Luminous bacteria are a group of microbes emitting bluish-green light as a result of luciferase reaction. Except for a few group, they are unique to marine environments and ubiquitously present in the ocean including deep sea. They have drawn attentions of microbiologists since the first description in 19(th) century. Why do most of them live in the sea? For what do they emit light? When and from which microbes, is the bioluminescence originated? These basic questions are still left unanswered.

Luminous bacteria are isolated from water column, particulate matter and the light organ of some fish. They are also easily found in the gut of marine animals. Such association with animals suggests that the interaction with animals may at least partly explain why they emit light. This further raises a question whether animals recognize only the presence of light or they recognize its color. There is, however, no microbial ecological work related to this intriguing question. In this work, I made two basic hypotheses; first, there are variations in the bacterial light emission spectra, and second, such spectra have ecological implications. The first purpose of this work was to verify these hypotheses. My second purpose was to clarify how bioluminescent spectra were related to the phylogeny of luminous bacteria. Finally, I intended to overview the origin and gene transfer among marine luminous bacteria. Phylogenetic, physiological, and biochemical analyses were done for the 777 luminous bacteria.

Chapter 1.

The distribution pattern of luminous bacteria of different phylogenetic group and their light emission spectra were investigated. Altogether 777 isolates were obtained from coastal and open ocean from surface to deep sea environments, and identified by using 16S rRNA sequences analysis. The data showed that Vibrio species are dominant at seashore and surface water of coastal area. Photobacterium species were mainly found in both coastal area and open ocean. Aliivibrio species were isolated from seashore in only winter season. These distribution patterns of each genus seem to be mainly controlled by sea temperature. The light emission spectra consist of 5 distinguishable types with different maximum wavelength and each type was unique to certain phylogenetic group (Vibrio, λ(max)~473 and 482 nm; Photobacterium, λ(max)~479 and 488 nm; Aliivibrio, λ(max)~485 nm). Among them, three types (λ(max)~482, 485 and 488 nm) showed Gaussian spectra, whereas the other two (λ(max)~473 and 479 nm) showed asymmetric one and have shorter maximum wavelength. In addition, the FWHM (full width at half maximum) of the former was more than 80 nm whereas those of the latter two were less than 75 nm. Considering on the related literature information, I concluded that the latter two were the blue-shifted light emission type. The distribution of the strains with blue-shifted light emission type did not seem to depend on the seawater temperature but rather on the light condition of the environment. These results verify my first hypothesis that there are variations of light emission spectra. Furthermore, It was revealed that those differences were reflected to the phylogenetic positions of each type.

Chapter 2.

At present, 19 species among 4 genera (Vibrio, Photobacterium, Aliivibrio, and Shewanella) have been known as marine luminous bacteria. During the course of investigation described in Chapter 1, the inconsistency and confusion of systematics of luminous bacteria became clear. Without robust and reliable identification scheme, it is difficult to deduce the phylogenetic position of my isolates and further discuss on the evolutionary processes. In this chapter, I tried to apply the latest molecular phylogenetic approach and identify my 5 isolates that seem to be ecologically or phylogenetically important. As a result, 5 new species, i.e., V. azureus, V. sagamiensis, P. aquimaris, A. marinus, and A. lajollensis were newly described and proposed. It is noteworthy that about 60% isolates at Sagami Bay belonging to new species.

Chapter 3.

The variations in the luminescent spectra may be ascribed to the structural differences of the molecules involved in the light emission. As they are encoded by lux genes, it is assumed that the variations may be correlated with the phylogeny of these genes. Therefore, luciferase alpha subunit gene (luxA) sequence and light emission spectra were analyzed concomitantly. Consequently, it was revealed that each species, except for P. leiognathi, formed individual clade and the strains sharing the same clade have same light emission spectra. Furthermore, the presence of the genes encoding the accessory fluorescent protein of P. phosphoreum was confirmed for all strains with blue-shifted light emission in the genus Photobacterium by PCR. Therefore, I concluded that light emission spectra were primarily determined by the lineage of phylogeny of luxA. The blue-shifted light emission among strains in the genus Photobacterium is explained by the presence of an accessory fluorescent protein. For other genera, however, it remains to be elucidated.

Chapter 4.

Chapter 1 and 3 clarified the variation of light emission spectra among marine luminous bacteria. So far, this is the first observation of blue-shifted light emission within genus Vibrio. Although the blue-shifted light emissions in Photobacterium seem to be explained by an accessory fluorescent protein, there has been no report on the presence of similar functional protein in the genus Vibrio. Therefore, I tried to detect an accessory fluorescent protein as substance responsible for blue-shifted light emission in Vibrio azureus, which is newly proposed species in Chapter 2. As a result, a blue fluorescent protein, which had a fluorescence spectrum similar to that of the in vivo light emission spectrum of the strain, was purified by the biochemical procedures using liquid chromatography analyses. Consequently, the data suggested that all blue-shifted light emissions of luminous bacteria are due to accessory fluorescent proteins.

Chapter 5.

The previous chapters clarified the correlation between luminescent spectra and their phylogenetic positions. On the other hand, luminous bacteria generally do not make single clade, but rather scattered in the phylogenetic tree. Taken together, it seems reasonable to assume that lux genes were acquired or lost during the process of evolution in Vibrionaceae. Also, it is expected that if any gene transfer events are involved, there may be a certain point that the evolution of species and that of lux genes do not agree with each other. In order to clarify the evolutionary process of bioluminescence among marine bacteria, I compared the differences between the phylogenetic trees constructed by both luxA and multilocus house-keeping genes. If any strain acquired luciferase gene from another strain by horizontal transfer, incongruence between the phylogenies of luxA and housekeeping genes may be seen among these strains. Results indicated that the scattered presence of lux genes in the phylogenetic tree may be explained by the gene deletion during the evolutionary process. However, there were also some cases which are explained reasonably if a gene transfer event is assumed. In addition, I examined the presence and phylogenetic analyses of genes encoding the accessory fluorescent protein. It is indicated that the accessory fluorescent protein of V. azureus was acquired from genus Photobacterium by horizontal transfer. These results imply that the diversification of luminous bacteria and their emission spectra are well explained by the evolutionary steps of both species and the related genes. What kind of actual event caused the gene loss and/or gene transfer remain to be elucidated.

This doctoral thesis is the first intensive examination of light emission spectra of marine luminous bacteria. I had made two hypotheses, i.e., first, there are variations in the bacterial light emission spectra, and second, such spectra have ecological implications. As was described in Chapter 1, there are 5 distinct types of the spectra, so the first one was confirmed. The second hypothesis was partly supported by the two findings. First, the strains with blue-shifted spectra showed characteristic distribution in the sea. Second, the luminous bacteria with blue-shifted spectra are quite widely distributed in marine environments and this shift seems to be attained by the accessory fluorescence protein. It is difficult to assume that those bacteria synthesize such compounds without any advantage. Then what are the ecological advantages of the modulation? There are two possibilities that are not exclusive each other. First, the bacteria of which luminescence penetrate most in the sea have selective advantages. Second, fish recognize the slight difference of the luminescence and preferentially ingest some types. My work at least supports the former possibility, and the latter needs fish physiologist to confirm it. In any case, these possibilities have never pointed out before because there has been no work on the luminescent spectra before. This work shows the ecological implication of not only luminescence spectra but also bacterial bioluminescence itself.

発光細菌は青白色の発光をおこなう細菌群で、その大部分が海に生息しており、現在までに4属(Vibrio,Photobacterium,Aliivibrio,shewanella)19種が知られる。これらの細菌は海水中に自由遊泳型として広く分布するとともに、一部は魚類などに共生している。しかしこれらの共生発光の例を除けば発光細菌がなぜ発光するのかは不明である。また、その解明のための糸口もはっきりしない。

発光細菌の発光はいわゆるルシフェリンールシフェラーゼ系の生化学的反応によるもので、そこに還元型のリボフラビン(riboflavin-5'-phosphate:FMNH2)、長鎖のアルデヒド、さらに分子状の酸素が関与している。発光細菌の世界色は全て480nm付近の青白い色に見えるが、その反応中心に関わる分子群によってその波長が決定されるとすれば、それらの分子のアミノ酸配列は多少なりとも発光波長に影響を及ぼすかもしれない。一方、大きな波長の変化を引き起こす場合には、それを変化させる別の物質の存在が予想される。しかし、このような考察のもとに、発光細菌の波長を詳細に調べる研究はこれまで全く行われてこなかった。これは見た目には発光波長が共通していること、また多少の違いが何らかの生態的意義を持つとは考えられなかったことによる。

吉澤君は第一章では、日本沿岸の近海および外洋域、ならびに地中海から発光細菌777株を分離し、その発光スペクトラムを詳細に測定した。その結果少なくとも5種類の異なる波長スペクトラムがあることを初めて明らかにした。そのうち2タイプはそれぞれ別のタイプの発光波長が短波長側にずれたようなパターン(Blue-shift type)を示していた。またその発光のピークと発光細菌の生息場所に関連があることも見出した。例えば相模湾では冬季と夏季では発光細菌の発光ピークがシフトする。また沿岸ではより長波長、外洋や深海ではより短波長側にシフトする傾向を示した。同時に、これらの細菌の同定を行い、それらの発光波長と系統群との間に関係があること、発光細菌の従来の同定法に問題があることを示した。第二章では、分離株の中から4株、保存株の中から1株を対象に、詳細な分子系統学的手法、生化学的手法を用いてそれらの同定を行い、それらが新種であることを解明し、それをIntemational Journal of Systematic and Evolutionary Microbiologyに投稿した。既に2株は受理されており、これは吉澤君が 命名した新種が公式に認められたことを示す。残りの3株についても投稿済みであり、新種となることがほぼ確実である。第三章では、こうして新たに記載された新種と50株の海洋性発光細菌を選びその系統について詳細な解析を行った。ルシフェラーゼ遺伝子(luxA)を用いて最新の分子系統技術で整理することにより、これまでの発光細菌の系統的研究には手法上の誤りが多く、混乱があることを確認した。さらに、Blue-shiftを示すタイプ、それ以外のタイプが系統上でクルードを作っていることがわかった。第四章では、発光波長のBlue-shiftは、特定のタンパクが関与しているとの仮定のもとに、そのタンパクの精製と発光特性の解析を行った。その結果、Vibrio azureus strain LC2-005株からBlue-shiftに関わるタンパクを精製し、その蛍光特性がこの株のin vivoの発光波長にぴったりと重なること、発光系を抽出してin vitroで発光波長を測定すると、長波長側にずれていることを明らかにした。この結果から、この蛍光タンパクは発光波長を短波長側にシフトさせる機能を持っていることを明らかにした。第五章では、発光細菌の系統解析および近縁の非発光性の細菌群との系統的関係を総合的に解析し、発光遺伝子が進化の過程でどこで生じ、その後どのような系統群に伝播して行ったのか、あるいは喪失したのか、さらに、波長を変化させる蛍光タンパクがやはり進化の過程でどのような変遷を遂げたのかについて考察した。また、最終的にこれらの結果を総合し、発光波長が視覚を持つ海洋生物との相互作用を通じて進化してきたこと、発光色というのが個々の水域の中で最も透過性のよい光として選択されてきたことを考察した。

これらの結果はほぼその全てが吉澤君のアイデアに基づき、本人の作業によって行われてきた。2009年2月2目、東京大学海洋研究所にて行われた審査においても、入念かつ詳細な発表と適切な質疑への対応が見られた。従って博士(環境学)の学位を授与できると認める。