volcanii (MULLAKHANBHAI and LARSEN, 1975; TORREBLANCA et aI., 1986), Haloarcula marismortui (OREN et aI., 1990), Halorubrum sodomense (MCGENITY et aI., 1995; OREN, 1983) and Halobaculum gomorrense (OREN et aI., 1995). In a previous study we proved that some enrichments obtained from Dead Sea water sam* Deceased on 6 February 1999 0723-2020100/23/03-376 $.15.00/0
pies used by Volcani in 1936 and kept by him during more than 50 years in closed bottles enabled the isolation of 22 extremely halophilic microorganisms that were characterized upon several phenotypic tests and the phylogenetic analysis of the 16S rRNA gene sequences of some representative strains (ARAHAL et aI., 1996). The aim of our study was to determine the taxonomic position of a large number of strains isolated from these enrichments from the Dead Sea and to compare them with the studies carried out by Volcani in the period 1936-1944 as well as with respect to the current classification of the family Halobacteriaceae.
Material and Methods Organisms and growth conditions
The strains used in this study were isolated from fifteen enrichments that were set up by Volcani in the 1930s using a Dead Sea water plus 1.0% (wtlvol) peptone medium. They were inoc-
Taxonomy of Dead Sea Halobacteria ulated with Dead Sea water samples collected in 1936 at surface level on the north basin of the Dead Sea, sampling at different spots near the mouth of Jordan river (VOLCANI 1940, 1944). These enrichments were kept in closed 500-ml bottles under aseptic conditions and stored in the dark in a dry place at 18-20 °C over the entire storage period. The isolation media and the maintenance conditions of the strains have been previously described (ARAHAl et aI., 1996). A total of 158 extremely halophilic isolates were obtained. For comparative purposes the following reference strains were used: Haloarcula argentinensis ATCC 29841 T (T = type strain), H. hispanica ATCC 33960T , H. japonica JCM 7785 T , H. marismortui ATCC 43049 T , H. mukohataei DSM 11483\ "H. sinaiiensis" ATCC 33800, H. vallismortis ATCC 29715 T , Halobacterium salinarum DSM 3754T , Halobaculum gomorrense DSM 9297T , Halococcus morrhuae CCM 53?T, H. saccharolyticus ATCC 4925 7T , Haloferax denitrificans DSM 4425 T , H. gibbonsii ATCC 33959T , H. mediterranei CCM 3361 T, H. volcanii NCIMB 2012 T , Halorubrum distributum VKM B-1733 T , H. lacusprofundi DSM 5036 T , H. saccharovorum NCIMB 2081 T , H. sodomense ATCC 33755 T , H. trapanicum NRC 3402JT, and Natrialba asiatica JCM 9576 T• Phenotypic characterization Tests for 126 characteristics, which included morphological, cultural, physiological, biochemical, nutritional and antibiotic susceptibility features, were carried out (Table 1). The methodology used has been described elsewhere (ARAHAl et aI., 1996; GERHARDT et aI., 1981; MONTERO et aI., 1988; NIETO et aI., 1987; RODRIGUEZ-VALERA et aI., 1980; TORREBlANCA et aI., 1986). The guidelines for the phenotypic characterization included into the minimal standards for description of new taxa in the order Halobacteriales, proposed by the subcommittee on the taxonomy of Halobacteriaceae, were also taken into consideration (OREN et aI., 1997). All tests were performed in media containing 25% (wt/vol) salts, at pH 7.2, and incubated at 37°C, unless otherwise indicated. Numerical analysis A total of 104 phenotypic traits out of the 126 studied were found to be differential features and were used for numerical analysis. The data were coded as 1 or 0 for positive and negative results, respectively; while noncompatible or missing data were coded as 9. For the estimation of the strain similarities the Jaccard coefficient (SJ) (JACCARD, 1908) was chosen and the cluster analysis was carried out using the unweighted pair group method of association (UGMA) (SNEATH and JOHNSON, 1972). Twenty strains were examined in duplicate in order to estimate the test error (SNEATH and JOHNSON, 1972). The cophenetic correlation was also evaluated (SNEATH and SOKAl, 1973). All computations were done using the NTSYS-pc version 1.80 program of EJ. ROHLF (1993) on a PC computer. DNA extraction and purification Cells from twenty-two representative strains selected for this study and sixteen reference strains were cultured at approximately the late exponential phase and then harvested by centrifugation. The cells were washed and suspended in 0.15 M NaCI-O.l M EDTA buffer (pH 8.0) (5 g wet weight in 50 ml of buffer). Lysis was accomplished at 60°C for 10 min adding sodium dodecyl sulfate at a final concentration of 2 % (wt/vol). The DNA was extracted and purified by the method of MARMUR (1961). Its purity was assessed from the A26(jA28o and A2301A26o extinction ratios (JOHNSON, 1994). DNA base composition The guanine-plus-cytosine (G+C) content of the DNA was calculated from the midpoint value of the thermal denaturation
377
profile (MARMUR and DoTY, 1962) obtained with a model UVVis 551S spectrophotometer (Perkin-Elmer Corp., Norwalk, Conn., USA) at 260 nm, programmed for temperature increases of 1.0 °C min-I. The G+C content was determined from the thermal denaturation temperature by using the equation of OWEN and HilL (1979). The G+C content of reference DNA from Escherichia coli NCTC 9001 was taken to be 51 mol% (OWEN and PITCHER, 1985). Preparation of 3H-labelled DNA and DNA-DNA hybridization experiments DNA was radioactively labelled by the multiprime system with a commercial kit (RPN 1601 Y; Amersham International, Amersham, England) , using [1',2',VH] dCTP (Amersham). The average specific activity obtained with this procedure was 8.4 x 10 6 cpm ).Ig-I of DNA. The labelled DNA was denatured before hybridization by heating at 100°C for 5 min and then placed on ice. DNA-DNA similarity was studied by the competition procedure described by JOHNSON (1994) . Competitor DNAs were sonicated (Braun Melsungen, Melsungen, Germany) at 50 W for two 15-s intervals. Membrane filters (HAHY; Millipore Corp., Bedford, Mass., USA) containing reference DNA (ca. 25 I1g cm-2) were placed in 5 ml screw cap vials which contained the labelled, sheared, denatured DNA and the denatured and sheared competitor DNA. The ratio of the concentration of competitor DNA to the concentration of labelled DNA was at least 150:1. The final reaction concentrations were 2 x SSC and 30% formamide, and the final volume was 140 ).II. The hybridization experiments were carried out under optimal conditions, with temperatures ranging between 54 and 58°C, which is within the limits of validity for the filter method (DE LEY and TIJTGAT, 1970). The vials were shaken slightly for 18 h in a water bath (Grant Instruments, Cambridge, England); these procedures were done in triplicate. After hybridization the filters were washed in 2 x SSC at the optimal renaturation temperature. The radioactivity bound to the filters was measured in a liquid scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif., USA), and the percentage of similarity was calculated as described by JOHNSON (1994). At least three independent determinations were carried out for each experiment, and the mean values are reported here.
Results Using the appropriate media and culture conditions, we were able to isolate a total of 158 extremely halophilic strains from the 56 years old enrichments obtained from Dead Sea water samples. All isolates required for optimal growth salt concentrations between 20 to 25% (wt/vol). They produced pink to red pigmented colonies on solid media. According to their phenotypic characteristics (Table 1) they were identified as members of the Halobacteriaceae (GRANT and LARSEN, 1989). All strains were Gram-negative, motile and catalase positive. They reduced nitrates and were able to grow on pyruvate and L-asparagine, but were not able to degrade DNA or Tween 80. They were Voges-Proskauer, Simmons citrate, phenylalanine deaminase and arginine dehydrolase negative. Acid was not produced from lactose or D-mannose and no growth was obtained on lactose, L-alanine, DLarginine, L-aspartic acid, L-phenylalanine, L-glutamine, L-proline, L-serine and L-threonine. All strains were resistant to chloramphenicol, erythromycin and neomycin.
378
D. R. ARAHAL et al.
Table 1. Phenotypic characteristics of the 158 extremely halophilic archaea which group into six phenons. The percentage of strains giving positive is indicated for each test. Characteristics:
Phenon No. strains
Pigmentation: Pink" Red" Gas vacuoles" Growth at % (w/v) salts:
3 5 10 15 25 30
B
C
D
28
7
E 24
F
18
38 62 0
22
29 71 0
100 0 0
17 83 0
0 100 100
6 49 89 97 46
0 6 28 50 100 100
4 4 89 100
0 8 67 100 100
36
0 0 86 100 100 100
92
0 0 44 67 100 44
0 7 79 100 0
0 22 61 100 0
0 0 14 100 7
71
100 100 86 0
79 100 100 100 0
78 100 67 0
19 85 100 99 18
0 56 100 100 28
29 86 100
96
78
14
100 100 86 0 0
100 79 38 0
100 56 11 0
24 35 18 24
83 6 11 0
18 82 0 0
14 0 0 86
96
56 33 0
0 1
0 0
11
4
0 0
79 0
44
64 26 67 0
17 22 0 0
86 18 57 0
100 71 0 0
21
79 97 57 31 99 36 1 0 4 6 63 76 6
83 100
21 100
29 86 14 86 86 100 100 71 0 29 100 86 100 0
75 100 8 54 100 17
11 11
92
0 0 67 89 100
0 14 100
4 100 100 100 100 0 42 100 100 100 100 88 42
A 72
11
78
0
96
9
Growth at pH:
5.5 6.0" 6.5 8.5 9.0
78
Growth at (0C):
20 25 55 60 65
96
Minimal Mg2+ requeriment (%):
0.05" O.l" 0.2 0.5* Anaerobic growth with: Nitrate Arginine Hydrolysis of: Gelatine Starch Casein Phosphatase* Acid production from: D-Arabinose D-Fructose D-Galactose Glycerol D-Glucose Maltose Sucrose" D-Trehalose D-Xylose Indol production ,. Methyl red Nitrite reduction HzS production Urease" Utilization of: Starch D-Arabinose D-Cellobiose D-Fructose" D-Galactose " D-Glucose* Inulin Lactose * Maltose D-Mannose D-Raffinose D-Rhamnose D-Ribose"
11
99 93 97 99 93 100 100 100 100 25 99 40 4
11
11
28 94 100
32 100 86 7 14
11
0 11
11
0 100 89 22 94
86 32 86 7 4
61
86 7 4 100 0 100 82 18 14 14 7 21 0
72
94 100 100 100 89 100 100 83 72
83 78
71
100 0 100 43 86 43 100 100 0
0 4 0
13
0 0
8 21 88 100 4 100 0
11
78
78 0 0 100
0 22 11 11
78
100 100 0 100 89 0 100 78
100 0 89 67 11 0 100
Taxonomy of Dead Sea Halobacteria
379
Table 1. (Continued). Characteristics:
Ph en on No. strains
Sucrose" Salicin" D-Trehalose" D-Xylose* Glycerol" D-Mannitol" myo- Inositol" D-Sorbitol <. Acetate" Butyrate" Citrate" Fumarate L-Glutamate':' Hippurate Malate* Propionate" Succinate* L-Isoleucine* L-Leucine" L-Ornithine " Susceptibility to antibiotics: Aphidicolin (5 pg/ml) Anysomicin (10 pg/ml) Bacitracin (10 U) Novobiocin (30 pg/ml) Rifampin (5 pg/ml) Susceptibility to heavy metals (mM): 1.0 • Ag: 0.5 0.1 0.05" 20.0 • As: 10.0 5.0 1.0 • Cd: 0.5 0.1 0.05 1.0 • Co: 0.5 0.1 5.0 • Cr: 2.5 1.0 2.5 • Cu: 1.0 0.5 0.01 • Hg: 0.005 2.5 • Ni: 1.0 0.5 20.0 • Pb: 10.0 5.0 2.5 0.5 • Zn: 0.1
E
A 72
B 18
C 28
D 7
24
85 100 96 33 42 72 100 0 54 63 0 0 14 0 0 13 0 14 0 92
83 83 100 67 100 39 100 0 94 11 0 0 83 0 0 0 17 17 0 89
0 0 0 0 0 43 14 0 0 0 0 0 0 0 0 0 0 100 0 0
100 0 71 0 29 100 0 0 100 0 0 14 0 0 71 100 100 0 0 0
100 100 100 100 100 75 0 88 100 100 29 96 100 25 92 100 100 0 8 33
22 11 0 22 100 0 100 0 100 11 100 22 100 0 100 67
0 83 93 93 0
0 61 100 89 0
7 71 100 100 21
29 71 100 100 86
0 96 79 100 0
0 100 78 100 11
6 39 56 0 0 39 61 0 49 51 0 49 50 1 74 26 0 0 74 26 83 17 28 72 0 0 43 47 10 60 40
33 67 0 0 0 67 33 0 56 44 0 6 78 17 28 72 0 0 78 22 78 22 67 33 0 0 67 33 0
0 0 25 75 4 79 18 11 68 21 0 14 71 14 14 64 21 68 29 4 86 14 14 61 25 68 29 4 0 82 18
0 86 14 0 29 71 0 14 86 0 0 86 14 0 86 14 0 0 86 14 100 0 86 14 0 57 29 14 0 100 0
0 29 71 0 71 29 0 0 83 17 0 25 75 0 17 71 13 0 79 21 83 17 29 71 0 29 71 0 0 67 33
0 0 0 100 78 22 0 0 0 78 22 0 78 22 0 89 11 0 89 11 100 0 0 56 44 0 0
78 22
F 9
78 100 100 100
78 22 89 11
* Differential characteristics between the phenons. All strains were Gram-negative, motile and yielded positive results to the following tests: growth at 20% (w/v) salts, and at pH 7.0 to 8.0, growth in the range from 30 to 50°C, catalase production, nitrate reduction, and utilization of pyruvate and L-asparagine. All strains were negative to: growth at 0.9% (w/v) total salts, growth at pH 5.0 and 9.5, growth at 15 and 70°C, DNAse, acid production from lactose and D-mannose, Voges-Proskauer, Simmons citrate, phenylalanine deaminase, arginine dehydrolase, hydrolysis of Tween 80, utilization of lactate, L-alanine, DL-arginine, L-aspartic acid, L-phenylalanine, L-glutamine, L-proline, L-serine and Lthreonine and susceptibility to chloramphenicol (30 pg/ml), erythromycin (15 pg/ml) and neomycin (30 pg/ml).
380
D. R. ARAHAL et al.
Reproducibility of phenotypic characters The inclusion of the pairs of randomly chosen duplicate strains in the analysis enabled experimental test error to be calculated. The probability (P) of an erroneous result averaged 4.04%. A small number of tests were responsible for most of the test error. These tests, with a test variance of 0.03, were acid production from maltose and sucrose, Voges-Proskauer, and utilization of D-cellobiose, D-trehalose, fumarate and malate as sole source of carbon and energy.
Numerical analysis The result of the numerical study of the strains grouped by means of the SJ coefficient and UPGMA clustering yielded the dendrogram shown in Fig. 1. The cophenetic correlation was 0.901. The SSM coefficient was also used and the clustering of strains was quite similar to that obtained with the SJ coefficient. In the dendrogram obtained, all the isolates grouped into six phenons at similarity levels ranging from 76 to 85% (Fig 1). Table 1 shows the characteristics which differentiate the six phenons studied from one another.
DNA base composition The G+C contents of the DNAs of the 22 strains chosen as representative isolates of the different phenons ranged from 56.5 to 66.9 mol% (Table 2).
DNA-DNA hybridization The results of the DNA-DNA hybridization experiments are shown in Table 2.
Discussion The 158 isolates of this study were obtained from Dead Sea brine samples kept by Volcani for almost 60 years and, since the isolation media were designed for extremely halophilic microorganisms, all of them were members of the halobacteria (GRANT and LARSEN, 1989). The ability of halo bacteria to survive for such a long period was proved in a previous study in which we isolated 22 halo bacteria from these samples (ARAHAL et aI., 1996). The purpose of this study was to know in detail the taxonomic affiliation of a large number of strains obtained from the 56-years-old Dead Sea brine samples, and to compare them with both, the current classification of halo bacteria, as well as the information available in 1936-1944, when the studies of Volcani were reported (VOLCANI 1936,1940,1943,1944). All 158 isolates were grouped, based on the numerical taxonomy study, into six phenons whose phenotypic features are shown in Table 1. Reference strains were not included in any phenon, although some of them were closely related to the phenons; this could be due to their different ability to use organic compounds and different
response to antibiotics and heavy metals. Phenons A, B and C showed features (Table 1) similar to those of the genus Haloarcula (GRANT and LARSEN, 1989). Phenon A comprised pleomorphic, motile cells, rods are rarely observed, and gas vacuoles are not produced. Strain Ell, which is included in this phenon, was used as a representative isolate in a previous study from the enrichments from Dead Sea brine samples (ARAHAL et aI., 1996) and its 16S rRNA sequence was more closely related to Haloarcula hispanica (with a 98.8% similarity). The phenotypic features of phenon A are indeed similar to those of H. hispanica (JUEZ et ai., 1986), except for growth at 65 DC, acid production from D-mannose and D-xylose, and use of salicin, arginine, asparagine and glutamine. The DNA hybridization study shows that the six representative strains chosen from phenon A constitute a homogeneous genotypic cluster, with DNA relatedness ranging from 82 to 100%. Besides, the results clearly support the assignment of phenon A to the species H. hispanica, showing a 76% DNA relatedness with H. hispanica ATCC 33960T (Table 2). With respect to phenons Band C, they could also be assigned to the species H. hispanica, showing differences in some physiological and biochemical features with respect to ph en on A (Table 1). DNA-DNA hybridization results obtained between representative strains from phenons Band C, and those from phenon A where always above 68% (Table 2). The G+C contents of the thirteen representative strains included into these three phenons varies from 56.5 to 63.5 mol%, a range that also comprise the G+C content of H. hispanica (61.8 mol%) (GUTrERREZ et ai., 1989). The close relationship of strains included in phenons A, Band C to H. hispanica is interesting, considering that this species was originally isolated from salterns located in Alicante (Spain) (JUEZ et ai., 1986), and has not been reported in studies carried out in the Dead Sea (VENTOSA and ARAHAL, 1999). Different geochemical conditions were present at the Dead Sea in 1936 and in recent periods, with an increase of the salinity and a higher Mgz+ concentration. Although H. hispanica was never described to occur in the Dead Sea, another species of this genus, Haloarcula marismortui (OREN et ai., 1990) was originally isolated from this habitat in the 1960's (GINZBURG et ai., 1970). Prior to its formal designation it was known as the "Halobacterium of the Dead Sea" and it resembled Flavobacterium (Halobacterium) marismortui, which was one of the extremely halophilic microorganisms described by VOLCANI (1940) and whose cultures were unfortunately lost. Phenons D and E comprise 7 and 24 isolates, respectively. They show similar phenotypic features. They are pleomorphic cells able to grow from 15 to 30% salts, from pH 6.0 to 8.5 and require 0.5% Mgz+ ions. They produce indole and HzS and are able to use a large number of compounds (Table 1). They clustered close to the Haloferax reference strains, indicating a similarity with species of this genus. Ph en on E - and also phenon D to a lower extent - showed very similar phenotypic features with Haloferax volcanii. H. volcanii NCIMB 2012T, that was used as reference strain, did not clustered within the
Taxonomy of Dead Sea Halohacteria
381
% Similarity
60
70
80
90
100
No. strains
Phenon
72
A
18
B
Haloarcula vallismortis Haloarcula hispanica
28
c
Halobacterium salinarum Halorubrum saccharovorum
7
D
24
E
Haloferax volcanii Haloferax gibbonsii Haloferax denitrijicans Haloferax mediterranei
9
F
Halorubrum lacusprofundi Halococcus morrhuae Halorubrum distributum Halococcus saccharolyticus Haloarcula japonica
Fig. 1. Simplified dendrogram showing the clustering of the strains into six phenons based on the SJ coefficient and UPGMA (unweighted average linkage clustering method) for the 158 extremely halophilic archaea and some reference strains of the Halobacteriaceae.
382
D. R. ARAHAL et al.
Table 2. DNA G+C contents and levels of DNA-DNA contents and levels of DNA-DNA relatednedd for some strains representatives of the different phenons obtained by numerical analysis, and of the extreme halophilic genera. Source of unlabelled DNA'f
G+C content (mol%)
% Homology with 3H-labelled DNA from strain: Ell
E2
329
E8
E1
E12
PhenonA: strain 342 strain 344 strain 349 strain 409 strain Ell strain 619
60.4 63.0 63.5 64.0 56.5 59.9
100 88 100 94 100 82
73 ND 70 ND 80 72
ND ND 100 ND 93 ND
ND
ND
11
11
10 11 10 ND
ND ND 42 ND
ND 48 ND ND 0 ND
PhenonB: strain E2 strain 408 strain 647
61.6 60.4 58.0
60 90 78
100 70 86
90 ND ND
18 ND ND
0 ND ND
0 ND ND
Phenon C: strain 320 strain 329 strain 427 strain 634
60.0 58.7 59.0 63.4
ND 73 ND ND
ND ND ND ND
72 100 100 100
ND 60 ND ND
ND ND ND ND
ND 0 ND ND
Phenon D: strain 638 strain E8
65.1 63.0
ND 31
ND 2
ND 42
98 100
68 61
ND 56
Phenon E: strain 719 strain 808 strain 824 strain E1
62.2 63.5 61.6 63.8
ND ND ND 0
ND 0 0 ND
ND ND ND 55
67 66 65 ND
100 78 100 100
ND ND ND 0
PhenonF: strain E12 strain 706 strain 810 Haloarcula argentinensis ATCC 29841 Haloarcula hispanica ATCC 33960 Haloarcula japonica JCM 7785 Haloarcula marismortui ATCC 43049 Haloarcula mukohatei DSMZ 11483 Haloarcula quadrata DSMZ 11927 "Haloarcula sinaiiensis" ATCC 33800 Haloarcula vallismortis ATCC 29715 Halobacterium salinarum DSMZ 3754 Halobaculum gomorrense DSMZ 9297 Haloferax denitrificans DSMZ 4425 Haloferax gibbonsii ATCC 33959 Haloferax mediterranei CCM 3361 Haloferax volcanii NCIMB 2012 Halorubrum lacusprofundi DSMZ 5036 Halorubrum saccharovorum ATCC 29252 Halorubrum sodomense ATCC 33755 Haloterrigena turkmenica JCM 9743 Natrialba asiatica JCM 9576
66.9 60.0 61.4 62.0' 61.8" 63.3' 62.0 b 65.0 a 60.1d 59.7 b 61.0 b 66.4 b 70.0' 64.2b 61.8 1 59.1b 64.9 h 65.3-65.8 g 64.3 b 66.0 b 59.8 h 60.3-63.1i
6 ND ND ND 76 0 0
ND ND ND 15 ND 57 0 43 50 0 0 53 0 20 0 0 16 0 0 0 ND 12
13 ND ND ND ND 30 0 42 2 1 26 0 0 0 54 14 38 0 0 3 30 2
ND ND ND 45 17 0 0 0 0 ND 0 17
ND ND ND 1 26 37 0 53 44 35 0 8 37 1 15 67 98 0 0 0 33 31
100 70 75 0 47 15 0 49 0 21 0 90 31 0 67 0 0 53 65 16 0 0
11 ND 21 6 0 36 ND 0 0 0 28 0 36 ND 0
0 0 29 3 92 7 25 0 0 21
,. ATCC - American Type Culture Collection; JCM - Japan Collection of Microorganisms; DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; CCM - Czechoslovak Collection of Microorganisms; NCIMB - National Collection of Industrial and Marine Bacteria. Data from: aIHARA et al. (1997), bGUTIERREZ et al. (1989), 'TAKASHINA et al. (1990), dOREN et al. (1999), eOREN et al. (1995), IJUEZ et al. (1986), gMCGENITY and GRANT (1995), hVENTOSA et al. (1999b), iKAMEKURA and DYALLSMITH (1995).
Taxonomy of Dead Sea Halobacteria phenon mainly because of its different ability to use compounds as carbon source. The G+C content of the four representative strains chosen from phenon E ranged from 61.6 to 63.8 mol%, which is slightly lower than the G+C content reported for H. volcanii (64.9 mol%) (GuTIERREZ et ai., 1989), but in the case of the two representatives of phenon D it was 63.0 and 65.1 mol% (Table 2). DNA-DNA hybridization experiments show that the four strains of phenon E constitute an homogeneous genotypic group (with DNA relatedness ranging from 78-100%) not related to any other halo bacteria, except to H. volcanii NCMIB 2012\ with a DNA relatedness of 98%, that clearly indicates the assignment of strains of phenon E to the species H. volcanii. These phenotypic and genotypic results correlate with the phylogenetic study based on the comparison of the 16S rRNA sequences, in which isolate E1, representative strain of phenon E, and strain E8, representative from phenon D, showed a 99.7% similarity with H. volcanii (ARAHAL et ai., 1996). It is interesting to note that the species H. volcanii was isolated from the Dead Sea and described in 1975 (MULLAKHANBHAI and LARSEN, 1975). It was named after B.E. Volcani as a recognition of his early studies in the Dead Sea. Although he did not isolate this species in his studies carried out from 1936 to 1944, we show here that H. volcanii was present in the Dead Sea samples that he used at that time. Independently from the molecular techniques used for the characterization of the isolates, this could be also explained by the different isolation procedures used in our studies. Ph en on F comprises nine red pigmented strains that have gas vacuoles and require 0.1 to 0.2% Mg2+ for growth. They are H 2S, phosphatase, urease and methyl red positive. They are not able to hydrolyze starch or casein. They are not able to produce acids from glucose and other sugars. Their features are similar to these of the genus Halobacterium (GRANT and LARSEN, 1989). The DNA-DNA hybridization data and the phylogenetic analysis based on the 16S rRNA sequence comparison of the strain representative of this group E12 (ARAHAL et aI., 1996) clearly support that this phenon belongs to the species Halobacterium salinarum. Among his extremely halophilic isolates, VOLCANI (1940) identified one as Flavobacterium (Halobacterium) trapanicum. Since the cultures were lost and the information available is very limited not much can be concluded, but we have found similarities between the description of that organism and the strains of phenon F. VOLCANI (1940) described also the presence of Micrococcus morrhuae and Sarcina morrhuae - now believed to be one single species, Halococcus morrhuae (VENTOSA and ARAHAL, 1999), but we failed in isolating members of the genus Halococcus, probably because it is present normally in hypersalinic habitats in very low proportions (MONTERO et ai., 1988) or because it could not survive until our days. Therefore, an important question in our work is, how did the microorganisms isolated by us manage to keep their viability after 56 years of preservation? In the literature there are abundant reports of long term survival
383
events, even up to 650 million years, which is more than 10 7 fold longer than the case described here. A broad review about the topic is that of KENNEDY et al. (1994), while that of GRANT et al. (1998) deals only with halobacteria. Despite the abundance of reports by many different authors about longevity of microorganisms and the care taken in the processing of the samples to avoid external contamination, still many objections arise against the claims of revival of long-term isolates microorganisms. Even assuming that the sterilization methods applied have an absolute effectiveness and only microorganisms indigenous to the samples are recovered, there is still the doubt whether microorganisms entered the sample at any time during the storage period, not reflecting, thus, the age of the sample. This can be specially problematic for the longest records whose sources include fossils, rock salt, soils, ice, etc. (KENNEDY et ai., 1994). So far, attempts to find a molecular evidence for longevity have been unsuccessful (GRANT et ai., 1998). In any case, there is very little resemblance between those ancient samples and the ones used in this study. The first and most obvious difference is, as it was mentioned before, the storage time. Besides, our source consist of laboratory material (enrichments prepared from Dead Sea water samples) which brings two important consequences: first, it is a closed system (500 ml-flasks) whose physical parameters were controlled and free from any external environmental change. Second, since the medium was prepared with sterile Dead Sea water enriched with peptone (1 %) (VOLCANI, 1940) and all flasks were incubated and yielded growth before storage, it cannot be expected that their biodiversity reflects that of the Dead Sea at that time. Even more, the samples used for the inoculation of the flasks were collected close to the mouth of the Jordan River at surface level. At that time the lake was stratified and the salinity of the upper layer was slightly lower than 27% (VENTOSA et aI., 1999a). Nowadays, the Dead Sea has evolved to a holomictic lake with a salinity close to 34%. Therefore, there are enough reasons that explain the differences between the diversity obtained by us and that found in other studies performed at the Dead Sea. Still the questions remain, which are the mechanisms that enable this sustained survival and, what is the possible growth rate during their storage? While in nature growth is usually sporadic or unbalanced (DAWES, 1984), in the laboratory it is an optimized situation, not only for the abundance of nutrients, but also for other factors (temperature, osmotic pressure, etc.). The lack of nutrients, and to a lower extent, other adverse environmental factors, can drastically slow down the growth rate (doubling time of months or even years). The term "starvation survival" has been coined to indicate an absence of growth resulting from an insufficient level of nutrients (MORITA, 1990). It is a physiological state in which the metabolism is suspended or arrested (LYNCH, 1990). In the enrichments of our study nutrients (1 % peptone added to sterile Dead Sea water) are not likely to be a limiting factor at least in an initial stage. Even assuming a depletion of nutrients, and since it is a closed system,
384
D. R. ARAHAL et al.
dead lysed cells can serve to feed other cells, a phenomenon known as cryptic growth (POSTGATE and HUNTER,
1962). Acknowledgments This study was supported by grants from the Ministerio de Educaci6n y Cultura, Spain (grants IFD97-1162 and PB981150); and from the Junta de Andalucia.
References ARAHAL, D.R., DEWHIRST, EE., PASTER, B.J., VOLCANI, B.E., VENTO SA, A. Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences. Appl. Env. Microbiol. 62,3779-3786 (1996). DAWES, E.A. Stress of unbalanced growth and starvation in microorganisms pp. 19-43. In: The revival of injured microbes (M.H.E. ANDREW, A.D. RUSSEL eds.) London, Academic Press 1984. DE LEY, l, TIJTGAT, R. Evaluation of membrane filter methods for DNA-DNA hybridization. Antonie Leeuwenhoek 36, 461-474 (1970). GERHARDT, P., MURRAY, R.G.E., COSTILOW, R.N., NESTER, E.W., WOOD, W.A., KRIEG, N.R., PHILLIPS, G.B. (eds.): Manual of methods for general bacteriology. Washington, D. e., American Society for Microbiology 1981. GINZBURG, M., SACHS, L., GINZBURG, B.Z. Ion metabolism in a Halobacterium. l. Influence of age of culture on intracellular concentrations. J. Gen. Physiol. 55, 187-207 (1970). GRANT, W.D., GEMMELL, R.T., MCGENITY, T.J. Halobacteria: the evidence for longevity. Extremophiles 2, 279-287 (1998) . GRANT, W. D., LARSEN, H.: Extremely halophilic Archaeobacteria. Order Halobacteriales ord. nov., pp. 2216-2233. In: Bergey's manual of systematic bacteriology (J.T. STALEY, M .P. BRYANT, N. PFENNIG, J.G. HOLT eds.), vol. 3. Baltimore, The Williams & Wilkins Co. 1989. GUTIERREZ, M.e., VENTOSA, A., RUIZ-BERRAQUERO, E DNADNA homology studies among strains of Haloferax and other halo bacteria. Curro Microbiol. 18,253-256 (1989). IHARA, K., WATANABE, S., TAMURA, T. Haloarcula argentinensis sp. nov. and Haloarcula mukohataei sp. nov., two new extremely halophilic archaea collected in Argentina. Int. J. Syst. Bacteriol. 47, 73-77 (1997). JACCARD, P. Nouvelles recherches sur la distribution florale. Bull. Soc. Vandoise Sci. Nat. 44, 223-270 (1908) . JOHNSON, J.L.: Similarity analysis of DNAs, pp. 655-681. In: Methods for general and molecular bacteriology (P. GERHARDT, R.G.E. MURRAY, W.A. WOOD, N.R. KRIEG eds.), Washington, D. e., American Society for Microbiology 1994. JUEZ, G., RODRIGUEZ-VALERA, E, VENTOSA, A., KUSHNER, D.J. Haloarcula hispanica spec. nov. and Haloferax gibbonsii spec. nov., two new species of extremely halophilic archaebacteria. Syst. Appl. Microbiol. 8, 75-79 (1986). KAMEKURA, M ., DYALL-SMITH, M .L. Taxonomy of the family Halobacteriaceae and the description of two new genera Halorubrobacterium and Natrialba. J. Gen. Appl. Microbiol. 41,333-350 (1995). KENNEDY, M.l, READER, S.L., SWIERCZYNSKI, L.M. Preservation records of microorganisms: evidence of the tenacity of life. Microbiol. 140,2513-2529 (1994) . LYNCH, J.M. Longevity of bacteria: considerations in environmental release. Curro Microbiol. 20, 387-389 (1990).
MARMUR, J. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. BioI. 3,208-218 (1961). MARMUR, J., DOTY, P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. BioI. 5, 109-118 (1962). MCGENITY, T. l, GRANT, W.D. Transfer of Halobacterium sacccharovorum, Halobacterium sodomense, Halobacterium trapanicum NRC 34021 and Halobacterium lacusprofundi to the genus Halorubrum gen. nov., as Halorubrum saecharovorum comb. nov., Halorubrum sodomense comb. nov., Halorubrum trapanicum comb. nov., and Halobacterium lacusprofundi comb. nov. Syst. Appl. Microbiol. 18,237-243 (1995). MONTERO, e.G., VENTOSA, A., RODRiGUEZ-VALERA, E, RUlzBERRAQUERO, F. Taxonomic study of non-alkaliphilic halococci. J. Gen. Microbiol. 134,725-732 (1988). MORITA, R. Y. The starvation-survival state of microorganisms in nature and its relationships to the bioavailable energy. Experientia 46, 813-817 (1990). MULLAKHANBHAI, M.E, LARSEN, H. Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch. Microbiol. 104,207-214 (1975). NIETO, J.l, VENTOSA, A., RUIZ-BERRAQUERO, E Susceptibility of halobacteria to heavy metals. Appl. Environ. Microbiol. 53, 1199-1202 (1987). OREN, A. Halobacterium sodomense sp. nov., a Dead Sea halobacterium with an extremely high magnesium requeriment. Int. J. Syst. Bacteriol. 33, 381-386 (1983). OREN, A.: The microbial ecology of the Dead Sea, pp. 193-229. In: Advances in microbial ecology (K.e. MARSHALL ed.), vol. 10. New York, Plenum 1988. OREN, A.: Ecology of extremely halophilic microorganisms, pp. 25-53. In: The biology of halophilic bacteria (R.H. VREELAND, L.l. HOCHSTEIN eds.), Boca Raton, CRC Press 1993. OREN, A., GINZBURG, M., GINZBURG, B.Z., HOCHSTEIN, L.l., VOLCANI, B.E. Haloarcula marismortui (Volcani) sp. nov., nom. rev., an extremely halophilic bacterium from the Dead Sea. Int. J. Syst. Bacteriol. 40, 209-210 (1990). OREN, A., VENTOSA, A., GRANT, W.D. Proposed minimal standards for the description of new taxa in the order Halobacteriales. Int. J. Syst. Bacteriol. 47,233-238 (1997). OREN, A., VENTOSA, A., GUTIERREZ, M.e., KAMEKURA, M. Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt). Int. J. Syst. Bacteri01. 49,1149-1155 (1999). OWEN, R.l, HILL, L.R.: The estimation of base compositions, base pairing and genome size of bacterial deoxyribonucleic acids, pp. 217-296. In: Identification methods for microbiologists. 2 nd ed. (EA. SKINNER, D.W. LOVELOCK eds.) London, Academic Press, 1979. OWEN, R.J., PITCHER, D.: Current methods for estimating DNA base composition and levels of DNA-DNA hybridization, pp. 67-93. In: Chemical methods in bacterial systematics (M. GOODFELLOW, E. MINNIKIN eds.), London, Academic Press 1985. POSTGATE, J.R., HUNTER, J.R. The survival of soil bacteria. J. Gen. Microbiol. 29,233-263 (1962). RODRIGUEZ-VALERA, E, RUIZ-BERRAQUERO, E, RAMOS-CORMENZANA, A. Isolation of extremely halophilic bacteria able to grow in defined inorganic media with single carbon sources. J. Gen. Microbiol. 119, 535-538 (1980). ROHLF, E].: NTSYS-pc. Numerical taxonomy and multivariate analysis system, version 1.80. New York, Exeter Software 1993. SNEATH, P.H.A., JOHNSON, R. The influence on numerical taxonomy similarities of errors in microbiological tests. J. Gen. Microbiol. 72, 377-392 (1972).
Taxonomy of Dead Sea Halobacteria SNEATH, P.H.A., SOKAL, R.R.: Numerical taxonomy. The principles and practice of numerical classification. San Francisco, Freeman, W.H. 1973. TAKASHINA, T., HAMAMOTO, T., OTOZAI, K., GRANT, W.D., HORIKOSHI, K. Haloarcula japonica sp. nov., a new triangular halophilic archaebacterium. Syst. App!. Microbio!. 13, 177-181 (1990). TORREBLANcA, M., RODRIGUEZ-VALERA, E, JUEZ, G., VENTO SA, A., KAMEKURA, M., KATES, M. Classification of non-alkaliphilic halo bacteria based on numerical taxonomy and polar lipid composition and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst. App!. Microbio!. 8, 89-99 (1986). VENTOSA, A., ARAHAL, D.R.: Microbial life in the Dead Sea, pp. 357-368. In: Enigmatic microorganisms and life in extreme environments (J. SECKBACH ed.) Netherlands, Kluwer Academic Publishers 1999. VENTO SA, A., ARAHAL, D.R., VOLCANI, B.E.: Studies on the microbiota of the Dead Sea - 50 years later, pp. 139-147. In: Microbiology and biogeochemistry of hypersaline environments (A. OREN ed.) Boca Raton, CRC Press 1999a. VENTO SA, A., GUTIERREZ, M.C., KAMEKURA, M., DYALL-SMITH, M.L. Proposal for the transfer of Halococcus turkmenikus,
385
Halobacterium trapanicum JCM 9743 and strain GSL 11 to Haloterrigena turkmenica gen. nov., comb. nov. Int. ]. Syst. Bacterio!' 49,131-136 (1999b). VOLCANI, B.E. (B. WILKANSKY) Life in the Dead Sea. Nature 138,467 (1936). VOLCANI, B.E. (B. ELAZARI-VOLCANI): Studies on the micro flora of the Dead Sea. Ph.D. thesis. The Hebrew University, Jerusalem 1940. VOLCANI, B.E. (B. ELAZARI-VOLCANI) Bacteria in the bottom sediments of the Dead Sea. Nature 152, 274-275 (1943). VOLCANI, B.E.: The microorganisms of the Dead Sea. In: Volume Consists of Papers Collected to Commemorate the 70th Aniversary of Dr. Chaim Weizmann, pp. 71-85. Daniel Sieff Institute, Rehovoth, Israel 1944.
Corresponding author: A. VENTO SA, Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain Telephone: +34 954556702, Fax: + 34 95462 8162, e-mail: [email protected]