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1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia
2 Australian Centre for Astrobiology, Macquarie University, Building E8C 153, Sydney, NSW 2109, Australia
3 Australian Food Safety Centre, University of Tasmania, Private Bag 54, Hobart, Tasmania 7001, Australia
4 Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken 278-0037, Japan
Correspondence
Brett A. Neilan
b.neilan{at}unsw.edu.au
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). There are 20 different genera of halophilic archaea comprising 60 species (http://www.the-icsp.org/taxa/halobacterlist.htm). Members of the genus Halococcus have been shown to exhibit diverse phenotypes (Montero et al., 1993) and to date four species (Grant et al., 2001; Kamekura, 1998; Oren et al., 1997) have been described: Halococcus morrhuae (Kocur & Hodgkiss, 1973), Halococcus saccharolyticus (Montero et al., 1989), Halococcus salifodinae (Denner et al., 1994) and Halococcus dombrowskii (Stan-Lotter et al., 2002). These Halococcus species are more tolerant than other halobacteria under low salinity (Grant et al., 2001) and are not lysed in distilled water or in N-lauroylsarcosine (Kamekura, 1998).
In a recent study, we characterized the microbial diversity of stromatolites in the hypersaline marine setting of Shark Bay, Western Australia (Burns et al., 2004), and reported the presence of halophilic archaea using culture-independent approaches. Due to the restricted flow of seawater into Hamelin Pool and the high net evaporation rates, the salinity of the surface water in Hamelin Pool is twice that of normal seawater (Arp et al., 2001). The living stromatolites are partially submerged in this hypersaline environment. Microbial communities present in these stromatolites must therefore be able to adapt to this hypersaline environment. Microfossils of ancient stromatolites over 3 billion years old are thought to be evidence of one of the earliest life forms on Earth (Byerly et al., 1986; Walter et al., 1980). The living stromatolites of Shark Bay represent analogues of these fossilized stromatolites, and are thus excellent natural laboratories for studying complex microbial communities that are involved in their formation. The 16S rRNA gene clone libraries constructed from the Shark Bay stromatolites consist of clones related to Cenarchaeales of the Crenarchaeota, and Methanomicrobiales and Halobacteriales of the Euryarchaeota. More specifically within the Halobacteria, clones clustering with the genera Haloferax, Halogeometricum, Halobacterium, Halosimplex and Halococcus were identified (Burns et al., 2004). This revealed significant archaeal diversity associated with these stromatolites. However, the physiological role of archaea in stromatolite systems remains unexplored. In this study, we report the isolation and characterization of a novel halophilic archaeon from these stromatolites. The phenotypic characteristics of the isolates, including lipid profiles, as well as their phylogenetic assignment, based on their 16S rRNA gene sequences, DNA base compositions and DNA–DNA hybridization are described. The two characterized strains represent a novel species within the genus Halococcus.
Samples of columnar stromatolites were collected from the intertidal region of Hamelin Pool, Shark Bay, during two field trips in 1996 and 2002. Approximately 1 cm2 of stromatolite was physically ground and suspended in 5 ml sterile 5 % (w/v) NaCl. The concentration of ions present in the seawater of Hamelin Pool is approximately twice that of seawater (Arp et al., 2001); therefore, for the isolation of halophilic archaea from the stromatolites, solidified DSM97 medium (DasSarma et al., 1995) was modified to mimic the salt ionic concentrations of Hamelin Pool. Media contained the following (g l–1): Casamino acids, 7.50; yeast extract, 10.0; trisodium citrate, 3.00; NaCI, 250; KCI, 2.00; MgCI2.6H2O, 7.23; MgSO4.7H2O, 20.0; FeSO4.7H2O, 0.05; MnSO4.H2O, 0.20; CaCI2.2H2O, 2.70. One hundred millilitres was inoculated with 100 µl of stromatolite suspension in duplicate. Addition of three antibiotics (streptomycin, penicillin and ampicillin) at 100 µg ml–1 prevented the overgrowth of bacteria (Wais, 1988). When growth was observed by turbidity, cultures were spread or streaked on agar media. Petri dishes were incubated in sealed containers at 37 °C for 2–3 weeks and pure cultures were obtained by passaging single colonies on the same medium. Partial sequence analysis of the 16S rRNA gene suggested that random isolates were very similar to Halobacterium strain NCIMB 718. Halobacterium strain NCIMB 718 has yet to be studied taxonomically; however, the 16S rRNA gene sequences deposited suggest that the strain belongs to the species Halococcus (Kamekura et al., 2004). Strain NCIMB 718 was also a Gram-negative coccus, further suggesting that it belongs to the genus Halococcus. In the present study, two isolates, 100NA1 and 100A6T, were selected for further characterization. Strain 100NA1 was isolated from the stromatolites collected during a field trip in 2002, whereas 100A6T was isolated from the stromatolites collected in 1996. Colonies on agar plates were approximately 1–2 mm in diameter and had bright-orange–pink pigmentation.
The two strains were non-motile cocci in liquid and solid cultures, as determined by both phase-contrast microscopy and electron microscopy (Fig. 1). The cells stained Gram-negative and occurred in pairs or tetrads in exponential phase, or as large clusters when the cells were in stationary phase. Thin sections revealed a thick cell envelope (Fig. 1) with an irregular outer layer. Scanning electron microscopy further supported the finding that the outer layer had an irregular consistency.
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. The tests were carried out according to the recommended minimal standard methods for the description of new taxa in the Halobacteriales (Oren et al., 1997). The requirement for NaCl was determined in media containing 10–30 % NaCl (w/v). The requirement for magnesium was determined in media containing 0.5–50 mM MgCl2 and MgSO4, at the optimum NaCl concentration for growth. Oxidase activity was detected by spotting a loopful of culture on a paper strip containing a 1 % solution of tetramethyl-p-phenylenediamine dihydrochloride. In particular, the two isolates and strain NCIMB 718 were negative for oxidase activity, whereas Hcc. morrhuae NRC 16008, Hcc. saccharolyticus ATCC 49257T, Hcc salifodinae DSM 8989T and Halobacterium salinarum NRC-1 were all positive. API ZYM (bioMérieux) was also used for the identification of other enzymic activites (Humble et al., 1977). Strips were inoculated with cells in the exponential phase of growth and were incubated at 37 °C for up to 24 h (Stan-Lotter et al., 2002). The API ZYM strips revealed that the two isolates had similar enzyme profiles to strain NCIMB 718, except for leucine arylamidase and trypsin. The two isolates were positive for leucine arylamidase and negative for trypsin.
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), except that the cells were broken before extraction by grinding with quartz sand in a mortar and pestle placed on ice. TLC was performed using Merck HPTLC silica gel 60 plates (art. 5641) in the solvent system chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.). One-dimensional TLC of polar lipids (Fig. 2) suggested that strain 100A6T contained phosphatidylglycerol and phosphatidylglyceromethylphosphate and four glycolipids (marked with circles). The main glycolipid identified by RF values and comparison with controls was S-DGA-1, a sulphated glycolipid that has been detected in other Halococcus species and in Halobacterium sp. NCIMB 718. The faster-moving and slowest-moving glycolipids have not been detected in Halococcus strains.
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). PCR was used for the amplification of the 16S rRNA gene as described by Burns et al. (2004), using the primers Arch 21F, Arch 958R and 1492Rc (DeLong, 1992). Automated sequencing was carried out as described previously (Neilan et al., 2002). BLAST searches were used to identify similar sequences from GenBank. DNA sequences were aligned by using the multiple sequence alignment tool from CLUSTAL_X and phylogenetic tree reconstructions were performed as described previously (Neilan et al., 2002). The G+C content was determined by using the thermal denaturation method and DNA–DNA reassociation was assessed as described previously (Bowman et al., 1998). Sequences of the 16S rRNA genes of strains 100A6T (1312 bases) and 100NA1 (1192 bases) were obtained. The highest identity match (98 %) of the two strains was with strain NCIMB 718, which has been suggested to belong to the genus Halococcus (Kamekura et al., 2004).
Alignment of the 16S rRNA gene sequences with all published sequences of haloarchaea clearly showed that the strains belonged to the genus Halococcus, as they possessed 19 out of 21 signature bases of this genus (Kamekura et al., 2004). The exceptions were bases at positions 116 and 772, where the signatures were C and T, respectively. The two strains isolated and strain NCIMB 718 possessed A at these positions, supporting the finding that the 16S rRNA genes of the two strains and strain NCIMB 718 showed the highest similarity (98 %). As discussed by Stan-Lotter et al. (2002), the genus Halococcus has two phylotypes. One phylotype contains Hcc. salifodinae and Hcc. saccharolyticus, whereas the other comprises Hcc. morrhuae, Hcc. dombrowskii and other coccoid strains. From the phylogenetic tree constructed, the two strains belonged to a separate lineage with strain NCIMB 718 (Fig. 3), within the genus Halococcus. However, between the two strains and NCIMB 718 there are sufficient levels of differences in their physiological and biochemical characteristics to render them as representing separate species.
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) and were similar to values found for Hcc. saccharolyticus ATCC 49257T (59.5 mol%) and strain NCIMB 718 (60.8 mol%). DNA–DNA hybridization between the two isolates showed that isolate 100A6T had a relatedness value of 94±8 % to isolate 100NA1. Because of this high relatedness value to each other, the two strains were classed as representing the same species and hence further hybridization with other strains was only carried out for strain 100A6T. Isolate 100A6T had 74±10 % relatedness to strain NCIMB 718, 17±6 % relatedness to Hcc. morrhuae NRC 16008 and a lower relatedness value of 11±7 % to Hcc. saccharolyticus ATCC 49257T. The relatedness value to Halogeometricum borinquense JCM 10706T was 13±10 %. DNA–DNA hybridization of strain 100A6T with Hcc. salifodinae DSM 8989T was not carried out in this study. However, Hcc. saccharolyticus ATCC 49257T was used as a representative of that cluster from the phylogenetic tree (Fig. 3
). The DNA–DNA hybridization values revealed that strain 100A6T was more closely related to strain NCIMB 718 than to any other Halococcus species. Strains are normally identified as representing different species when any two strains have less than 70 % DNA–DNA hybridization values (Wayne et al., 1987) and the hybridization value between strain 100A6T and strain NCIMB 718 was between 64 and 84 %. Therefore, based on DNA–DNA hybridization values alone, it is difficult to determine whether strain 100A6T and strain NCIMB 718 represent two separate species. However, biochemical tests have shown distinct differences in properties between strains 100A6T and NCIMB 718, indicating that strain 100A6T does not belong to the same species as strain NCIMB 718 and that both strains are distantly related to the rest of the Halococcus species.
SDS gel electrophoresis was performed as described by Stan-Lotter et al. (2002). SDS-PAGE of whole-cell proteins can be used as a rapid method for distinguishing between bacterial species (Jackman, 1987). Strains 100A6T and 100NA1 had protein profiles that were very similar to each other (Fig. 4). However, they did not resemble the profile of strain NCIMB 718. This result differed from the 16S rRNA gene analysis and DNA–DNA hybridization results, which indicated that strains 100A6T and 100NA1 were most similar to strain NCIMB 718.
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). The optimum NaCl concentration for growth was 15 % (w/v), which contrasts with values of 20–30 % (w/v) for all of the other recognized Halococcus species. Of particular significance, strain 100A6T was oxidase-negative, whereas all recognized Halococcus species are oxidase-positive. Most of the aerobic halophilic archaea are also oxidase-positive (Grant et al., 2001). Strain 100A6T was also indole-negative, whereas the other Halococcus species tested were indole-positive. In addition, strain 100A6T was capable of hydrolysing starch, in contrast to the other Halococcus species. All Halococcus species tested were positive for sulphide reduction except strain 100A6T. Strains 100A6T and NCIMB 718 were negative for gelatin liquefaction, whereas the other Halococcus species were positive. Hence, the above differences, 16S rRNA gene sequences, DNA–DNA hybridization and protein profiles support our proposal of a novel species, Halococcus hamelinensis sp. nov. Lipid profiles of strain 100A6T showed that the isolate had a similar lipid profile to those of all other Halococcus species; however, strain 100A6T also contained an unknown glycolipid (X), which has not been identified in other Halococcus species.
Strains 100A6T and 100NA1 were similar to many halobacteria that inhabit extreme conditions (Kamekura, 1998; Ochsenreiter et al., 2002; Vreeland et al., 2000, 2002; Wais, 1988); however, they were isolated from an environment where no archaea had been shown previously. As they were isolated from stromatolites collected several years apart, but were not identified in seawater surrounding the stromatolite (data not shown), we propose that this archaeon may be intrinsically associated with these stromatolites and thus may potentially play a role in some aspect of stromatolite formation. However, in situ studies such as fluorescence in situ hybridization need to be carried out to clarify the association of archaea with stromatolites. Mechanisms of osmotolerance by strain 100A6T in this hypersaline environment, as well as interactions of this archaeon with other micro-organisms, are also currently under investigation.
Description of Halococcus hamelinensis sp. nov.
Halococcus hamelinensis (ha.me.li'nen.sis. N.L. masc. adj. hamelinensis pertaining to Hamelin Pool, where the type strain was isolated).
Cells are cocci with a diameter of 0.8–1.2 µm and occur singly, in pairs, tetrads or as irregular clusters. Gram-negative, non-motile and strictly aerobic. Small, bright-orange–pink, circular colonies are formed after 1 week incubation at 37 °C on agar media. The optimum NaCl concentration for growth is 15 % (w/v), with a doubling time of 13–15 h; capable of growth in 12.5–30 % (w/v) NaCl. The optimum temperature and pH range for growth are 37 °C and pH 4–9. 0.5–50 mM MgCl2 is required for growth. Oxidase-negative and catalase-positive. Hydrolyses starch. Indole is not produced. Negative for sulphide reduction, urease and gelatin liquefaction. Utilizes glucose, sucrose, xylose, maltose, trehalose and glycerol as complex carbon sources in the presence of 0.1 % yeast extract; and glucose, mannitol, galactose, sucrose, xylose, maltose, trehalose, glycerol and ethanol as a single carbon source. Strong acidification occurs for sucrose, xylose, maltose and trehalose, whereas only slight acidification of glucose and glycerol occurs. Sensitive to rifampicin, novobiocin and bacitracin, but resistant to kanamycin, tetracycline, streptomycin, neomycin and penicillin (50 µg ml–1). DNA G+C content is 60.0–60.5 mol% (Tm). Main polar lipid is S-DGA-1, a sulphated glycolipid detected in all strains of the genera Haloferax and Halococcus.
The type strain, 100A6T (=JCM 12892T=ACM 5227T), was isolated from stromatolites of Shark Bay, Hamelin Pool, Western Australia.
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ACKNOWLEDGEMENTS |
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