| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Istituto di Chimica Biomolecolare, Comprensorio ex Olivetti, via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
2 CSIC, Estacion Experimental del Zaidin, Department of Earth Sciences and Environmental Chemistry, Profesor Albareda 1, 18008 Granada, Spain
3 Istituto di Ricerche di Biologia Molecolare Angeletti IRBM, Pomezia, Roma, Italy
Correspondence
Barbara Nicolaus
bnicolaus{at}icmib.na.cnr.it
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-hydroxybutyric acid; S-DGD, mannose-6-sulfate(1-2)-glucose glycerol diether; S2-DGD, mannose-2,6-disulfate(1-2)-glucose glycerol dietherThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FP1T is AM285297.
A thin-layer chromatogram of total polar lipids and a table giving 13C NMR assignments for polar lipids from strain FP1T and related species are available with the online version of this paper.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Oren et al., 1997; Grant, 2004; Fendrihan et al., 2006). Several saline and hypersaline environments in Spain have been studied and many halophiles have been isolated from them. A few studies have described halophilic bacteria from the Fuente de Piedra saline lake in the province of Malaga, southern Spain (Martinez-Canovas et al., 2004; Martinez-Checa et al., 2005; Mata et al., 2006), but no extremely halophilic archaea have been described from this lake.
In the present study, a novel member of the genus Haloterrigena is proposed on the basis of conventional physiological, biochemical and chemical characteristics and through phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization results. At the time of writing, the genus Haloterrigena comprises five species of extremely halophilic archaea, Haloterrigena turkmenica (Ventosa et al., 1999), Haloterrigena thermotolerans (Montalvo-Rodriguez et al., 2000), Haloterrigena saccharevitans (Xu et al., 2005a), Haloterrigena longa and Haloterrigena limicola (Cui et al., 2006). Owing to taxonomic problems arising within the genera Haloterrigena and Natrinema (Montalvo-Rodriguez et al., 2000; Xin et al., 2000; Tindall, 2003; Xu et al., 2005b; Wright, 2006; Castillo et al., 2006), a combination of other morphological and chemotaxonomic characters such as lipid analyses have been used to distinguish between species of these two genera.
Strain FP1T was isolated from samples collected during summer 2005 from Fuente de Piedra saline lake, Malaga province, southern Spain (37° 6' N 4° 44' W).
Strain FP1T was isolated from a saltern crystallizer pond by the dilution-plating technique. This strain represented the predominant organism in the enrichment and was the only colony-forming organism at the highest dilutions. Enrichment medium (medium 1; DSM 372) contained the following components (per litre): 5.0 g yeast extract (Oxoid), 5.0 g Casamino acids (Oxoid), 3.0 g trisodium citrate (Applichem), 2.0 g KCl (Applichem), 20 g MgSO4.7H2O (Carlo Erba), 200 g NaCl (Applichem), 0.36 µg MnCl2.4H2O (J. T. Baker) and 0.05 g FeSO4.7H2O (Carlo Erba). The pH of medium 1 was 7.0. Growth on single carbon sources (medium 2) was tested on liquid media containing (per litre) 200 g NaCl (Applichem), 2.0 g KCl (Applichem), 1 g MgSO4.7H2O (Carlo Erba), 16.4 g MgCl2.6H2O (Riedel-de Haën), 0.2 g NaHCO3 (J. T. Baker), 2.29 g CaCl2.2H2O (J. T. Baker), 152 mg NH4Cl (Applichem), 33 mg K2HPO4 (Applichem), 0.26 mg FeCl2.4H2O (J. T. Baker) and 10.0 g of the test compound. Solid media were prepared by the addition of 1.8 % (w/v) agar.
Haloterrigena turkmenica JCM 9191T, Haloterrigena thermotolerans DSM 11552T, Haloterrigena saccharevitans JCM 12889T, Haloterrigena limicola JCM 13563T and Natrinema pellirubrum JCM 10476T (McGenity et al., 1998), obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSMZ) and from the Japan Collection of Microorganisms (JCM), were grown in the media suggested by the culture collections (DSMZ medium no. 372 for Htg. turkmenica and Htg. thermotolerans; JCM medium no. 168 for Htg. saccharevitans, Htg. limicola and N. pellirubrum). Haloferax mediterranei CCM 3361T was grown as described by Lanzotti et al. (1988).
Cell morphology was determined by phase-contrast microscopy (Zeiss). Colony morphology was analysed on solid medium via a stereomicroscope (M8; Leica). Tolerance to NaCl and MgSO4 and growth at various temperatures and pH were tested in medium 1. All growth tests were performed at the optimal growth temperature (50 °C) for 3 days. Sensitivity of the strain to antibiotics was tested by using medium 1 with agar (1.8 %, w/v) and Sensi-discs (6 mm; Oxoid) (Romano et al., 1993). Phenotypic tests were performed according to the proposed minimal standards for the description of novel taxa of the order Halobacteriales (Oren et al., 1997). Gelatin hydrolysis was determined as described by Oren et al. (2002). Casein hydrolysis and oxidase, tyrosinase, aminopeptidase (Bactident-Merck) and catalase activities were tested in medium 1 as described by Oren et al. (1997). For nitrate reduction, medium 1 plus 0.1 % (w/v) KNO3 was employed. Hydrolysis of hippurate was tested in medium 1 plus hippurate (1 %, w/v) (Poli et al., 2006). For the indole test, the novel strain was grown at pH 7.0 in medium 1. Gram-staining was performed according to Dussault (1955). The KOH test was performed according to Halebian et al. (1981). Hydrolysis of N'-benzoyl-arginine-p-nitroaniline (BAPNA) stereoisomers was tested as described by Oren & Galinski (1994). Intracellular solutes and poly-hydroxybutyric acid (PHB) were extracted according to Motta et al. (2004) and Sykes (1971), respectively. Cell mass for quinone and lipid analyses was obtained from cultures of test strains grown under their optimal growth conditions for 24 h. Lipid analysis, lipid hydrolysis and identification of core lipids were performed as reported by Nicolaus et al. (2001). Quinones were analysed by LC/MS on a reversed-phase column by Electron Impact MS (EI/MS) and H1 NMR spectra. Phospholipids and glycolipids were separated by TLC on silica gel plates (10x10 cm; 10x25 cm; 0.25 mm, F254; Merck) and were analysed according to Nicolaus et al. (2001). Complex lipids of Hfx. mediterranei CCM 3361T were used as reference (Lanzotti et al., 1988).
The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) and the values given are the mean of three independent analyses of the same DNA sample. DNA was hydrolysed and the resultant nucleotides were analysed by HPLC. The DNA was isolated as described by Poli et al. (2006).
The 16S rRNA gene sequence was determined by direct sequencing of the PCR product. Genomic DNA extraction, amplification of the 16S rRNA gene and purification of the PCR products were carried out as described by Reed et al. (2006) with the primers 5'-CTGGTTGATCCTGCCAG-3' and 5'-ACGGCTACCTTGTTACGACTT-3'. Purified PCR products were sequenced by the DSMZ with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer's recommendations. Sequence reactions were electrophoresed by using the Applied Biosystems 373A DNA sequencer.
The sequence of the novel strain was compared with closely related sequences of reference organisms from the FASTA network service. Sequence data were aligned with the CLUSTAL W 1.8 program (Chenna et al., 2003). Phylogenetic analysis was performed by using the PHYLIP package, version 3.6 (Felsenstein, 2004) and with the neighbour-joining method within the MEGA3 program package (Kumar et al., 2004). DNA–DNA hybridizations were performed at 48.5 °C according to Ezaki et al. (1989) and the levels of DNA–DNA relatedness were calculated according to Goris et al. (1998). Hybridizations were carried out between strain FP1T and related species (Htg. thermotolerans DSM 11552T, Htg. saccharevitans JCM 12889T and Htg. limicola JCM 13563T).
Cells of strain FP1T were coccoid, Gram-negative and able to grow over a restricted range of salinities (2.2–4.0 M NaCl). Colonies on agar medium were light red-pigmented. Strain FP1T did not require magnesium for growth and was unable to assimilate sugars (glucose, sucrose or maltose). Detailed results of morphological analyses, antibiotic sensitivity tests and biochemical tests for strain FP1T are given in the species description below.
In a phylogenetic tree based on 16S rRNA gene sequences, strain FP1T clustered with recognized species of the genus Haloterrigena (Fig. 1). 16S rRNA gene sequence similarities between strain FP1T and Htg. limicola JCM 13563T, Htg. thermotolerans DSM 11552T, Htg. saccharevitans JCM 12889T, Htg. turkmenica JCM 9101T and Htg. longa JCM 13562T were 98.9, 96.2, 95.6, 95.5 and 94.6 %, respectively, but the novel strain was also related to Natrinema versiforme JCM 10478T and Natrinema altunense JCM 12890T (96.1 and 95.3 % sequence similarity, respectively). The position of Natrinema ejinorense in this phylogenetic tree should be resolved in the near future (Fig. 1).
|
; Montalvo-Rodriguez et al., 2000; Xu et al., 2005a; Cui et al., 2006), whereas strain FP1T lacks this component. Species of the genus Natrinema have been shown to possess unknown glycolipids and sulfated glycolipids (McGenity et al., 1998; Xin et al., 2000; Xu et al., 2005b) (see Supplementary Fig. S1 in IJSEM Online). Polar lipids were identified by 1H and 13C NMR spectra (see Supplementary Table S1) (De Rosa et al., 1988; Lanzotti et al., 1988, 1989). LC/MS as well as EI/MS analyses of the quinone content of strain FP1T revealed the presence of two menaquinones, MK8 and MK8(H2). Strain FP1T accumulated PHB under optimal growth conditions. The above results indicated that strain FP1T was a member of the genus Haloterrigena. However, it could be distinguished from recognized species of the genus Haloterrigena on the basis of several phenotypic characteristics (Table 1).
|
On the basis of the phylogenetic, genotypic and chemotaxonomic data presented here, we suggest that strain FP1T should be classified as the type strain of a novel species within the genus Haloterrigena, for which the name Haloterrigena hispanica sp. nov. is proposed.
Description of Haloterrigena hispanica sp. nov.
Haloterrigena hispanica (his.pa'ni.ca. L. fem. adj. hispanica of Hispania, from where the organism was originally isolated).
Cells are Gram-negative, coccoid (1.5–2.0 µm in diameter) and become oval in stationary cultures. Colonies on complex agar medium with 3.4 M NaCl are light red, elevated and circular. Growth occurs at NaCl concentrations of 2.2–4.0 M, at Mg2+ concentrations of 0–0.4 M, at pH values in the range 6.5–8.5 and at temperatures of 37–60 °C. The optimal NaCl concentration, Mg2+ concentration, pH and temperature for growth are 3.4 M, 0.2 M, pH 7.0 and 50 °C, respectively. Chemo-organotrophic and aerobic. Oxidase- and catalase-positive. Indole formation is positive. Nitrate reduction is observed but nitrite reduction is not. Gelatin, starch, casein, and Tweens 20, 40 and 60 are not hydrolysed. The following substrates are utilized for growth: glycerol, sodium acetate, sodium propionate and sodium citrate. Glucose, mannose, rhamnose, fructose, sucrose, galactose, trehalose, ribose, xylose, cellobiose and arabinose are not utilized for growth. Sensitive to (µg per disc) fusidic acid (10), bacitracin (10) and novobiocin (30). Resistant to the following antibiotics (µg per disc): streptomycin (25), tetracycline (50), nystatin (100), vancomycin (30), kanamycin (30), lincomycin (10), neomycin (30), erythromycin (30), ampicillin (25), chloramphenicol (50) and penicillin (10 U). Major polar lipids are C20C20 and C20C25 derivatives of PG, PGP-Me and S-DGD. The DNA G+C content is 62.0 mol% (Tm).
The type strain, FP1T (=DSM 18328T=ATCC BAA-1310T), was isolated from Fuente de Piedra salt lake, southern Spain.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31, 3497–3500.
Cui, H. L., Tohty, D., Zhou, P. J. & Liu, S. J. (2006). Haloterrigena longa sp. nov. and Haloterrigena limicola sp. nov., extremely halophilic archaea isolated from a salt lake. Int J Syst Evol Microbiol 56, 1837–1840.
De Rosa, M., Gambacorta, A., Grant, W. D., Lanzotti, V. & Nicolaus, B. (1988). Polar lipids and glycine betaine from haloalkaliphilic archaeabacteria. J Gen Microbiol 134, 205–211.
Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felsenstein, J. (2004). PHYLIP (phylogeny inference package), version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fendrihan, S., Legat, A., Pfaffenhuemer, M., Gruber, C., Weidler, G., Gerbl, F. & Stan-Lotter, H. (2006). Extremely halophilic archaea and the issue of long-term microbial survival. Rev Environ Sci Biotechnol 5, 203–218.[CrossRef]
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]
Grant, W. B. (2004). Introductory chapter: half a lifetime in Soda Lakes. In Halophilic Microorganisms, pp. 17–22. Edited by A. Ventosa. Heidelberg: Springer.
Halebian, S., Harris, B., Finegold, S. M. & Rolfe, R. (1981). Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria. J Clin Microbiol 13, 444–448.
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]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lanzotti, V., Nicolaus, B., Tincone, A. & Grant, W. D. (1988). The glycolipid of Halobacterium saccharovorum. FEMS Microbiol Lett 55, 223–228.[CrossRef]
Lanzotti, V., Nicolaus, B., Tincone, A., De Rosa, M., Grant, W. D. & Gambacorta, A. (1989). A complex lipid with a cyclic phosphate from the archaebacterium Natronococcus occultus. Biochim Biophys Acta 1001, 31–34.
Martinez-Canovas, M. J., Bejar, V., Martinez-Checa, F. & Quesada, E. (2004). Halomonas anticariensis sp. nov., from Fuente de Piedra, a saline-wetland wildfowl reserve in Malaga, southern Spain. Int J Syst Evol Microbiol 54, 1329–1332.
Martinez-Checa, F., Bejar, V., Llamas, I., Del Moral, A. & Quesada, E. (2005). Alteromonas hispanica sp. nov., a polyunsaturated-fatty-acid-producing, halophilic bacterium isolated from Fuente de Piedra, southern Spain. Int J Syst Evol Microbiol 55, 2385–2390.
Mata, J. A., Bejar, V., Llamas, I., Arias, S., Bressollier, P., Tallon, R., Urdaci, M. C. & Quesada, E. (2006). Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis. Res Microbiol 157, 827–835.[Medline]
McGenity, T. J., Gemmell, R. T. & Grant, W. D. (1998). Proposal of a new halobacterial genus Natrinema gen. nov., with two species Natrinema pellirubrum nom. nov. and Natrinema pallidum nom. nov. Int J Syst Bacteriol 48, 1187–1196.
Montalvo-Rodriguez, R., Lopez-Garriga, J., Vreeland, R. H., Oren, A., Ventosa, A. & Kamekura, M. (2000). Haloterrigena thermotolerans sp. nov., a halophilic archaeon from Puerto Rico. Int J Syst Evol Microbiol 50, 1065–1071.[Abstract]
Motta, A., Romano, I. & Gambacorta, A. (2004). Rapid and sensitive method for osmolyte determination. J Microbiol Methods 58, 289–294.[CrossRef][Medline]
Nicolaus, B., Manca, M. C., Lama, L., Esposito, E. & Gambacorta, A. (2001). Lipid modulation by environmental stresses in two models of extremophiles isolated from Antarctica. Polar Biol 24, 1–8.[Medline]
Oren, A. & Galinski, E. A. (1994). Hydrolysis of N'-benzoyl-arginine-p-nitroanilide stereoisomers as a phenotypic test: a study of Gram-positive halotolerant bacteria. Syst Appl Microbiol 17, 7–10.[Medline]
Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposal of minimal standards for the description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233–238.
Oren, A., Elevi, R., Watanabe, S., Ihara, K. & Corcelli, A. (2002). Halomicrobium mukohataei gen. nov., comb. nov., and emended description of Halomicrobium mukohataei. Int J Syst Evol Microbiol 52, 1831–1835.[Abstract]
Poli, A., Esposito, E., Lama, L., Orlando, P., Nicolaus, G., de Appolonia, F., Gambacorta, A. & Nicolaus, B. (2006). Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica). Syst Appl Microbiol 29, 300–307.[CrossRef][Medline]
Reed, A. J., Lutz, R. A. & Vetriani, C. (2006). Vertical distribution and diversity of bacteria and archaea in sulphide and methane-rich cold seep sediments located at the base of the Florida Escarpment. Extremophiles 10, 199–211.[CrossRef][Medline]
Romano, I., Manca, M. C., Lama, L., Nicolaus, B. & Gambacorta, A. (1993). A method for antibiotic assay on Sulfolobales. Biotechnol Tech 7, 439–440.[CrossRef]
Sykes, J. (1971). Methods in microbiology. In Centrifugal Techniques for the Isolation and Characterization of Sub-cellular Components from Bacteria, pp. 189–193. Edited by J. R. Norris & D. W. Ribbons. London: Academic Press.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Tindall, B. J. (2003). Taxonomic problems arising in the genera Haloterrigena and Natrinema. Int J Syst Evol Microbiol 53, 1697–1698.
Ventosa, A., Gutiérrez, M. C., Kamekura, M. & Dyall-Smith, M. L. (1999). Proposal to transfer Halococcus turkmenicus, Halobacterium trapanicum JCM 9743 and strain GSL-11 to Haloterrigena turkmenica gen. nov., comb. nov. Int J Syst Bacteriol 49, 131–136.
Wright, A. D. (2006). Phylogenetic relationships within the order Halobacteriales inferred from 16S rRNA gene sequences. Int J Syst Evol Microbiol 56, 1223–1227.
Xin, H., Itoh, T., Zhou, P., Suzuki, K., Kamekura, M. & Nakase, T. (2000). Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China. Int J Syst Evol Microbiol 50, 1297–1303.[Abstract]
Xu, X. W., Liu, S. J., Tohty, D., Oren, A., Wu, M. & Zhou, P. J. (2005a). Haloterrigena saccharevitans sp. nov., an extremely halophilic archaeon from Xin-Jiang, China. Int J Syst Evol Microbiol 55, 2539–2542.
Xu, X. W., Ren, P.-G., Liu, S.-J., Wu, M. & Zhou, P. J. (2005b). Natrinema altunense sp. nov., an extremely halophilic archaeon isolated from a salt lake in Altun Mountain in Xin-Jiang, China. Int J Syst Evol Microbiol 55, 1311–1314.
This article has been cited by other articles:
|
|
 |
|
  S. W. Roh, Y.-D. Nam, H.-W. Chang, K.-H. Kim, Y. Sung, M.-S. Kim, H.-M. Oh, and J.-W. Bae Haloterrigena jeotgali sp. nov., an extremely halophilic archaeon from salt-fermented food Int J Syst Evol Microbiol, September 1, 2009; 59(9): 2359 - 2363. [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]
|
|
|
|
|
|
M. C. Gutierrez, A. M. Castillo, M. Kamekura, and A. Ventosa Haloterrigena salina sp. nov., an extremely halophilic archaeon isolated from a salt lake Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2880 - 2884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Romano, I. Finore, G. Nicolaus, F. J. Huertas, L. Lama, B. Nicolaus, and A. Poli Halobacillus alkaliphilus sp. nov., a halophilic bacterium isolated from a salt lake in Fuente de Piedra, southern Spain Int J Syst Evol Microbiol, April 1, 2008; 58(4): 886 - 890. [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 | |
