Masahiro Kamekura

Classification of Non-alkaliphilic Halobacteria Based on Numerical Taxonomy and Polar Lipid Composition, and Description of Haloarcula gen. nov. and Haloferax gen. nov.

Summary A large number of halobacteria have been isolated from different hypersaline environments at different geographical sites and studied together with type culture collection strains with respect to several phenotypic features. Numerical processing of the data resulted in thirteen phenons. A representative of each was studied to determine its polar lipid composition. Our results show the existence of at least three groups with the taxonomic level of genus. Results obtained by other authors with regard to the genotypic relationships among halobacteria support this. Besides the existing genus Halobacterium we propose the two new genera Haloarcula and Haloferax for the nomenclatural accomodation of the three groups.

Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China.

A novel extremely halophilic archaeon, strain XF10T, was isolated from a salt lake in China. This organism was neutrophilic, non-motile and pleomorphic, and was rod, coccus or irregularly shaped. It required at least 1.5 M NaCl for growth and grew in a wide range of MgCl2 concentrations (0.005-0.5 M). Lipid extract of whole cells contained two glycolipids with the same chromatographic properties as two unidentified glycolipids found in the two described Natrinema species, Natrinema pellirubrum and Natrinema pallidum. Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain XF10T clustered with the two described Natrinema species and several other strains (strains T5.7, GSL-11 and Haloterrigena turkmenica JCM 9743) with more than 98.1% sequence similarities, suggesting that strain XF1OT belongs to the genus Natrinema. Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain XF10T and the Natrinema species supported the conclusion that strain XF10T is a novel species within the genus Natrinema. The name Natrinema versiforme sp. nov. is proposed for this strain. The type strain is XF10T (=JCM 10478T=AS 1.2365T=ANMR 0149T).

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

The 16S rRNA genes of three species of the genus Natronobacterium (Natronobacterium gregoryi, Natronobacterium pharaonis, and Natronobacterium vacuolatum) were sequenced and compared to that of the previously sequenced species Natronobacterium magadii. The sequences revealed that Natronobacterium pharaonis was phylogenetically distinct from the other members of the genus and also from other recognized genera of the family Halobacteriaceae. However, Natronobacterium vacuolatum and Natronobacterium magadii were found to be most closely related to the genera Halorubrum and Natrialba, respectively. An unidentified haloalkaliphile, strain SSL1, was also closely related to Natronobacterium magadii and Natrialba asiatica. On the basis of phylogenetic tree reconstructions, signature bases specific for individual genera, and sequences of spacer regions between 16 and 23S rRNA genes, we propose the following changes: Natronobacterium pharaonis to be transferred to Natronomonas gen. nov. as Natronomonas pharaonis gen. nov., comb. nov.; Natronobacterium vacuolatum to be transferred to the genus Halorubrum as Halorubrum vacuolatum comb. nov.; and Natronobacterium magadii to be transferred to the genus Natrialba as Natrialba magadii.

Halosimplex carlsbadense gen. nov., sp. nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate.

Abstract. A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico. This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone. The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth. Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel. The microbe is unique in that it has three dissimilar 16S rRNA genes. Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%–93% similarity to the other two. Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus. All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea. The name suggested for this new genus and species is Halosimplex carlsbadense. The type strain is 2–9-1T (= ATCC BAA-75 and JCM 11222) as written in the formal description.

Proposal to transfer Halococcus turkmenicus, Halobacterium trapanicum JCM 9743 and strain GSL-11 to Haloterrigena turkmenica gen. nov., comb. nov.

The 16S rRNA gene sequences of Halococcus saccharolyticus and Halococcus salifodinae were closely related (94.5-94.7% similarity) to that of Halococcus morrhuae, the type species of the genus Halococcus. However, Halococcus turkmenicus was distinct from the other members of this genus, with low 16S rRNA similarities when compared to Halococcus morrhuae (88.7%). On the basis of phylogenetic tree reconstruction, detection of signature bases and DNA-DNA hybridization data, it is proposed to transfer Halococcus turkmenicus to a novel genus, Haloterrigena, as Haloterrigena turkmenica gen. nov., comb. nov., and to accommodate Halobacterium trapanicum JCM 9743 and strain GSL-11 in the same species. On the basis of morphological, cultural and 16S rRNA sequence data, it is also proposed that the culture collection strains of Halobacterium trapanicum NCIMB 767, ATCC 43102 and JCM 8979 should be renamed as Halococcus sp.

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

BackgroundGenerally, extremophiles have been deemed to survive in the extreme environments to which they had adapted to grow. Recently many extremophiles have been isolated from places where they are not expected to grow. Alkaliphilic microorganisms have been isolated from acidic soil samples with pH 4.0, and thermophiles have been isolated from samples of low temperature. Numerous moderately halophilic microorganisms, defined as those that grow optimally in media containing 0.5–2.5 Molar (3–15%) NaCl, and halotolerant microorganisms that are able to grow in media without added NaCl and in the presence of high NaCl have been isolated from saline environments such as salterns, salt lakes and sea sands. It has tacitly been believed that habitats of halophiles able to grow in media containing more than 20% (3.4 M) are restricted to saline environments, and no reports have been published on the isolation of halophiles from ordinary garden soil samples.ResultsWe demonstrated that many halophilic bacteria that are able to grow in the presence of 20% NaCl are inhabiting in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo, Japan. Analyses of partial 16S rRNA gene sequences of 176 isolates suggested that they were halophiles belonging to genera of the family Bacillaceae, Bacillus (11 isolates), Filobacillus (19 isolates), Gracilibacillus (6 isolates), Halobacillus (102 isolates), Lentibacillus (1 isolate), Paraliobacillus (5 isolates) and Virgibacillus (17 isolates). Sequences of 15 isolates showed similarities less than 92%, suggesting that they may represent novel taxa within the family Bacillaceae.ConclusionThe numbers of total bacteria of inland soil samples were in a range from 1.4 × 107/g to 1.1 × 106/g. One tenth of the total bacteria was occupied by endospore-forming bacteria. Only very few of the endospore-forming bacteria, roughly 1 out of 20,000, are halophilic bacteria. Most of the halophilic bacteria were surviving as endospores in the soil samples, in a range of less than 1 to about 500/g soil. Samples collected from seashore in a city confronting Tokyo Bay gave the total numbers of bacteria and endospores roughly 1000 time smaller than those of inland soil samples. Numbers of halophilic bacteria per gram, however, were almost the same as those of inland soil samples. A possible source of the halophilic endospore originating from Asian dust storms is discussed.

Bacillus halophilus sp. nov., a Moderately Halophilic Bacillus Species

Summary Bacillus halophilus, a new moderately halophilic (optimum growth at 15% salt) species, is described. This species was isolated from rotting wood from the Pacific Ocean. The G+C mol % (Tm) of its deoxyribonucleic acid is 51.5. The type strain is N23-2 (= ATCC 49085 = DSM 4771 = CCM 4074).

Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt)

The motile, predominantly square-shaped, red archaeon strain 801030/1T, isolated from a brine pool in the Sinai peninsula (Egypt), was characterized taxonomically. On the basis of its polar lipid composition, the nucleotide sequences of its two 16S rRNA genes, the DNA G+C content (60.1 mol%) and its growth characteristics, the isolate could be assigned to the genus Haloarcula. However, phylogenetic analysis of the two 16S rRNA genes detected in this organism and low DNA-DNA hybridization values with related Haloarcula species showed that strain 801030/1T is sufficiently different from the recognized Haloarcula species to warrant its designation as a new species. A new species, Haloarcula quadrata, is therefore proposed, with strain 801030/1T (= DSM 11927T) as the type strain.

Micrococcus halobius sp. n.

A moderately halophilic coccus is described which possesses morphological and physiological characteristics most nearly corresponding to those of members of the genus Micrococcus. The organism was isolated from unrefined solar salt and is a moderate halophile: no growth occurs in media without added NaCl, but good growth is observed in media containing 1 to 4 m NaCl or KCl. The characteristics of this organism are sufficiently discrete to suggest that it be placed in a new species, for which the name Micrococcus halobius is proposed. The type strain is 28-3 (= ATCC 21727).

Haloterrigena salina sp. nov., an extremely halophilic archaeon isolated from a salt lake

A novel extremely halophilic strain, designated XH-65(T), isolated from the salt lake Xilinhot in Inner Mongolia, PR China, was subjected to a polyphasic taxonomic characterization. Strain XH-65(T) is neutrophilic, non-motile and requires at least 2.5 M NaCl for growth, with an optimum at 3.4 M NaCl, and grows at pH 6.0-9.0, with optimum growth at pH 7.5. Strain XH-65(T) grows at 25-50 degrees C, with optimal growth at 37 degrees C. Magnesium is not required for growth. On the basis of 16S rRNA gene sequence analysis, strain XH-65(T) was shown to belong to the genus Haloterrigena and was related to Haloterrigena turkmenica VKM B-1734(T) (98.1 % sequence similarity), Haloterrigena saccharevitans AB14(T) (96.9 %), Haloterrigena thermotolerans PR5(T) (96.3 %), Haloterrigena limicola AX-7(T) (95.8 %) and Haloterrigena hispanica FP1(T) (95.7 %). DNA-DNA hybridization revealed 37 % relatedness between strain XH-65(T) and Htg. turkmenica VKM B-1734(T). The polar lipid composition revealed the presence of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and mannose-2,6-disulfate (1-->2)-glucose glycerol diether (S(2)-DGD). The results of the DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain XH-65(T) from the six Haloterrigena species with validly published names. Therefore, strain XH-65(T) represents a novel species, for which the name Haloterrigena salina sp. nov. is proposed, with the type strain XH-65(T) (=CGMCC 1.6203(T) =JCM 13891(T)).