Phylogenomics of Haloarchaea: The Controversy of the Genera Natrinema-Haloterrigena.

The haloarchaeal genera Natrinema and Haloterrigena were described almost simultaneously by two different research groups and some strains studied separately were described as different species of these genera. Furthermore, the description of additional species were assigned to either Natrinema or Haloterrigena, mainly on the basis of the phylogenetic comparative analysis of single genes (16S rRNA gene and more recently rpoB' gene), but these species were not adequately separated or assigned to the corresponding genus. Some studies suggested that the species of these two genera should be unified into a single genus, while other studies indicated that the genera should remain but some of the species should be reassigned. In this study, we have sequenced or collected the genomes of the type strains of species of Natrinema and Haloterrigena and we have carried out a comparative genomic analysis in order to clarify the controversy related to these two genera. The phylogenomic analysis based on the comparison of 525 translated single-copy orthologous genes and the Overall Genome Relatedness Indexes (i.e., AAI, POCP, ANI, and dDDH) clearly indicate that the species Haloterrigena hispanica, Haloterrigena limicola, Haloterrigena longa, Haloterrigena mahii, Haloterrigena saccharevitans, Haloterrigena thermotolerans, and Halopiger salifodinae should be transferred to the genus Natrinema, as Natrinema hispanicum, Natrinema limicola, Natrinema longum, Natrinema mahii, Natrinema saccharevitans, Natrinema thermotolerans, and Natrinema salifodinae, respectively. On the contrary, the species Haloterrigena turkmenica, Haloterrigena salifodinae, and Haloterrigena salina will remain as the only representative species of the genus Haloterrigena. Besides, the species Haloterrigena daqingensis should be reclassified as a member of the genus Natronorubrum, as Natronorubrum daqingense. At the species level, Haloterrigena jeotgali and Natrinema ejinorense should be considered as a later heterotypic synonyms of the species Haloterrigena (Natrinema) thermotolerans and Haloterrigena (Natrinema) longa, respectively. Synteny analysis and phenotypic features also supported those proposals.

Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt.

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5-4.6 M NaCl (optimum, 3.6 M) at pH 6.0-8.0 (optimum, pH 7.0) and at 25-50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4 %) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33-85.96 % in ANIb and 30.4-32.9 % using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula, for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

Salinarchaeum chitinilyticum sp. nov., a chitin-degrading haloarchaeon isolated from commercial salt.

Two chitin-degrading halophilic archaeal strains, MC-74T and MC-23, were isolated from commercial salt samples. Cells were motile, rod-shaped and stained Gram-negative. Colonies were vermillion-pigmented. Strains MC-74T and MC-23 were able to grow with 1.5-5.1 M NaCl (optimum, 2.6-3.1 M) at pH 6.0-10.0 (optimum, pH 7.0) and at 20-50 °C (optimum, 40 °C). The orthologous 16S rRNA gene sequence similarity between the two strains was 99.8 %, and the closest phylogenetic relative was Salinarchaeum laminariae JCM 17267T with 99.3-99.5 % similarity. The level of DNA-DNA relatedness between the two strains was 93 and 94 % (reciprocally), and those between the two strains and Salinarchaeumlaminariae JCM 17267T were 35-36 % and 38-39 % (reciprocally). The polar lipids of both strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Glycolipids were not detected. Based on the phenotypic and phylogenetic analyses, the strains represent a novel species of the genus Salinarchaeum, for which the name Salinarchaeum chitinilyticum sp. nov. is proposed. The type strain is MC-74T (=JCM 19597T=KCTC 4262T), isolated from solar salt produced in France. Strain MC-23, isolated from a commercial solar salt sample produced in China, is an additional strain of the species.

Haloparvum alkalitolerans sp. nov., alkali-tolerant haloarchaeon isolated from commercial salt.

A Gram-stain-negative, rod-pleomorphic, aerobic, halophilic archaeon, strain MK62-1T, was isolated from commercial salt made from seawater in the Philippines. Strain MK62-1T was able to grow at 2.1-4.7 M NaCl (with optimum at 2.1-2.6 M NaCl), pH 6.5-9.5 (optimum, pH 7.0-7.5) and 20-55 °C (optimum, 45-50 °C). Based on the orthologous 16S rRNA gene sequence, the closest relative was Haloparvum sedimenti JCM 30891T with 99.2 % similarity. The RNA polymerase subunit B' gene sequence also showed the highest similarity (97.4 %) to that of Haloparvum sedimenti DYS4T. The DNA G+C content of MK62-1T was 70.1 mol%, while that of Haloparvum sedimenti JCM 30891T was 69.5 mol% by the HPLC method. The levels of DNA-DNA relatedness between MK62-1T and Haloparvum sedimenti JCM 30891T were 60.6 and 60.8 % (reciprocally). The major polar lipids of the isolate were C20C20 archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Based on the phenotypic and phylogenetic analyses, it is proposed that the isolate represents species within the genus Haloparvum, for which the name Haloparvum alkalitolerans sp. nov. is proposed. The type strain is MK62-1T (=JCM 30442T =KCTC 4214T).

Halopiger thermotolerans sp. nov., a thermo-tolerant haloarchaeon isolated from commercial salt.

Three thermo-tolerant halophilic archaeal strains, SR-441T, SR-412 and SR-188, were isolated from commercial salt samples. Cells were non-motile pleomorphic rod-shaped, and stained Gram-negative. Colonies were pink-pigmented. The three strains were able to grow with 1.7-4.6 M NaCl (optimum, 2.5 M), at pH 6.5-9.0 (optimum, pH 8.0) and at 35-60 °C (optimum, 45 °C). The orthologous 16S rRNA gene sequence similarities amongst the three strains were 98.8-99.3 %, and the level of DNA-DNA relatedness was 71-74 and 72-75 % (reciprocally). The closest relative was Halopiger aswanensis JCM 11628T with 98.6 %-99.1 % similarity in the orthologous 16S rRNA gene sequences, followed by two more Halopiger species, Halopiger xanaduensis JCM 14033T (98.5 %-99.1 %) and Halopiger salifodinae JCM 9578T (95.5 %-95.6 %). DNA-DNA relatednesses between the three strains and H. aswanensis JCM 11628T and H. xanaduensis JCM 14033T were 61 and 54 %, respectively. The polar lipids of the three novel strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and bis-sulfated diglycosyl archaeol-1. The most distinctive feature of the three strains was the ability to grow at 60 °C, while the maximum growth temperature of H. aswanensis is 55 °C. Based on phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halopiger, for which the name Halopiger thermotolerans sp. nov. is proposed. The type strain is SR-441T (=JCM 19583T=KCTC 4248T) isolated from solar salt produced in Australia. SR-412 (=JCM 19582) and SR-188 (=JCM 19581) isolated from commercial salt samples are additional strains of the species.

Halorubrum gandharaense sp. nov., an alkaliphilic haloarchaeon from commercial rock salt.

A Gram-stain-negative, non-motile, pleomorphic rod-shaped, orange-red-pigmented, facultatively aerobic and haloalkaliphilic archaeon, strain MK13-1T, was isolated from commercial rock salt imported from Pakistan. The NaCl, pH and temperature ranges for growth of strain MK13-1T were 3.0-5.2 M NaCl, pH 8.0-11.0 and 15-50 °C, respectively. Optimal growth occurred at 3.2-3.4 M NaCl, pH 9.0-9.5 and 45 °C. Addition of Mg2+ was not required for growth. The major polar lipids of the isolate were C20C20 and C20C25 archaeol derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Glycolipids were not detected. The DNA G+C content was 64.1 mol%. The 16S rRNA gene sequence of strain MK13-1T was most closely related to those of the species of the genus Halorubrum, Halorubrum luteum CECT 7303T (95.9% similarity), Halorubrum alkaliphilum JCM 12358T (95.3%), Halorubrum kocurii JCM 14978T (95.3%) and Halorubrum lipolyticum JCM 13559T (95.3%). The rpoB' gene sequence of strain MK13-1T had < 90% sequence similarity to those of other members of the genus Halorubrum. Based on the phylogenetic analysis and phenotypic characterization, strain MK13-1T may represent a novel species of the genus Halorubrum, for which the name Halorubrum gandharaense sp. nov. is proposed, with the type strain MK13-1T ( = JCM 17823T = CECT 7963T).

Halococcus agarilyticus sp. nov., an agar-degrading haloarchaeon isolated from commercial salt.

Two agar-degrading halophilic archaeal strains, 62 E(T) and 197 A, were isolated from commercial salt samples. Cells were non-motile cocci, approximately 1.2-2.0 µm in diameter and stained Gram-negative. Colonies were pink-pigmented. Strain 62 E(T) was able to grow with 24-30% (w/v) NaCl (optimum, 27%), at pH 6.5-8.5 (optimum, pH 7.5) and at 22-47 °C (optimum, 42 °C). The 16S rRNA gene sequences of strains 62 E(T) and 197 A were identical, and the level of DNA-DNA relatedness between them was 90 and 90% (reciprocally). The closest relative was Halococcus saccharolyticus JCM 8878(T) with 99.7% similarity in 16S rRNA orthologous gene sequences, followed by Halococcus salifodinae JCM 9578(T) (99.6%), while similarities with other species of the genus Halococcus were equal to or lower than 95.1%. The rpoB' gene tree strongly supported that the two strains were members of the genus Halococcus . Mean DNA-DNA relatedness between strain 62 E(T) and H. saccharolyticus JCM 8878(T) and H. salifodinae JCM 9578(T) was 46 and 44%, respectively. The major polar lipids were archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, derived from both C20C20 and C20C25 archaeol, and sulfated diglycosyl archaeol-1. Several unidentified glycolipids were present. Based on the phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halococcus , for which the name Halococcus agarilyticus sp. nov. is proposed. The type strain is 62 E(T) ( = JCM 19592(T) =KCTC 4143(T)).

Halocalculus aciditolerans gen. nov., sp. nov., an acid-tolerant haloarchaeon isolated from commercial salt.

Three halophilic archaeal strains, MH2-243-1(T), MH2-93-1 and MH2-91-1 were isolated from commercial salt samples from Japan, Australia, and Bolivia. Strain MH2-243-1(T) was able to grow in the presence of 12-30% (w/v) NaCl (optimum, 18% NaCl), at pH 4.5-7.0 (optimum, pH 6.0) and at 20-60 °C (optimum, 40 °C). Strains MH2-91-1 and MH2-93-1 grew in slightly different ranges. The orthologous 16S rRNA gene sequences of the three strains were almost identical (99.8-99.9% similarities), and the closest relative was Salarchaeum japonicum JCM 16327(T) with 94.2-94.3% 16S rRNA gene sequence similarities, followed by strains of members of the closely related genera Halobacterium and Halarchaeum . The RNA polymerase subunit B' gene (rpoB') sequence also showed the highest similarity (86.6%) to that of Salarchaeum japonicum JCM 16327(T). The DNA G+C contents of strains MH2-243-1(T), MH2-93-1 and MH2-91-1 were 68.5, 68.8 and 68.3 mol%, respectively. DNA-DNA relatedness values amongst the three strains were 97-99%. The polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and at least seven unidentified glycolipids. The polar lipid composition differed from those of Salarchaeum japonicum and species of the genera Halobacterium and Halarchaeum . Based on the phenotypic and phylogenetic analyses, it is proposed that the isolates represent a novel species of a new genus, for which the name Halocalculus aciditolerans gen. nov., sp. nov. is proposed. The type strain of the type species is MH2-243-1(T) ( = JCM 19596(T) =KCTC 4149(T)) isolated from solar salt produced in Japan. MH2-93-1 ( = JCM 19595) and MH2-91-1 ( = JCM 19594) are additional strains of the type species.

Halarchaeum grantii sp. nov., a moderately acidophilic haloarchaeon isolated from a commercial salt sample.

Three moderately acidophilic, halophilic archaeal strains, MH1-243-3T, MH1-243-5 and MH1-243-6, were isolated from a commercial salt sample made from seawater in Okinawa, Japan. Cells of the three strains were pleomorphic and stained Gram-negative. Colonies of the strains were orange-red-pigmented. Strain MH1-243-3T was able to grow at 15-27 % (w/v) NaCl (optimum 24 °C), at pH 4.5-6.5 (pH 5.5) and at 35-50 °C (45 °C). Strains MH1-243-5 and MH1-243-6 grew within slightly different ranges (shown in text). The 16S rRNA gene sequences of the three strains were identical, and the closest phylogenetic relative was Halarchaeum salinum MH1-34-1T with 97.0 % similarity. The rpoB' gene sequences of the three strains were also identical, and the closest phylogenetic relative was Halarchaeum acidiphilum JCM 16109T with 92.0 % similarity. The DNA G+C content of MH1-243-3T, MH1-243-5 and MH1-243-6 was 65.2 mol%. The levels of DNA-DNA relatedness amongst the three strains were 84.1-99.8 %, while that between MH1-243-3T and H. salinum MH1-34-1T was 30.6 % and 31.6 % (reciprocally), and those between MH1-243-3T and type strains of other species in the genus Halarchaeum were 42.3-29.4 %. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates should represent a novel species of the genus Halarchaeum, for which the name Halarchaeum grantii sp. nov. is proposed. The type strain is MH1-243-3T ( = JCM 19585T = KCTC 4142T), isolated from commercial sea salt produced in Okinawa, Japan. MH1-243-5 ( = JCM 19586) and MH1-243-6 ( = JCM 18422) are additional strains of the species.

Thermophilic and halophilic ß-agarase from a halophilic archaeon Halococcus sp. 197A.

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a ß-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65-80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.

Halarchaeum nitratireducens sp. nov., a moderately acidophilic haloarchaeon isolated from commercial sea salt.

Two halophilic moderately acidophilic archaeal strains, MH1-136-2(T) and MH1-370-1 were isolated from commercial salt samples made from seawater in Japan and Indonesia, respectively. Cells of the two strains were pleomorphic and Gram-stain-negative. Strain MH1-136-2(T) was pink pigmented, while MH1-370-1 was orange-red pigmented. Strain MH1-136-2(T) was able to grow at 9-30 % (w/v) NaCl (with optimum, 21 % NaCl, w/v) at pH 4.5-6.2 (optimum, pH 5.2-5.5) and at 18-55 °C (optimum, 45 °C). Strain MH1-370-1 was able to grow at 12-30 % (w/v) NaCl (optimum, 18 %, w/v) at pH 4.2-6.0 (optimum, pH 5.2-5.5) and 20-50 °C (optimum, 45 °C). Strain MH1-136-2(T) required at least 1 mM Mg(2+), while MH1-370-1 required at least 10 mM for growth. Both strains reduced nitrate and nitrite under aerobic conditions. The 16S rRNA gene sequences of strains MH1-136-2(T) and MH1-370-1 were identical, and the closest relative was Halarchaeum rubridurum MH1-16-3(T) with 98.3 % similarity. The level of DNA-DNA relatedness between these strains was 90.9 % and 92.4 % (reciprocally), while that between MH1-136-2(T) and Halarchaeum acidiphilum MH1-52-1(T), Halarchaeum salinum MH1-34-1(T) and Halarchaeum rubridurum MH1-16-3(T) was 37.7 %, 44.3 % and 41.1 % (each an average), respectively. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates represent a novel species of the genus Halarchaeum, for which the name Halarchaeum nitratireducens sp. nov. is proposed. The type strain is MH1-136-2(T) ( = JCM 16331(T) = CECT 7573(T)) isolated from solar salt produced in Japan.

Halomicroarcula pellucida gen. nov., sp. nov., a non-pigmented, transparent-colony-forming, halophilic archaeon isolated from solar salt.

A novel halophilic strain, BNERC31(T), was isolated from solar salt, 'Sel marin de Guérande', imported from France. Colonies on agar medium containing soluble starch, sodium citrate, sodium glutamate and inorganic salts were non-pigmented and transparent, while cells obtained by centrifuging liquid cultures were red-pigmented. Cells of strain BNERC31(T) were non-motile, pleomorphic, stained Gram-negative and lysed in distilled water. Growth occurred with 20-30 % (w/v) NaCl (optimum, 25 %, w/v), with 0-500 mM MgCl2 (optimum, 10 mM), at pH 6.0-8.5 (optimum, pH 7.0) and at 25-55 °C (optimum, 40 °C). Growth was dependent on soluble starch, and inhibited completely by 0.5 % organic nutrients, such as Casamino acids or yeast extract. The DNA G+C content was 64.1 mol%. Strain BNERC31(T) possessed at least two heterogeneous 16S rRNA genes, and the sequence of the orthologous gene (preceded by the dihydroorotate oxidase gene, pyrD) showed the highest similarity (96.5 %) to that of Haloarcula marismortui JCM 8966(T). The RNA polymerase subunit B' gene sequence showed the highest similarity (91.7 %) to that of Haloarcula amylolytica JCM 13557(T). The polar lipids of strain BNERC31(T) were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, diglycosyl diether and sulfated diglycosyl diether, similar to those of species of the genus Halomicrobium. The phenotypic and phylogenetic characteristics showed that strain BNERC31(T) differed from species of the genera Haloarcula and Halomicrobium and indicated that it represents a novel species in a new genus, for which the name Halomicroarcula pellucida gen. nov., sp. nov. is proposed. The type strain of the type species is BNERC31(T) ( = JCM 17820(T) = CECT 7537(T)).

Halarchaeum rubridurum sp. nov., a moderately acidophilic haloarchaeon isolated from commercial sea salt samples.

Six halo-acidophilic archaeal strains were isolated from four commercial salt samples obtained from seawater in the Philippines, Indonesia (Bali) and Japan (Okinawa) on agar plates at pH 4.5. Cells of the six strains were pleomorphic, and stained Gram-negative. Two strains were pink-red pigmented, while four other strains were orange-pink pigmented. Strain MH1-16-3(T) was able to grow at 9-30% (w/v) NaCl [with optimum at 18% (w/v) NaCl], at pH 4.5-6.8 (optimum, pH 5.5) and at 20-50 °C (optimum, 42 °C). The five other strains grew at slightly different ranges. The six strains required at least 1 mM Mg(2+) for growth. The 16S rRNA gene sequences of the six strains were almost identical, sharing 99.9 (1-2 nt differences) to 100% similarity. The closest relatives were Halarchaeum acidiphilum MH1-52-1(T) and Halarchaeum salinum MH1-34-1(T) with 97.7% similarity. The DNA G+C contents of the six strains were 63.2-63.7 mol%. Levels of DNA-DNA relatedness amongst the six strains were 79-86%, while those between MH1-16-3(T) and H. acidiphilum MH1-52-1(T) and H. salinum MH1-34-1(T) were both 43 and 45% (reciprocally), respectively. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the six isolates represent a novel species of the genus Halarchaeum, for which the name Halarchaeum rubridurum sp. nov. is proposed. The type strain is MH1-16-3(T) ( =JCM 16108(T) =CECT 7535(T)).

Natronoarchaeum philippinense sp. nov., a haloarchaeon isolated from commercial solar salt.

A Gram-staining-negative, pleomorphic, aerobic, halophilic archaeon, designated strain 294-194-5(T), was isolated in Japan from commercial solar salt imported from the Philippines. Colonies of strain 294-194-5(T) were translucent and red. Strain 294-194-5(T) was able to grow at 20-50 °C (optimum, 37-45 °C), with 14-30 % (w/v) NaCl (optimum, 18 %), and at pH 6.5-8.5 (optimum, pH 8.0). MgCl2 was not required for growth. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain 294-194-5(T) was most closely related to Natronoarchaeum mannanilyticum YSM-123(T) (96.8-97.1 % sequence similarities). The major polar lipids of the novel strain were the C20C20 and C20C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester and the same glycolipids (disulfated diglycosyl diether and one unidentified glycolipid) as detected in N. mannanilyticum YSM-123(T). The DNA G+C content of strain 294-194-5(T) was 63.0 mol%. The DNA-DNA relatedness values between the novel strain and N. mannanilyticum YSM-123(T)were 46.5 % and 48.5 % (reciprocal). Based on these data, strain 294-194-5(T) represents a novel species of the genus Natronoarchaeum, for which the name Natronoarchaeum philippinense sp. nov. is proposed. The type strain is 294-194-5(T) ( = JCM 16593(T) = CECT 7630(T)).

Halobaculum magnesiiphilum sp. nov., a magnesium-dependent haloarchaeon isolated from commercial salt.

Two extremely halophilic archaea, strains MGY-184(T) and MGY-205, were isolated from sea salt produced in Japan and rock salt imported from Bolivia, respectively. Both strains were pleomorphic, non-motile, Gram-negative and required more than 5 % (w/v) NaCl for growth, with optimum at 9-12 %, in the presence of 2 % (w/v) MgCl2 . 6H2O. In the presence of 18 % (w/v) MgCl2 . 6H2O, however, both strains showed growth even at 1.0 % (w/v) NaCl. Both strains possessed two 16S rRNA genes (rrnA and rrnB), and they revealed closest similarity to Halobaculum gomorrense JCM 9908(T), the single species with a validly published name of the genus Halobaculum, with similarity of 97.8 %. The rrnA and rrnB genes of both strains were 100 % similar. The rrnA genes were 97.6 % similar to the rrnB genes in both strains. DNA G+C contents of strains MGY-184(T) and MGY-205 were 67.0 and 67.4 mol%, respectively. Polar lipid analysis revealed that the two strains contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester derived from C20C20 archaeol. The DNA-DNA hybridization value between the two strains was 70 % and both strains showed low levels of DNA-DNA relatedness (48-50 %) with Halobaculum gomorrense JCM 9908(T). Physiological and biochemical characteristics allowed differentiation of strains MGY-184(T) and MGY-205 from Halobaculum gomorrense JCM 9908(T). Therefore, strains MGY-184(T) and MGY-205 represent a novel species of the genus Halobaculum, for which the name Halobaculum magnesiiphilum sp. nov. is proposed; the type strain is MGY-184(T) ( = JCM 17821(T) = KCTC 4100(T)).

Halarchaeum salinum sp. nov., a moderately acidophilic haloarchaeon isolated from commercial sea salt.

Three halophilic archaeal strains, MH1-34-1(T), MH1-16-1 and MH1-224-5 were isolated from commercial salt samples produced from seawater in Indonesia, the Philippines and Japan, respectively. Cells of the three strains were pleomorphic and stained Gram-negative. Strain MH1-34-1(T) was orange-red pigmented, while MH1-16-1 and MH1-224-5 were pink-pigmented. Strain MH1-34-1(T) was able to grow at 12-30 % (w/v) NaCl (with optimum at 18 % NaCl, w/v) at pH 4.5-7.2 (optimum, pH 5.2-5.5) and at 15-45 °C (optimum, 42 °C). Strains MH1-16-1 and MH1-224-5 grew in slightly different ranges. These strains required at least 1 mM Mg(2+) for growth. The 16S rRNA gene sequences of strains MH1-34-1(T), MH1-16-1 and MH1-224-5 were almost identical (99.8-99.9 % similarities), and the closest relative was Halarchaeum acidiphilum MH-1-52-1(T) with 98.4 % similarities. The DNA G+C contents of MH1-34-1(T), MH1-16-1 and MH1-224-5 were 59.3, 60.8 and 61.0 mol%, respectively. The level of DNA-DNA relatedness amongst the three strains was 90-91 %, while that between each of the three strains and Halarchaeum acidiphilum MH1-52-1(T) was 51-55 %. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates should represent a novel species of the genus Halarchaeum, for which the name Halarchaeum salinum sp. nov. is proposed. The type strain is MH1-34-1(T) ( = JCM 16330(T) = CECT 7574(T)).

Gene orders in the upstream of 16S rRNA genes divide genera of the family Halobacteriaceae into two groups.

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.

Natribacillus halophilus gen. nov., sp. nov., a moderately halophilic and alkalitolerant bacterium isolated from soil.

A moderately halophilic and alkalitolerant bacterium, designated strain HN30(T), was isolated from garden soil in Japan. Cells of strain HN30(T) were motile, endospore-forming, aerobic, rod-shaped and gram-positive, and contained A1γ meso-diaminopimelic acid-type murein. Growth occurred in 7-23 % (w/v) NaCl (optimum, 10-15 %, w/v), at pH 6.5-10.0 (optimum, pH 8.0-8.5) and at 20-40 °C (optimum, 30 °C). The isoprenoid quinone was menaquinone-7. The polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(16 : 0) and C(16 : 0). The DNA G+C content of strain HN30(T) was 47 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HN30(T) was most closely related to Geomicrobium halophilum BH1(T) (93 % sequence similarity). 16S rRNA gene sequence similarities with other recognized species were less than 89 %. Phylogenetic and phenotypic characteristics indicated that strain HN30(T) represents a novel species in a new genus, for which the name Natribacillus halophilus gen. nov., sp. nov. is proposed; the type strain is HN30(T) ( = JCM 15649(T) = DSM 21771(T)).

Halobacterium piscisalsi Yachai et al. 2008 is a later heterotypic synonym of Halobacterium salinarum Elazari-Volcani 1957.

Halobacterium piscisalsi was proposed by Yachai et al. (2008), with a single strain, HPC1-2(T) (= BCC 24372(T) = JCM 14661(T) = PCU 302(T)), which was isolated from fermented fish (pla-ra) in Thailand. According to Yachai et al. (2008), the strain was closely related to Halobacterium salinarum based on 16S rRNA gene sequence comparisons and could be differentiated by low DNA-DNA relatedness values and different biochemical profiles compared with other species of the genus. The reanalysis of the 16S rRNA gene sequences and the DNA-DNA relatedness among H. piscisalsi JCM 14661(T) and H. salinarum strains JCM 8978(T), R1 and NRC-1 revealed that they all had exactly the same 16S rRNA gene sequence and shared more than 70 % DNA-DNA relatedness. In addition, the full-length DNA-dependent RNA polymerase subunit B (RpoB) protein sequence of H. piscisalsi JCM 14661(T) (607 amino acids) was the same as that of H. salinarum JCM 8978(T) and showed 94.7 and 96.7 % similarities with those of Halobacterium noricense JCM 15102(T) and Halobacterium jilantaiense JCM 13558(T), respectively. Despite the different biochemical properties described by Yachai et al. (2008), the characteristic phenotypic properties of H. piscisalsi agreed with those in the description of H. salinarum emended by Gruber et al. (2004). Therefore, H. piscisalsi Yachai et al. (2008) should be regarded as a later heterotypic synonym of H. salinarum Elazari-Volcani 1957.

A multilocus sequence analysis approach to the phylogeny and taxonomy of the Halobacteriales.

Members of the order Halobacteriales are obligate extreme halophiles that belong to the domain Archaea. The classification of the Halobacteriales currently relies on a polyphasic approach, which integrates phenotypic, genotypic and chemotaxonomic characterization. However, the most utilized genetic marker for phylogeny, the 16S rRNA gene, has multiple drawbacks for use with the Halobacteriales: the species of many genera exhibit large intragenic differences between multiple ribosomal RNA operons, the gene is too conserved to discriminate reliably at the species level and it appears to be the most frequently recombined gene between closely related species. Moreover, the Halobacteriales is a rapidly expanding group due to recent successes at cultivating novel strains from a diverse set of hypersaline environments; a fast, reliable, inexpensive, portable molecular method for discriminating species is required for their investigation. Recently, multilocus sequence analysis (MLSA) has been shown to be an effective tool for strain identification and taxonomic designation, even for those taxa that experience frequent lateral gene transfer and homologous recombination. In this study, MLSA was utilized for evolutionary and taxonomic investigation of the Halobacteriales. Efficacy of the MLSA approach was tested across a hierarchical gradient using 52 halobacterial strains, representing 33 species (including names without standing in nomenclature) and 14 genera. A subset of 21 strains from the genus Haloarcula was analysed separately to test the sensitivity and relevance of the MLSA approach among closely related strains and species. The results demonstrated that MLSA differentiated individual strains, reliably grouped strains into species and species into genera and identified potential novel species and also family-like relationships. This study demonstrates that MLSA is a rapid and informative molecular method that will probably accommodate strain analysis at any taxonomic level within the Halobacteriales.