Toru Mizuki

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.

Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

We immobilize alpha-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzymes activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems (mu-TAS).

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)).

The Potential Use of Signature Bases from 16S rRNA Gene Sequences To Aid the Assignment of Microbial Strains to Genera of Halobacteria

In the second edition of Bergey’s Manual of Systematic Bacteriology, Volume I, published in May 2001, the extremely halophilic, aerobic Archaea are classified within the domain Archaea, phylum AII Euryarchaeota, class III Halobacteria, order I Halobacteriales, family Halobacteriaceae. Currently, Halobacteriaceae comprises 17 valid genera (as of March 2003). The family is extraordinary in that it contains so many genera. In other families in the domain Archaea the highest number is the eight genera of the family Desulfurococcaceae. When the many unclassified halobacterial isolates are taken into consideration, it is clear that halobacterial diversity extends even further.

Ureases of extreme halophiles of the genus Haloarcula with a unique structure of gene cluster.

We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method. Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts. Only 4 strains of the genus Haloarcula, Har. aidinensis, Har. hispanica, Har. japonica, and Har. marismortui were finally shown as the urease producers. A partially purified urease from Har. hispanica was a typical halophilic enzyme in that it showed maximum activity at 18–23% NaCl and lost the activity irreversibly in the absence of NaCl. Partial genes (1596 bp) of the urease encoding from upstream of the β subunit down to the N-terminal 139 amino acids of the α subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains. Strains of other genera, which were urease-negative, did not yield PCR products. The deduced amino acid sequences of the β subunit and partial α subunit were similar to each other (92–100% similarities) and to those from other organisms. Analysis of the draft genome sequence of Har. marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was β-α-γ-UreG-UreD-UreE-UreF. This order is quite unique, since in other microorganisms the order is γ-β-α-UreE-UreF-UreG-UreD in most cases. No open reading frames were detected in the PCR-amplified upstream of the β subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.

Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

Organic Solvent Tolerance of Halophilic Archaea

Organic solvent tolerance was tested in type strains of type species of the sixteen genera of Halobacteriaceae, the halophilic archaea. Most of the strains were observed to grow in the presence of hexylether (log P ow=5.1), but none grew in the presence of n-octane (log P ow=4.9) except Halogeometricum borinquense JCM 10706T and Halorubrum saccharovorum JCM 8865T. On the other hand, two strains, Haloarcula spp. OHF-1 and 2 isolated from a French solar salt were found to show stronger tolerance even to isooctane (log P ow=4.8). Growth of some strains was retarded by the presence of n-decane but reached to the same cell densities at late stationary phase. Final cell densities of some strains were greatly repressed by the presence of the solvent.

Activity of Lipase and Chitinase Immobilized on Superparamagnetic Particles in a Rotational Magnetic Field

We immobilize hydrolases such as lipase and chitinase on superparamagnetic particles, which are subjected to a rotational magnetic field, and measure the activities of the enzymes. We find that the activities of lipase and chitinase increase in the rotational magnetic field compared to those in the absence of a magnetic field and reach maximum at certain frequencies. The present methodology may well be utilized for the design and development of efficient micro reactors and micro total analysis systems (μ-TASs).

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)).