Several classes of microorganisms have been recognized as extremophiles inhabiting in an extreme environments of heat or cold, pH, salinity, pressure, and even radiation. These extremophiles, having many interesting biological secrets, provide a novel source of discoveries in applied and basic sciences. We studied a new frontier in extremophiles, i.e., microorganisms tolerating toxic levels of boron.

　Boron (B), a non-metal micronutrient, has been known to be essential for plants since long. Some animals and unicellular eukaryotes also require B but the level of requirement differs among various organisms. On the other hand, B is toxic to living cells when present above a certain threshold. Environmental B toxicity occurs in many parts of the world and B contaminated soils are difficult to ameliorate. Due to its toxic effects for microorganisms, B has been used as a food preservative and also as an insecticide against cockroaches. The problem of both B deficiency and toxicity becomes two-folds because of its limit being narrow for deficiency and toxicity.

　Substantial variation in tolerance to high B has been reported among plant species and it is possible that microorganisms also differ greatly in B tolerance. Based on this hypothesis, we isolated and identified several B-tolerant bacterial strains from normal soil of greenhouse area (The University of Tokyo) and a naturally high B-containing soil of Hisarcik area (Kutahya Province, Turkey). Organisms that grow on soils naturally high in a particular element such as B, are of great interest biologically for their ability to function under such extreme conditions and also as a source of tolerance related gene(s). This thesis reports isolation, identification and characterization of highly B-tolerant bacteria, especially this represents the first report, to our knowledge, of the novel species of bacteria that requires B for its growth and that can tolerate more than 450 mM B. Additionally, a B toxicity tolerance mechanism has also been demonstrated.

Boron-tolerant species

　The strains isolated from normal greenhouse soil were found to tolerate only up to 150 mM B; however, those isolated from the soil of Turkey that is naturally high in B-containing minerals could grow at 450 mM B, an extremely high concentration of B. Phylogenetic analyses based on comparative 16S rRNA gene sequence data demonstrated that these B-tolerant strains belong to six genera; Rhodococcus, Arthrobacter, Chimaereicella, Gracilibacillus, Lysinibacillus and Bacillus. The Bacillus species were found to be the highest B-tolerant and could grow in TSB medium containing more than 450 mM B (Fig. 1); followed by Gracilibacillus, Chimaereicella, Lysinibacillus, Rhodococcus and Arthrobacter tolerating B concentration of 450, 300, 150, 100 and 80 mM B, respectively. It is also observed that the neighboring species in their respective clusters could not tolerate toxic levels of B as much as these novel taxa.

　Based upon phylogenetic analyses, DNA-DNA homology, phenotypic and chemotaxonomic data, the isolated strains belong to the genera; Bacillus (three strains), Gracilibacillus (one strains), and Chimaereicella (one strains), have been characterized as B. boroniphilus sp. nov., G boraciitolerans sp. nov., and C. boritolerans sp. nov. (Ahmed et al., 2006a, b, & c), respectively. These species were isolated from soil of Hisarcik area (Turkey) that was reported to be naturally high in B-minerals.

　So far a peptidoglycan consisting of Lys-Asp has not been reported for any other endospore-forming species of the Bacillus group 2 except the novel B-tolerant strains of L. boronitolerans sp. nov. (three strains) along with the neighboring clade consisting of B. fusiformis and B. sphaericus. Therefore, on the basis of Lys-Asp in the cell wall peptidoglycans and other chemotaxonomic data, this B-tolerant species has been assigned to a new genus, Lysinibacillus boronitolerans gen. nov. sp. nov (Ahmed et al., 2006d). This chemotaxonomic data and phylogenetic analyses also demonstrated that B. fusiformis and B. sphaericus should be transferred to the genus Lysinibacillus as L. fusiformis comb. nov. and L. sphaericus comb. nov., respectively.

　The other eight B-tolerant strains belonging to the genera; Rhodococcus (six strains) and Arthrobacter (two strains) have the highest 16S rRNA gene sequence similarity (> 99%) with the closed relatives in their respective clusters and therefore, have not been included in the characterization studies.

Boron-requiring species

　Although essentiality of B has been established for plants and for some animals, however, B has not yet been reported to be essential for Bacteria, except for cyanobacteria. During the course of these studies, a bacterial species, B. boroniphilus sp. nov. has been identified with the unique feature that it requires boron for its growth (Fig. 1). The fact that the novel strain requires B as an essential nutrient while others do not is not unusual because B requirement differs from species to species as was clear from an evolutionary study of the acquisition of an essential role for B in the metabolism of plants.

　Our data also showed a decrease in growth even at 20 and 50 mM B levels after several hours but at high B levels, the growth remains constant at stationary phase with a slight increase, indicating that the strains are borophilus one.

Mechanism of boron tolerance

　Studies of tolerance to B toxicity in the B-tolerant strains demonstrated that these B-tolerant strains maintained significantly lower B concentration in the cells in comparison to sensitive strain i.e. Bacillus subtilis as a control (Fig. 2). Critical analysis of data showed an apparent negative correlation between the protoplasmic boron concentration and the degree of tolerance to a high external boron concentration (Fig. 2). Time course B uptake studies of B-tolerant strain showed rapid uptake of B but was able to maintain steady-state cellular soluble B concentration four-folds less than the sensitive strain (Fig. 3). Further analysis showed a decrease in cellular soluble B concentration, suggesting that efflux and/or exclusion of B is a mechanism of tolerance against high external concentration of B in prokaryotes.

General conclusions

　Bacillus boroniphilus could not grow without B (Fig. 1), suggesting that B is essential for the growth, for some unknown functions. It is hypothesized that B may be needed as a structural component of cell wall as is the case in plants where it forms esters with a cis-diol moiety in rhamnogalacturonan-II (RG-II) that is required for stabilization and integrity; it is also clear from our data that when B in the medium is used up during the initial few hours of growth, the bacterium could not survive, supporting our conclusion. Although B salts have often been included in microbial growth media, but B functions are not yet clear in prokaryotes. Identification of B-requiring strain will provide enormous information to understand the biochemistry of B in living cells.

　Finally, the findings of B tolerance and essentiality for the novel strain provide a genetic resource to identify the gene(s) responsible for the mechanism of B tolerance in bacteria because of its small genome size. Such gene(s) may be useful for cloning in other organisms especially crop species that are grown on high B soils.

Figure 1. Growth curves of Bacillus boroniphilus sp. nov. grown at different levels of B supply in TSB medium.

Figure 2. Uptake of boron in cells at two levels (10 and 50 mM) of boron supply for one hour. Data are means with error bars (± SD) for four independent replications. d.w., dry weight.

Figure 3. Time-course of B influx and efflux in the cells at 10 mM of boron supply. Data are means with error bars (± SD) for four independent replications. d.w., dry weight.

　ホウ素は植物の必須元素であると共に高濃度に存在すると毒性を示す。また、ホウ素は溶脱しやすく、世界の多雨地域には欠乏症が、小雨地域には過剰症が広がり、農業生産の低下要因となっている。このような問題の解決には、植物におけるホウ素の輸送機構を明らかにするとともに、ホウ素過剰や欠乏に耐性を示す植物の作出が望まれている。しかしその一方で、ホウ素の植物における輸送の制御は、ホウ酸の高い膜透過性が問題となる。ホウ素と微生物の関わりについてはあまり研究されていないが、ホウ酸に耐性な微生物などを単離することができれば、植物の研究では得られない生物とホウ酸の関わりを明らかにすることができると期待される。本論文は、高濃度のホウ酸に耐性な細菌を土壌より検索し、4種の新種の細菌を同定するとともに、ホウ酸要求性の細菌を単離したものである。シロイヌナズナにおける輸送機構を明らかにすると共に、ホウ素過剰や欠乏に耐性を示すシロイヌナズナの策出に成功したものである。

　本論文は、全部で8個の章からなり、第一章の要約、第二章の序論に続いて、第3章から第6章までのそれぞれが、4種の新種の細菌の単離と同定を述べており、第7章はホウ素過剰耐性の機構についての解析、最後の題8章において総合的な結論を述べている。

　第3章では土壌より単離したホウ素過剰耐性菌の一つについて、16SリボゾームRNA配列の解析と各種生理実験、DNA-DNAハイブリダイゼーションの結果等を元に、Bacillus. boroniphilusと名付けた新種の細菌であることを示した。この細菌は、飽和濃度に近い450mMのホウ酸を含む培地でも良好な生育を示した。また、この細菌はホウ素欠除培地では生育することができず、細菌では初めて同定されたホウ素要求性の細菌であった。

　第4章では、土壌より単離したホウ素過剰耐性菌の一つについて、16SリボゾームRNA配列の解析と各種生理実験、DNA-DNAハイブリダイゼーションの結果等を元に、Gracilibacillus boraciitoleransと名付けた新種の細菌であることを示した。この細菌は、飽和濃度に近い450mMのホウ酸を含む培地でも良好な生育を示した。

　第5章では、土壌より単離したホウ素過剰耐性菌の一つについて、16SリボゾームRNA配列の解析と各種生理実験、DNA-DNAハイブリダイゼーションの結果等を元に、Chimaereicella boritoleransと名付けた新種の細菌であることを示した。この細菌は、300mMのホウ酸を含む培地でも良好な生育を示した。

　第6章では、土壌より単離したホウ素過剰耐性菌の一つについて、16SリボゾームRNA配列の解析と各種生理実験、DNA-DNAハイブリダイゼーションの結果等を元に、Lysinibacillus boronitoleransと名付けた新種の細菌であることを示した。この細菌は、150mMのホウ酸を含む培地でも良好な生育を示した。

　第7章では、単離された各種菌株のホウ素含量、ホウ素濃度の経時的変化を通じて、ホウ酸耐性の程度と細胞内のホウ素濃度には逆相関があること、ホウ酸耐性株には、ホウ素の排出能力があることを示唆した。

　以上、本論文は、これまで全く未知であった、高濃度のホウ酸に耐性の微生物を単離、同定するとともに、ホウ素要求性細菌を同定したものである。また、高濃度のホウ素耐性がホウ素の細胞からの排出によるものであることを示唆した。これらの成果は生物におけるホウ素輸送の分子機構の多様性を示すものであり、極めて高い貢献をしている。

　よって、審査委員一同は、本論文を博士(農学)の学位論文として高く価値あるものと認めた。