| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Departamento de Microbiologia, Facultad de Farmacia, Campus Universitario de Cartuja s/n 18071, Granada, Spain
2 Institut de Nutrition de l'Alimentation et des Technologies Agro-Alimentaires, Université Mentouri, Constantine, Algeria
3 Institut des Sciences de la Nature, Faculté des Sciences, Université Mentouri, Constantine, Algeria
Correspondence
Mercedes Monteoliva-Sánchez
mmonteol{at}ugr.es
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). The current divisions are based on 16S rRNA gene sequences and chemotaxonomic criteria, particularly polar lipid composition. To date, 20 genera of haloarchaea are recognized.
Members of the genus Halorubrum (McGenity & Grant, 1995, 2001) are widely distributed in hypersaline environments and play important roles in the carbon and nitrogen cycles in this ecosystem. Currently, the genus Halorubrum comprises 12 species with validly published names: Halorubrum sodomense (Oren, 1983), Halorubrum lacusprofundi (Franzmann et al., 1988), Halorubrum saccharovorum (Tomlinson & Hochstein, 1976), Halorubrum trapanicum (Petter, 1931; McGenity & Grant, 1995), Halorubrum tebenquichense (Lizama et al., 2002), Halorubrum tibetense (Fan et al., 2004), Halorubrum terrestre (Ventosa et al., 2004), Halorubrum xinjiangense (Feng et al., 2004) and Halorubrum alkaliphilum (Feng et al., 2005). Oren & Ventosa (1996) reclassified Halorubrobacterium coriense (Kamekura & Dyall-Smith, 1995) and Halorubrobacterium distributum (Zvyagintseva & Tarasov, 1987; Kamekura & Dyall-Smith, 1995) as Halorubrum coriense and Halorubrum distributum, respectively. Natronobacterium vacuolatum (Mwatha & Grant, 1993) was also reclassified as Halorubrum vacuolatum by Kamekura et al. (1997).
In this paper, a novel halophilic archaeon designated strain 5.1T belonging to the genus Halorubrum is described. Strain 5.1T was isolated from water samples collected from Ezzemoul sabkha, in north-east Algeria. Halophilic medium modified from the formulation of Oren (1983) was used, comprising (per litre distilled water): 125 g NaCl, 100 g MgCl2.6H2O, 5 g K2SO4, 0.1 g CaCl2.2H2O, 1 g yeast extract, 1 g Casamino acids and 2 g soluble starch. The pH was adjusted to 7.0 and incubation was at 37 °C. The following reference strains were used for comparative purposes: Hrr. sodomense DSM 3755T, Hrr. saccharovorum DSM 1137T, Hrr. lacusprofundi DSM 5036T, Hrr. coriense DSM 10284T, Hrr. tebenquichense CECT 5317T, Hrr. trapanicum NRC 34021T, Hrr. distributum JCM 9100T, Hrr. xinjiangense JCM 12388T and Hrr. terrestre JCM 10247T. The reference strains were cultured in medium HE, at a final concentration of approximately 25 % (w/v) salts (Torreblanca et al., 1986) and supplemented with 0.1 % (w/v) glucose.
Phenotypic analysis of strain 5.1T was performed according to the proposed minimal standard for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Cell morphology was examined using transmission electron microscopy. Gram staining was performed by using acetic-acid-fixed samples, as described by Dussault (1955). Growth rates in various salt concentrations were determined in medium supplemented with 0, 7.5, 10, 15, 20, 25 or 30 % (w/v) NaCl and the requirement for Mg2+ was tested using agar plates containing final concentrations of 0, 0.005, 0.01, 0.02, 0.1, 0.5, 0.7 or 0.8 M MgCl2. The pH range for growth was determined using solid medium at pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0. Temperatures tested included 4, 22, 30, 37, 40, 50, 55 and 60 °C. Anaerobic growth in the presence of L-arginine and nitrate was tested as described by Hartmann et al. (1980). Controls without arginine and nitrate were included and incubation was performed in the dark. Reduction of nitrate was detected by using the sulfanilic acid and 
-naphthylamine reagent (Smibert & Krieg, 1981). Tests for formation of indole and hydrolysis of starch and aesculin were performed as described by González et al. (1978). Hydrolysis of gelatin and Tween 80 was tested as outlined by Gutiérrez & González (1972). Catalase and oxidase activities were tested according to Gerhardt et al. (1994). Utilization of carbohydrates, sugar alcohols, amino acids and organic acids were carried out as described by Oren et al. (1997). Acid production from carbohydrates and sugar alcohols was detected using a phenol red solution. Antibiotic susceptibility was determined by spreading bacterial suspensions on culture plates and applying disks impregnated with the antibiotics tested (Bonelo et al., 1984).
Lipids were extracted from the strains using a modified Bligh and Dyer extraction procedure (Kates, 1972). The lipids were separated by thin-layer chromatography with single development on silica gel in a chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.) solvent system. In addition, two-dimensional chromatography was performed using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first dimension and chloroform/methanol/acetic acid/water (80 : 12 : 15 : 4, by vol.) in the second dimension. Glycolipid spots were detected by spraying the plates with 0.5 % -naphthol in 50 % methanol, followed by 5 % sulfuric acid in ethanol and heating at 150 °C. Phospholipids were visualized with an ammonium molybdate/sulfuric acid spray.
Genomic DNA was extracted and purified by using the method of Lind & Ursing (1986). Purity was assessed from A260/A280 and A230/A260 ratios. The G+C content was determined from the mid-point value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962), with a Perkin Elmer UV-Vis Lambda 3B spectrophotometer at 260 nm, programmed for temperature increases of 1.0 °C min–1. The Tm was determined by using the graphic method described by Ferragut & Leclerc (1976) and the G+C content was calculated from this temperature by using the equation of Owen & Hill (1979). DNA–DNA hybridization studies were performed as described by Ziemke et al. (1998). DNA was double-labelled using DIG-11-dUTP and biotin-16-dUTP (Boehringer Mannheim). The labelling reaction was carried out using a Boehringer Mannheim nick-translation kit.
16S rRNA genes were amplified by PCR with a primer set designed to complement the highly conserved regions of the Halobacterium salinarum and Halorubrum species 16S rRNA genes: forward primer F8 (5'-TTGATCCTGCCGGAGGCCATTG-3') and reverse primer R1462 (5'-ATCCAGCGCAGATTCCCCTAC-3'), corresponding to positions 8–30 and 1462–1441, respectively. PCR and sequencing were performed as described previously (Lizama et al., 2002). Multiple sequence alignments were performed using CLUSTAL W 1.8 (Thompson et al., 1994). A phylogenetic tree was constructed by using the neighbour-joining method with the MEGA 3 program package (Kumar et al., 2004).
On the basis of pigmentation, NaCl-dependent growth, antibiotic susceptibility and 16S rRNA gene sequences, strain 5.1T was identified as representing a member of the family Halobacteriaceae. Colonies on standard growth agar medium were circular, convex and with entire margins. Light-red-coloured colonies, about 0.5–1.0 mm in diameter, were formed after 7 days of incubation at 37 °C. Cells of strain 5.1T were motile, single, small rods. Pleomorphic forms (irregular rods and coccoid and triangular forms) were also observed in liquid culture. Mean cell dimensions of the rod forms were 0.6x1.5–3.0 µm (Fig. 1). Cells stained Gram-negative and lysed in distilled water. Strain 5.1T grew in media containing 15–25 % (w/v) NaCl, with optimum growth at 20 % (w/v) NaCl. The strain grew at pH 6.5–9.0, with optimum growth at pH 7.0–7.5. Strain 5.1T grew at temperatures between 22 and 50 °C. Mg2+ was required for growth and optimum growth occurred at 37–40 °C.
|
Strain 5.1T contained C20C20 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate and a single glycolipid, chromatographically identical to S-DGD-3 of Hrr. coriense DSM 10284T.
The 16S rRNA gene sequence of strain 5.1T was determined and comparison with the 16S rRNA gene sequences of members of the family Halobacteriaceae placed strain 5.1T in the genus Halorubrum (Fig. 2). Strain 5.1T was related to Hrr. coriense (97 % 16S rRNA gene sequence similarity), Hrr. sodomense (97 % similarity), Hrr. trapanicum (96 %), Hrr. distributum (96 %), Hrr. xinjiangense (96 %), Hrr. terrestre (95 %), Hrr. tebenquichense (94 %), Hrr. saccharovorum (93 %) and Hrr. lacusprofundi (93 %).
|
.
|
Description of Halorubrum ezzemoulense sp. nov.
Halorubrum ezzemoulense (ez.ze.mou.len'se. N.L. neut. adj. ezzemoulense pertaining to Ezzemoul sabkha, where the type strain was isolated).
Cells stain Gram-negative, are motile, lyse in distilled water and are pleomorphic. Cells occur mostly as single rods and exhibit a range of morphological types, including irregular rod-shaped, cocci and triangular forms. Rods are 0.6x1.5–3.0 µm. Colonies on standard agar growth medium are 0.5–1.0 mm in diameter, circular, convex, entire and red. Chemo-organotrophic, aerobic growth occurs at 15–25 % (w/v) NaCl, pH 6.5–9.0 and 22–50 °C. Optimum NaCl concentration, pH and temperature for growth are 20 % (w/v), pH 7.0–7.5 and 37–40 °C, respectively. Mg2+ is required for growth. Catalase- and oxidase-positive. Anaerobic growth with nitrate or L-arginine does not occur. Nitrate is reduced to nitrite. Indole is not produced. Gelatin is not liquefied. Starch, aesculin and Tween 80 are not hydrolysed. Lactose, xylose, rhamnose, galactose, raffinose, cellobiose, mannose, fructose, starch, inositol, sorbitol, dulcitol, adonitol, salicin, cellulose, malonate, fumarate, formate and amino acids are not utilized. Glucose, sucrose, maltose, arabinose, mannitol, glycerol, acetate, oxalate, citrate and malate are used as carbon sources. Acids are produced from glucose, lactose, xylose, galactose and rhamnose. Sensitive to novobiocin and bacitracin, but resistant to penicillin, chloramphenicol, ampicillin, rifampicin, streptomycin and neomycin. Polar lipids are C20C20 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Also contains phosphatidylglycerol sulfate and a sulfated diglycosyl diether. The G+C content of genomic DNA of the type strain is 61.9 mol% (Tm).
The type strain, 5.1T (=CECT 7099T=DSM 17463T), was isolated from Ezzemoul sabkha in Algeria.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
Elshahed, M. S., Najar, F. Z., Roe, B. A., Oren, A., Dewers, T. A. & Krumholz, L. R. (2004). Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring. Appl Environ Microbiol 70, 2230–2239.
Fan, H., Xue, Y., Ma, Y., Ventosa, A. & Grant, W. D. (2004). Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon from Lake Zabuye in Tibet, China. Int J Syst Evol Microbiol 54, 1213–1216.
Feng, J., Zhou, P.-J. & Liu, S.-J. (2004). Halorubrum xinjiangense sp. nov., a novel halophile isolated from saline lakes in China. Int J Syst Evol Microbiol 54, 1789–1791.
Feng, J., Zhou, P., Zhou, Y.-G., Liu, S.-J. & Warren-Rhodes, K. (2005). Halorubrum alkaliphilum sp. nov., a novel haloalkaliphile isolated from a soda lake in Xinjiang, China. Int J Syst Evol Microbiol 55, 149–152.
Ferragut, C. & Leclerc, H. (1976). Etude comparative des méthodes de détermination du Tm de l'ADN bactérien. Ann Microbiol 127A, 223–235 (in French).
Franzmann, P. D., Stackebrandt, E., Sanderson, K., Volkman, J. K., Cameron, D. E., Stevenson, P. L., McMeekin, T. A. & Burton, H. R. (1988). Halobacterium lacusprofundi sp. nov., a halophilic bacterium isolated from Deep Lake, Antarctica. Syst Appl Microbiol 11, 20–27.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
González, C., Gutiérrez, C. & Ramírez, C. (1978). Halobacterium vallismortis sp. nov. An amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 24, 710–715.[Medline]
Grant, W. D., Kamekura, M., McGenity, T. J. & Ventosa, A. (2001). Order 1. Halobacteriales Grant and Larsen 1989b, 495VP (effective publication: Grant and Larsen 1989a, 2216). In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 294–334. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
Gutiérrez, C. & González, C. (1972). Method for simultaneous detection of proteinase and esterase activities in extremely halophilic bacteria. Appl Microbiol 24, 516–517.[Medline]
Hartmann, R., Sickinger, H.-D. & Oesterhelt, D. (1980). Anaerobic growth of halobacteria. Proc Natl Acad Sci U S A 77, 3821–3825.
Kamekura, M. & Dyall-Smith, M. L. (1995). Taxonomy of the family Halobacteriaceae and the description of two new genera Halorubrobacterium and Natrialba. J Gen Appl Microbiol 41, 333–350.[CrossRef]
Kamekura, M., Dyall-Smith, M. L., Upasani, V., Ventosa, A. & Kates, M. (1997). Diversity of alkaliphilic halobacteria: proposals for transfer of Natronobacterium vacuolatum, Natronobacterium magadii, and Natronobacterium pharaonis to Halorubrum, Natrialba, and Natronomonas gen. nov., respectively, as Halorubrum vacuolatum comb. nov., Natrialba magadii comb. nov., and Natronomonas pharaonis comb. nov., respectively. Int J Syst Bacteriol 47, 853–857.
Kates, M. (1972). Techniques of Lipidology. New York: Elsevier.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA 3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lind, E. & Ursing, J. (1986). Clinical strains of Enterobacter agglomerans (synonyms: Erwinia herbicola, Erwinia milletiae) identified by DNA-DNA-hybridization. Acta Pathol Microbiol Immunol Scand [B] 94, 205–213.[Medline]
Lizama, C., Monteoliva-Sánchez, M., Suárez-García, A., Rosselló-Mora, R., Aguilera, M., Campos, V. & Ramos-Cormenzana, A. (2002). Halorubrum tebenquichense sp. nov., a novel halophilic archaeon isolated from the Atacama Saltern, Chile. Int J Syst Evol Microbiol 52, 149–155.[Abstract]
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
McGenity, T. J. & Grant, W. D. (1995). Transfer of Halobacterium saccharovorum, Halobacterium sodomense, Halobacterium trapanicum NRC 34021 and Halobacterium lacusprofundi to the genus Halorubrum gen. nov., as Halorubrum saccharovorum comb. nov., Halorubrum sodomense comb. nov., Halorubrum trapanicum comb. nov., and Halorubrum lacusprofundi comb. nov. Syst Appl Microbiol 18, 237–243.
McGenity, T. J. & Grant, W. D. (2001). Genus VII. Halorubrum McGenity and Grant 1996, 362VP (effective publication: McGenity and Grant 1995, 241). In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 320–324. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
Mwatha, W. E. & Grant, W. D. (1993). Natronobacterium vacuolata sp. nov., a haloalkaliphilic archaeon isolated from Lake Magadi, Kenya. Int J Syst Bacteriol 43, 401–404.
Oren, A. (1983). Halobacterium sodomense sp. nov., a Dead Sea halobacterium with an extremely high magnesium requirement. Int J Syst Bacteriol 33, 381–386.
Oren, A. & Ventosa, A. (1996). A proposal for the transfer of Halorubrobacterium distributum and Halorubrobacterium coriense to the genus Halorubrum as Halorubrum distributum comb. nov. and Halorubrum coriense comb. nov., respectively. Int J Syst Bacteriol 46, 1180.
Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233–238.
Owen, R. J. & Hill, L. R. (1979). The estimation of base compositions, base pairing and genome size of bacterial deoxyribonucleic acids. In Identification Methods for Microbiologists, pp. 277–296. Edited by F. A. Skinner & D. W. Lovelock. London: Academic Press.
Petter, H. F. M. (1931). On bacteria of salted fish. Proc K Ned Akad Wet 34, 1417–1423.
Smibert, R. M. & Krieg, N. R. (1981). General characterization. In Manual of Methods for General Bacteriology, pp. 409–443. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Tomlinson, G. A. & Hochstein, L. I. (1976). Halobacterium saccharovorum sp. nov., a carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 22, 587–591.[Medline]
Torreblanca, M., Rodríguez-Valera, F., Juez, G., Ventosa, A., Kamekura, M. & Kates, M. (1986). Classification of non-alkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst Appl Microbiol 8, 89–99.
Ventosa, A., Gutiérrez, M. C., Kamekura, M., Zvyagintseva, I. S. & Oren, A. (2004). Taxonomic study of Halorubrum distributum and proposal of Halorubrum terrestre sp. nov. Int J Syst Evol Microbiol 54, 389–392.
Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.
Zvyagintseva, I. S. & Tarasov, A. L. (1987). Extreme halophilic bacteria from saline soils. Mikrobiologiia 56, 839–844 (in Russian).
This article has been cited by other articles:
|
|
 |
|
  R. L. Mancinelli, R. Landheim, C. Sanchez-Porro, M. Dornmayr-Pfaffenhuemer, C. Gruber, A. Legat, A. Ventosa, C. Radax, K. Ihara, M. R. White, et al. Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece Int J Syst Evol Microbiol, August 1, 2009; 59(8): 1908 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Oren, D. R. Arahal, and A. Ventosa Emended descriptions of genera of the family Halobacteriaceae Int J Syst Evol Microbiol, March 1, 2009; 59(3): 637 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. T. Pesenti, M. Sikaroodi, P. M. Gillevet, C. Sanchez-Porro, A. Ventosa, and C. D. Litchfield Halorubrum californiense sp. nov., an extreme archaeal halophile isolated from a crystallizer pond at a solar salt plant in California, USA Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2710 - 2715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Gutierrez, A. M. Castillo, E. Pagaling, S. Heaphy, M. Kamekura, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, et al. Halorubrum kocurii sp. nov., an archaeon isolated from a saline lake Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2031 - 2035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Castillo, M. C. Gutierrez, M. Kamekura, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Halorubrum ejinorense sp. nov., isolated from Lake Ejinor, Inner Mongolia, China Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2538 - 2542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-L. Cui, Z.-Y. Lin, Y. Dong, P.-J. Zhou, and S.-J. Liu Halorubrum litoreum sp. nov., an extremely halophilic archaeon from a solar saltern Int J Syst Evol Microbiol, October 1, 2007; 57(10): 2204 - 2206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-W. Xu, Y.-H. Wu, H.-b. Zhang, and M. Wu Halorubrum arcis sp. nov., an extremely halophilic archaeon isolated from a saline lake on the Qinghai-Tibet Plateau, China Int J Syst Evol Microbiol, May 1, 2007; 57(5): 1069 - 1072. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
