Haloferax elongans sp. nov. and Haloferax mucosum sp. nov., isolated from microbial mats from Hamelin Pool, Shark Bay, Australia

Extremely halophilic archaea were cultivated from smooth and pustular microbial mats collected from Hamelin Pool, Shark Bay, Western Australia. On the basis of morphology, two phenotypes were present and 16S rRNA gene sequence analysis indicated that all strains were most closely related to members of the genus Haloferax (98.1–99.4 % similarity). One representative strain from each phenotype was selected for further taxonomic characterization. Strain SA5^T, isolated from the smooth mat, formed small (∼1 mm diameter), red, translucent colonies on agar medium and strain PA12^T, isolated from the pustular mat, formed large (3–5 mm diameter), pink, mucoid, domed colonies. Both strains grew in media with 1.7–5.1 M NaCl, required at least 0.2 M Mg²⁺ for growth and had pH optima of 7.4. The 16S rRNA gene similarity between strains SA5^T and PA12^T was 97.1 %. Physiological properties, G+C content and polar lipid composition supported placement of both strains in the genus Haloferax. Phenotypic analysis indicated that the two strains were distinct from each other and from all other members of the genus. This was confirmed by the low DNA–DNA relatedness between strains SA5^T and PA12^T (18–30 %) and between both strains and all other recognized Haloferax species. Two novel species of the genus Haloferax are proposed to accommodate these novel isolates, Haloferax elongans sp. nov. (type strain SA5^T=JCM 14791^T=ATCC BAA-1513^T=UNSW 104100^T) and Haloferax mucosum sp. nov. (type strain PA12^T=JCM 14792^T=ATCC BAA-1512^T=UNSW 104200^T).

---

Abbreviations: DGA-1, diglycosyl archaeol-1; PG, phosphatidylglycerol; PGP-Me, phosphatidylglycerophosphate methyl ester; PGS, phosphatidylglycerosulfate; S-DGA-1, sulfated diglycosyl archaeol-1.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Haloferax elongans sp. nov. SA5^T and Haloferax mucosum sp. nov. PA12^T are DQ860977 and DQ860980, respectively.

Phase-contrast micrographs of late-exponential-phase cells of SA5^T and PA12^T (Fig. S1), SDS-PAGE patterns of whole-cell proteins (Fig. S2) and TLC analysis of polar lipids (Fig. S3) of SA5^T and PA12^T and reference taxa are available with the online version of this paper.

Hamelin Pool, Shark Bay, Australia, is a hypersaline embayment in which the mean salt concentration is roughly double that of seawater (0.852 M Na⁺, 0.090 M Mg²⁺, 0.018 M Ca²⁺, 1.01 M Cl^–; Arp et al., 2001), with morphologically diverse microbial mats and stromatolites (lithified mats) forming in the intertidal regions. These microbial communities are exposed to salt, desiccation and UV stress, suggesting that they may be habitats for novel biodiversity, and are important for our understanding of stromatolite formation processes. During our surveys of the microbial diversity of the microbial mats and stromatolites of Hamelin Pool (Burns et al., 2004; Allen, 2006; Leuko et al., 2007), 13 archaeal strains were isolated from the smooth and pustular mats of the pool. The smooth mat has an even, light brown/green-pigmented surface and displays defined laminations corresponding to distinct green (cyanobacterial), pink (unknown) and black (sulfate-reducing) layers, whereas the pustular mat has an irregular, dark purple/brown-pigmented surface comprised predominantly of cyanobacteria and with little observable lamination (Logan et al., 1974; Allen, 2006). The isolates formed two clusters (or groups) within the genus Haloferax on the basis of cell morphology and 16S rRNA gene sequence analysis.

Currently, there are nine recognized species of Haloferax: Haloferax volcanii (Mullakhanbhai & Larsen, 1975), Haloferax mediterranei (Rodriguez-Valera et al., 1983), Haloferax denitrificans (Tomlinson et al., 1986), Haloferax gibbonsii (Juez et al., 1986), Haloferax alexandrinus (Asker & Ohta, 2002), Haloferax lucentense (Gutierrez et al., 2002), Haloferax sulfurifontis (Elshahed et al., 2004), Haloferax prahovense (Enache et al., 2007) and Haloferax larsenii (Xu et al., 2007). In this paper, taxonomic characterization of a representative isolate from each of the clusters described above is presented and two novel species are proposed.

A halophilic archaeal medium [containing (g l^–1): Casamino acids, 7.5; yeast extract, 10.0; trisodium citrate, 3.00; NaCl, 150; KCl, 2.00; MgSO₄ . 7H₂O, 20.0; MgCl₂ . 6H₂O, 7.23; CaCl₂ . 2H₂O, 2.70; FeSO₄ . 7H₂O, 0.05; MnSO₄ . H₂O, 0.0002; adjusted to pH 7.4; Goh et al., 2006] was inoculated with portions of smooth or pustular mat collected from Hamelin Pool in April 2004. Plates were incubated aerobically at 37 °C in the dark and colonies with pink or red morphology appeared after 2 weeks. The strains were purified by repeated streaking for single colonies and purity was confirmed by uniform cell and colony morphology and by 16S rRNA gene amplification and sequencing. DNA extraction and 16S rRNA gene amplification methods have been described previously (Allen, 2006).

On the basis of 16S rRNA gene sequences and colony and cell morphologies, the 13 isolates formed two groups. Seven strains from the smooth mat, designated strains SA1–SA7, and one strain from the pustular mat, designated strain PA15, formed small (1 mm), red, translucent, convex colonies on agar medium. This group of strains all formed pleomorphic rods (see supplementary Fig. S1 available with the online version of this paper) and analysis of their partial 16S rRNA gene sequences indicated they were most closely related to Hfx. larsenii (99.0–99.4 % similarity). Strain SA5^T was selected as a representative strain for further taxonomic characterization.

Five strains, designated PA12–14, PA16 and PA17, isolated from the pustular mat formed pink–red, entire, opaque, domed, mucoid colonies, 3–5 mm in diameter and exhibited pleomorphic cell morphology (see supplementary Fig. S1 available with the online version of this paper). Analysis of their partial 16S rRNA gene sequences indicated they were most closely related to Hfx. mediterranei (98.1–98.8 % similarity). Strain PA12^T was selected as a representative strain for further taxonomic characterization.

Characterization of strains SA5^T and PA12^T was carried out according to the proposed minimal standards for the description of novel taxa in the order Halobacteriales (Oren et al., 1997). Morphology, nutritional requirements, growth characteristics, miscellaneous biochemical activities and sensitivity to antimicrobial agents were determined as outlined previously (Goh et al., 2006; Allen, 2006). Hfx. volcanii, Hfx. mediterranei and Hfx. lucentense were used as controls for the biochemical tests. Generation times for SA5^T and PA12^T were determined by the method of Robinson et al. (2005) at 53 and 48 °C, respectively. The NaCl, pH and temperature ranges for growth, sensitivity to antibiotics and carbon source utilization results are presented in the species descriptions. Selected characteristics that distinguish strains SA5^T and PA12^T from other Haloferax species are shown in Table 1.

Table 1. Phenotypic and other characteristics that distinguish the Hamelin Pool isolates SA5T and PA12T from other Haloferax species Taxa: 1, SA5T (data from this study); 2, PA12T (data from this study); 3, Hfx. mediterranei JCM 8866T (Rodriguez-Valera et al., 1983); 4, Hfx. volcanii JCM 8879T (Mullakhanbhai & Larsen, 1975); 5, Hfx. lucentense JCM 9276T (Gutierrez et al., 2002); 6, Hfx. denitrificans JCM 8864T (Tomlinson et al., 1986); 7, Hfx. gibbonsii JCM 8863T (Juez et al., 1986); 8, Hfx. alexandrinus JCM 10717T (Asker & Ohta, 2002); 9, Hfx. sulfurifontis JCM 12327T (Elshahed et al., 2004); 10, Hfx. prahovense JCM 13924T (Enache et al., 2007); 11, Hfx. larsenii JCM 13917T (Xu et al., 2007). Additional data are from Tindall (1992), Tindall et al. (1989), Ventosa (2001) and Robinson et al. (2005). +, Positive; –, negative; ±, variable; ND, not determined; w, weakly positive.

Of note, strain PA12^T was oxidase-negative and strain SA5^T exhibited an oxidase-variable response (Table 1). The first oxidase-negative halophilic archaeon to be identified (Halococcus hamelinensis) was isolated recently from Hamelin Pool stromatolites (Goh et al., 2006). This suggests that the oxidase-negative phenotype may be a selective advantage in the Hamelin Pool environment. Furthermore, both strains required higher salt levels for growth than are found in Hamelin Pool seawater. Survival and growth of these strains within the Hamelin Pool environment may be facilitated by the intertidal location of the smooth and pustular mats, as regular cycles of desiccation may result in concentrated salt pockets within the fabric of the microbial mats.

Whole-cell protein profiles were determined by SDS-PAGE (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) and both strains showed different patterns compared to known representatives of the same genus (see supplementary Fig. S2 available with the online version of this paper).

Polar lipids were extracted from SA5^T, PA12^T and reference strains by the method of Kamekura (1993). TLC and staining of the lipids was performed as outlined previously (Usami et al., 2005). Both strains possessed sulfated diglycosyl archaeol-1 (S-DGA-1) and lacked phosphatidylglycerosulfate (PGS) (see supplementary Fig. S3 available with the online version of this paper), as is characteristic for species of the genus Haloferax (Kamekura et al., 2004).

Phylogenetic trees were reconstructed from 16S rRNA gene sequences by the neighbour-joining method and the maximum-likelihood algorithm using ARB (Ludwig et al., 2004). Both the neighbour-joining tree (Fig. 1) and the maximum-likelihood tree (not shown) indicated that strain SA5^T clustered most closely with Hfx. larsenii and strain PA12^T clustered most closely with Hfx. mediterranei. All signature bases of the genus Haloferax (Kamekura et al., 2004) were conserved in the 16S rRNA genes of SA5^T and PA12^T and sequence similarities with Halogeometricum borinquense, their closest relative outside the genus Haloferax, were 91.8 and 92.9 %, respectively.

(30K): Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between strains SA5T and PA12T and related taxa. Bootstrap values >50 % (1000 resamplings) are shown. Bar, 0.01 substitutions per nucleotide position. Halobacterium salinarum DSM 671 was used as the outgroup (not shown). GenBank accession numbers are indicated for each strain.

DNA G+C content was determined by the HPLC method of Tamaoka & Komagata (1984) and DNA–DNA hybridization was performed by the fluorometric method of Ezaki et al. (1989). The two isolates had low DNA–DNA relatedness to each other and to all recognized Haloferax species (33 % or less; Table 1), suggesting that strains SA5^T and PA12^T both represent novel species.

Phylogenetic, phenotypic and chemotaxonomic data indicate that strains SA5^T and PA12^T are members of the genus Haloferax. The DNA–DNA hybridization data, whole-cell protein profiles and phenotypic characteristics (Table 1) justify the creation of two novel species within the genus Haloferax to accommodate these strains, for which the names Haloferax elongans sp. nov. and Haloferax mucosum sp. nov., respectively, are proposed.

Description of Haloferax elongans sp. nov.
Haloferax elongans (e.lon'gans. N.L. part. adj. elongans elongating/extending, referring to the capacity of the species to form very long rods).

Cells are pleomorphic, with irregular short rods and pleomorphic forms common during stationary phase and in suboptimal media. During exponential growth under optimum conditions, cells elongate to up to 12 µm long. Rotational motility is observed in elongated cells. Gram-negative. Colonies on complex agar medium are small (1–2 mm diameter), red, entire, translucent and convex. Growth occurs in 1.7–5.1 M NaCl, with optimum growth at 2.6–3.4 M NaCl. Growth occurs in media containing at least 0.2 M Mg²⁺ or more, with an optimum of 0.4 M. At least 0.04 M Mg²⁺ is required for maintenance of native cell morphology and cell lysis occurs in NaCl solutions of less than 0.85 M. Grows at pH 7.0–9.0 and 30–55 °C, with optimum growth at pH 7.4 and 53 °C. The generation time at 53 °C is 32±3 min. Chemo-organotrophic. Aerobic. Oxidase-variable and catalase-positive. Nitrate is not reduced to nitrite. Does not produce gas from nitrate. Indole is produced from tryptophan. H₂S is not produced from thiosulfate. Anaerobic growth is not observed with either nitrate, DMSO or arginine. Gelatin, casein, starch and Tween 80 are hydrolysed. The following substrates are used as carbon sources: glucose, glycerol, maltose, sucrose and trehalose. Acid is produced from glucose, glycerol, maltose, sucrose and trehalose. The following substrates are not used as carbon sources: ethanol, fumarate, galactose, glycine, lactose, mannitol, mannose, raffinose, xylose, L-arginine, L-histidine, L-lysine, L-ornithine and D-sorbitol. Sensitive to novobiocin (50 µg ml^–1). Resistant to ampicillin, bacitracin, chloramphenicol, cycloheximide, erythromycin, gentamicin, neomycin, penicillin, streptomycin and tetracycline (all at 50 µg ml^–1), ciprofloxacin (2.5 µg per disk), norfloxacin (10 µg per disk) and rifampicin (1 µg per disk). Lipids S-DGA-1, diglycosyl archaeol-1 (DGA-1), phosphatidylglycerophosphate methyl ester (PGP-Me) and phosphatidylglycerol (PG) are present, but PGS is not present.

The type strain is SA5^T (=JCM 14791^T=ATCC BAA-1513^T=UNSW 104100^T), isolated from a smooth mat, Hamelin Pool, Shark Bay, Western Australia. The DNA G+C content of the type strain is 61.4 mol%.

Description of Haloferax mucosum sp. nov.
Haloferax mucosum (mu.co'sum. L. neut. adj. mucosum slimy, a property of the colonies).

Cells are pleomorphic and non-motile. Gram-negative. Colonies on complex agar medium are 3–5 mm in diameter, pink–red, entire, opaque, mucoid and domed. Growth occurs in 1.7–5.1 M NaCl, with optimum growth at 2.6–3.4 M. Growth occurs in media containing at least 0.2 M Mg²⁺ or more. Greater than 0.04 M Mg²⁺ is required for maintenance of native cell morphology and cell lysis occurs in NaCl solutions of 0.85 M or less. Grows at pH 6.0–10.0 and 23–55 °C, with optimum growth at pH 7.4 and 42–53 °C. The generation time at 48 °C is 57±3 min. Chemo-organotrophic. Aerobic. Oxidase-negative and catalase-positive. Nitrate is not reduced to nitrite. Does not produce gas from nitrate. Indole is produced from tryptophan. H₂S is not produced from thiosulfate. Anaerobic growth with nitrate, DMSO or arginine does not occur. Gelatin and casein are hydrolysed, but starch and Tween 80 are not. The following substrates are used as carbon sources: glucose, glycerol, maltose, sucrose and trehalose. Acid is produced from glucose, glycerol, maltose, sucrose and trehalose. The following substrates are not used as carbon sources: ethanol, fumarate, galactose, glycine, lactose, mannitol, mannose, raffinose, xylose, L-arginine, L-histidine, L-lysine, L-ornithine and D-sorbitol. Sensitive to novobiocin (50 µg ml^–1). Resistant to ampicillin, bacitracin, chloramphenicol, cycloheximide, erythromycin, gentamicin, neomycin, penicillin, streptomycin and tetracycline (all at 50 µg ml^–1), ciprofloxacin (2.5 µg per disk), norfloxacin (10 µg per disk) and rifampicin (1 µg per disk). Lipids S-DGA-1, DGA-1, PGP-Me and PG are present, but PGS is not present.

The type strain is PA12^T (=JCM 14792^T=ATCC BAA-1512^T=UNSW 104200^T), isolated from a pustular mat, Hamelin Pool, Shark Bay, Western Australia. The DNA G+C content of the type strain is 60.8 mol%.