Akinobu Echigo

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.

Halarchaeum acidiphilum gen. nov., sp. nov., a moderately acidophilic haloarchaeon isolated from commercial solar salt.

A novel halophilic archaeon, strain MH1-52-1(T), was isolated from solar salt imported from Australia. Cells were pleomorphic, non-motile and Gram-negative. Strain MH1-52-1(T) required at least 3.0 M NaCl and 1 mM Mg(2+) for growth. Strain MH1-52-1(T) was able to grow at pH 4.0-6.0 (optimum, pH 4.4-4.5) and 15-45 °C (optimum, 37 °C). The diether phospholipids phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, derived from both C(20)C(20) and C(20)C(25) archaeol, were present. Four unidentified glycolipids were also detected. The 16S rRNA gene sequence showed the highest similarity to that of Halobacterium noricense A1(T) (91.7%); there were lower levels of similarity to other members of the family Halobacteriaceae. The G+C content of its DNA was 61.4 mol%. Based on our phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolate should be classified as a representative of a new genus and species, for which the name Halarchaeum acidiphilum gen. nov., sp. nov. is proposed. The type strain of Halarchaeum acidiphilum is MH1-52-1(T) (=JCM 16109(T) =DSM 22442(T) =CECT 7534(T)).

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 SA5T, isolated from the smooth mat, formed small ( approximately 1 mm diameter), red, translucent colonies on agar medium and strain PA12T, 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 SA5T and PA12T 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 SA5T and PA12T (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 SA5T=JCM 14791T=ATCC BAA-1513T=UNSW 104100T) and Haloferax mucosum sp. nov. (type strain PA12T=JCM 14792T=ATCC BAA-1512T=UNSW 104200T).

Natronoarchaeum mannanilyticum gen. nov., sp. nov., an aerobic, extremely halophilic archaeon isolated from commercial salt

Strain YSM-123(T) was isolated from commercial salt made from Japanese seawater in Niigata prefecture. Optimal NaCl and Mg(2+) concentrations for growth were 4.0-4.5 M and 5 mM, respectively. The isolate was a mesophilic and slightly alkaliphilic haloarchaeon, whose optimal growth temperature and pH were 37 °C and pH 8.0-9.0. Phylogenetic analysis based on 16S rRNA gene sequence analysis suggested that strain YSM-123(T) is a member of the phylogenetic group defined by the family Halobacteriaceae, but there were low similarities to type strains of other genera of this family (≤90 %); for example, Halococcus (similarity <89 %), Halostagnicola (<89 %), Natronolimnobius (<89 %), Halobiforma (<90 %), Haloterrigena (<90 %), Halovivax (<90 %), Natrialba (<90 %), Natronobacterium (<90 %) and Natronococcus (<90 %). The G+C content of the DNA was 63 mol%. Polar lipid analysis revealed the presence of phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, disulfated diglycosyl diether and an unknown glycolipid. On the basis of the data presented, we propose that strain YSM-123(T) should be placed in a new genus and species, Natronoarchaeum mannanilyticum gen. nov., sp. nov. The type strain of Natronoarchaeum mannanilyticum is strain YSM-123(T) (=JCM 16328(T) =CECT 7565(T)).

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)).

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)).

Halostagnicola alkaliphila sp. nov., an alkaliphilic haloarchaeon from commercial rock salt.

A Gram-negative, pleomorphic, aerobic, haloalkaliphilic archaeon, strain 167-74(T), was isolated from commercial rock salt imported into Japan from China. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain 167-74(T) is closely related to Halostagnicola larsenii XH-48(T) (98.3 %) and Halostagnicola kamekurae 194-10(T) (97.2 %). The major polar lipids of the isolate were C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. A glycolipid was not detected, in contrast to the two existing, neutrophilic species of the genus Halostagnicola. The DNA G+C content of strain 167-74(T) was 60.7 mol%. and it gave DNA-DNA reassociation values of 19.5 and 18.8 %, respectively, with Hst. larsenii JCM 13463(T) and Hst. kamekurae 194-10(T). Therefore, strain 167-74(T) represents a novel species, for which the name Halostagnicola alkaliphila sp. nov. is proposed, with the type strain 167-74(T) ( = JCM 16592(T)  = CECT 7631(T)).

Geomicrobium halophilum gen. nov., sp. nov., a moderately halophilic and alkaliphilic bacterium isolated from soil

Two moderately halophilic and alkaliphilic bacteria, designated strains BH1(T) and HN5, were isolated from forest soil and garden soil, respectively, in Japan. Cells of strains BH1(T) and HN5 were non-motile, aerobic, bean-shaped, formed irregular clusters with 2-20 cells, Gram-positive and contained A1gamma, meso-diaminopimelic acid-type murein. Spore formation was not detected. Growth occurred in 5-25 % (w/v) NaCl (optimum, 10-15 %, w/v), at pH 6.0-10.0 (optimum, pH 8.5-9.0) and at 20-40 degrees C (optimum, 30 degrees C). The predominant isoprenoid quinones were menaquinone-7 and menaquinone-6. The phospholipids were diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids were i-C(15 : 0), i-C(17 : 0) and i-C(18 : 0). The DNA G+C content of strains BH1(T) and HN5 was 45 and 46 mol%, respectively. The 16S rRNA gene sequences of the two strains were 99.9 % similar. DNA-DNA hybridization results indicated high levels of relatedness (88 and 85 % reciprocally). Similarities with recognized species were less than 90.2 %. The phylogenetic and phenotypic characteristics indicated that strains BH1(T) and HN5 represent a novel species in a new genus, for which the name Geomicrobium halophilum gen. nov., sp. nov. is proposed. The type strain is BH1(T) (=JCM 15647(T)=DSM 21769(T)).

Halostagnicola kamekurae sp. nov., an extremely halophilic archaeon from solar salt

A novel extremely halophilic archaeon, strain 194-10(T), was isolated from a solar salt sample imported into Japan from the Philippines. Strain 194-10(T) was pleomorphic, neutrophilic and mesophilic and required at least 10 % (w/v) NaCl but no MgSO(4) . 7H(2)O for growth; it exhibited optimal growth at 15 % (w/v) NaCl and 60 mM MgSO(4) . 7H(2)O. Strain 194-10(T) grew at 20-45°C (optimum, 30°C) and pH 6.0-9.0 (optimum, pH 6.5-7.0). The G+C content of its DNA was 59.8 mol%. 16S rRNA gene sequence analysis revealed closest proximity to Halostagnicola larsenii XH-48(T) (98.5 % similarity), the sole representative of the genus Halostagnicola. Polar lipid analysis revealed that strain 194-10(T) contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester (the latter derived from both C(20)C(20) and C(20)C(25) archaeol) and several unidentified glycolipids. The results of DNA-DNA hybridization (20.7 % relatedness between Hst. larsenii JCM 13463(T) and strain 194-10(T)) and physiological and biochemical characteristics allowed differentiation of strain 194-10(T) from Hst. larsenii XH-48(T). Therefore, strain 194-10(T) represents a novel species of the genus Halostagnicola, for which the name Halostagnicola kamekurae sp. nov. is proposed, with the type strain 194-10(T) (=DSM 22427(T) =JCM 16110(T) =CECT 7536(T)).

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.