1. Introduction

Microorganisms play an enormous role in the underlying processes of material cycle on earth especially in the transfer of energy through natural ecosystems and in the cycling of bio-chemically important elements. In natural ecosystem, microorganisms are found in a variety of forms of consortium with a wide range of associations from parasitism to symbiosis. However, there have been only a small number of studies that succeeded in revealing some aspects of associations between microorganisms.

In soil ecosystems, the interactions of bacteria and plants have been well studied especially on those in the rhizosphere, the area in the soil where microorganisms are influenced by plant roots. The concept of the "phycosphere" is a pelagic analogy to the rhizosphere. The phycosphere is the zone surrounding an algal cell within which microorganisms are influenced by algal products. Not a small number of microalgae are living in soil, and the phycosphere in soil may represent a good microhabitat for soil bacteria. Studies on the associations between green microalgae (the phylum Chlorophyta, kingdom Plantae) and bacteria are considered to be of great value, because the form is not only a microorganism-microorganism association but, at the same time, may also be a primitive plant-microorganisms association. However, not much information on these associations has been accumulated to date. Furthermore, there is limited information available on which bacteria are living in the green algal phycosphere.

Under the above circumstance, bacteria were isolated from green microalgal-bacterial consortia and identified in this study. Then the effects of the isolates on the growth of green microalgae were examined. In addition, genes involved in vitamin B12 (cobalamin) synthesis of a selected isolate were identified, and the transcriptional activity of one of the genes of the isolate co-cultivated with a cobalamin-requiring green microalga was examined, since cobalamin has been reported to be a key of some marine algal-bacterial associations.

2. Composition of cultivable bacteria in green algal-bacterial consortia

Chlorella spp. (Chlorophyta) are one group of the representative soil algae. Three non-axenic strains of Chlorella spp., C2, C6, and CSB-227, were used as algal-bacterial consortia in this study. Chlorella sp. C2 and C6 were originally isolated from soil and had been maintained without purification. Chlorella vulgaris CSB-227 was established by inoculating soil into an axenic strain C. vulgaris NIES-227 (NIES, National Institute fro Environmental Studies). Twenty-three bacterial strains were isolated from these three consortia, and identified as members of the genera Caulobacter, Brevundimonas, Polaromonas Variovorax, Ensifer, Shinella, Aminobacter, Microbacterium, Bacillus, and Emticicia, based on the neighbor-joining trees constructed with almost full length of 16S rRNA gene sequences. Several taxonomic properties placed the strains belonging to the genera Caulobacter and Variovorax as novel species, for which scientific names, Caulobacter aquatilis and Variovorax aquatilis were proposed.

3. Effect of co-cultivation with bacteria on algal growth

One strain each of nine bacterial genera was selected and individually co-cultivated with each of the two strains of microalgae, C. vulgaris NIES-227 and Monomastix minuta NIES-255 (Chlorophyta), to examine the effects of the bacteria on the growth of the algae. Prior to this experiment, M. minuta NIES-255 was screened in this study as a vitamin B12 -requiring green alga from axenic green algal collections maintained in the Microbial Culture Collection of NIES. Brevundomonas C2e1 statistically significantly increased the culture lifetime of C. vulgaris NIES 227, and decreased the die-off rate of M. minuta NIES-255. Bacillus strain CSBb decreased the die-off rate of M. minuta NIES-255 but did not affect on the growth of C. vulgaris NIES-227. On the other hand, the culture lifetime of M. minuta NIES-255 was statistically significantly decreased when co-cultivated with the Caulobacter C2a1, Aminobacter C6b, Ensifer CSBa, and Variovorax C6d. The latter two strains also statistically significantly decreased the lifetime of C. vulgaris NIES-227. Other strains showed no effect on the growth of the two algae.

Among the above results, the effect of Brevundimonas C2e1 on the growth of C. vulgaris NIES-227 was prominent. Therefore, another strain of Brevundimonas, DC2a-G2, isolated by Ueda (2010, master's thesis) from another algal-bacterial consortium was added, and the effect of Brevundimonas C2e1 and DC2a-G2 on the growth of five strains of Chlorella, Chlorella sorokiniana NIES-2167, C. sorokiniana NIES-2168, C. vulgaris NIES-2170, Chlorella sp. NIES-217, and Chlorella elkhatiense NIES-2250, was examined. As the results, Brevundimonas C2e1 statistically significantly increased the culture lifetime of the C. vulgaris NIES-2170, decreased the culture lifetime of C. sorokiniana NIES-2168, inhibited the growth of C. sorokiniana NIES-2167, and showed no effect on the growth of Chlorella sp. NIES-2171 and C. elkhatiense NIES-2250. Another strain, Brevundimonas DC2a-G2, statistically significantly inhibited the growth of C. sorokiniana NIES-2167 and showed no effect on the growth of the other four algae.

4. Identification and transcriptional activity of cobalamin biosynthesis genes of Ensifer CSBa

In an inorganic medium without cobalamin, each of the above nine strains of bacteria and cobalamin-requiring M. minuta NIES-255 were co-cultivated, for the purpose of screening bacteria that synthesis and release cobalamin to the alga. Three strains, Ensifer CSBa, Bacillus CSBb, and Aminobacter C6b, enabled M. minuta NIES-255 to grow, indicating that these three bacteria supplied cobalamin to the alga, although the growth rate of the alga co-cultivated with Aminobacter C6b was very small. It was surprising that out of nine bacterial strains isolated from cobalamin-independent algae, three could synthesize cobalamin. Among the three, Ensifer CSBa was subjected to a further study, because the genus Ensifer includes populations that have a symbiotic association with higher plants, and Ensifer bacteria have been repeatedly detected from green algal-bacterial consortia in previous studies.

Draft genome analysis was done for Ensifer CSBa, and the genes involved in cobalamin synthesis (cob genes) were identified. It was revealed that Ensifer CSBa harbored 22 putative cob genes in total, and each of them were homologous to those found in a cobalamin- synthesizing bacterium, Pseudomonas denitrificans SC510 Rif and/or SBL27 Rif. Based on the composition of the cob genes, it was suggested that Ensifer CSBa synthesizes cobalamin by the oxygen-dependent (aerobic) pathway as P. denitrificans SC510 Rif and/or SBL27 Rif does. There are two pathways in the biosynthesis of cobalamin; one is aerobic and another is anaerobic. The anaerobic pathway has been well studied in Salmonella typhimurium, Bacillus megaterium and Propionibacterium freudenreichii, but the whole gene set involved in the aerobic pathway has so far been revealed only in P. denitrificans SC510 Rif and/or SBL27 Rif.

Based on the DNA sequence of one of the cob genes, cobT, specific PCR primers for the gene were designed. Real-time quantitative reverse transcription PCR analysis was performed targeting cobT of Ensifer CSBa co-cultivated with M. minuta NIES-255 in an inorganic medium with and without cobalamin. It was revealed that the presence/absence of cobalamin in the medium did not affect the transcriptional activity of cobT of Ensifer CSBa co-cultivated with a cobalamin-requiring alga, M. minuta NIES-255.

5. Conclusion

In this study, the cultivable bacterial composition of green microalgal-bacterial consortia were identified, and two novel bacterial species names were taxonomically proposed. The bacterial isolates showed culture lifetime-increasing, culture die-off rate-decreasing, or growth inhibiting effects on microalgae, depending on the combination of a bacterial and an algal strain. Totally 22 putative genes involved in the aerobic pathway of cobalamin synthesis (cob genes) were identified with an isolate, Ensifer CSBa. The presence/absence of cobalamin in the medium did not affect the transcriptional activity of cobT of Ensifer CSBa co-cultivated with a cobalamin-requiring alga. It seems that keys to the green microalgal-bacterial associations are still more complex. Further studies are necessary to reveal the mechanisms with compounds produced by algae/bacteria other than cobalamin as the targets.

環境中の微生物は、様々な共同体を築いて生活しており、その機能や生態を解明するためには、微生物の相互作用を知ることが重要である。しかし、これまでの多くの取り組みにもかかわらず、微生物相互作用に関する知見の蓄積は十分でない。本研究は、以下の3点を目的としている。すなわち、(1) 微細藻類と細菌の共培養系から細菌を分離、同定し、必要に応じてその分類学的性状を明らかにすること、(2) 得られた細菌培養株と微細緑藻の純粋培養株を様々な組み合わせで共培養し、共存する細菌が藻類の生育に与える影響を明らかにすること、(3) コバラミン(ビタミンB12)生産細菌とコバラミン要求性藻類とを共培養したときに、培地中のコバラミンの有無がこの細菌のコバラミン生産に与える影響を明らかにすること。

第1章では、微生物相互作用に関するこれまでの知見を概説したのち、研究材料として微細藻類を用いる利点を挙げつつ、本研究の目的を述べている。

第2章と第3章では、明暗条件下、無機塩類培地中で、代表的な土壌藻類の一つであるChlorella属緑藻と共存して培養されている土壌由来の細菌を分離し、種または属レベルの同定と分類を行っている。4門10属にまたがる計23株の細菌を得て、関連研究との比較考察から、特定の分類群の細菌が藻類と共同体を形成しやすい可能性を指摘した。また、分離株に含まれていた4つの新種候補の形態学的性状、生化学的性状、生理学的性状、および分子遺伝学的性状を解析し、そのうちCaulobacter属およびVariovorax属の細菌について、新種記載するために必要なデータを得てCaulobacter aquatilisおよびVariovorax aquatilisと命名した。

第4章では、共培養される細菌が緑藻の生育に与える影響を試験している。緑藻としては、コバラミン非要求性緑藻Chlorella vulgaris NIES-227株およびコバラミン要求性緑藻Monomastix minuta NIES-255株を、また細菌としては上述の分離細菌から1属につき1株ずつ選んで用いた。明暗条件下、C培地中(コバラミンを含む)での共培養試験の結果、C. vulgaris NIES-227の生育速度は共培養される細菌の影響を受けなかったが、その生存期間は一部の細菌の影響を受けた。M. minuta NIES-255は共培養される細菌によって死滅速度に差を生じた。また、コバラミンを除いたC培地中で細菌と緑藻M. minuta NIES-255を共培養することにより、3株の細菌がこの緑藻の生育を可能とすることを見いだし、これらの細菌がコバラミンを生産し緑藻に供給していると推測した。これらの細菌の1つEnsifer属細菌CSBa株は、コバラミンを十分に含む通常のC培地中ではC. vulgaris NIES-227の生育速度を増大しないが、コバラミンを除いたC培地中ではその生育速度を統計的に有意に増大した。よって、一部の藻類で知られているようにC. vulgaris NIES-227はコバラミン依存性および非依存性の両方の代謝経路を有しており、コバラミン非要求性であっても細菌からのコバラミン供給を受けて生育が促進されると推論した。

第5章では、まずEnsifer属細菌CSBaが保有すると考えられたコバラミン合成系遺伝子の同定を行っている。この細菌のゲノムのドラフト解析を行い、解読された塩基配列から遺伝子解析を行って、この細菌が好気的コバラミン合成に関わる22の遺伝子(cob遺伝子群)を保有していることを明らかにした。つづいて、C培地およびコバラミンを除いたC培地で、明暗条件下、このEnsifer属細菌CSBaとM. minuta NIES-255を共培養したときの、細菌のcob遺伝子群の一つcobTの転写活性をリアルタイムPCRにより解析し比較している。共培養において、Ensifer属細菌CSBaはM. minuta NIES-255の分泌物を生育基質として利用し、M. minuta NIES-255は特にコバラミンを除いたC培地においてはEnsifer属細菌CSBaが分泌するコバラミンを利用していると考えられる。解析の結果、共培養されている状態でも、Ensifer属細菌CSBaのcobTは、培地中のコバラミンの有無にかかわらず同程度の転写活性をもつことが示された。

以上、本研究は、微生物の分類、生態、および応用にとって重要で新規性の高い知見を提供しており、また今後の微生物相互作用研究の足がかりとなる有用な材料を提供しており、学術的、応用的に貢献するところが少なくない。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。