劍尖槍鎖管發光器官內共生發光細菌之鑑定及特性分析研究

Abstract

前人研究 (Fukasawa & Dunlap, 1986) 指出共生於劍尖槍鎖管 [Uroteuthis (Photololigo) edulis] 發光器官內的發光細菌為Photobacterium leiognathi，然而本研究有新的發現。本研究從臺灣北部海域釣取劍尖槍鎖管，並就其發光器官內的共生細菌進行基因、生理和生化特性的探討。「16S rDNA」和「luxF」兩種基因的序列比對和特徵分析的結果顯示，25株分離自五尾劍尖槍鎖管的發光細菌中，有24株為Photobacterium mandapamensis，另外1株為P. leiognathi。因此，劍尖槍鎖管發光器官內的共生細菌應大多數為P. mandapamensis，僅少數發光器官內有P. mandapamensis和P. leiognathi同時共生 (cosymbiosis) 的情形。本研究分離的24株P. mandapamensis中，有7株的發光基因操縱組 (lux operon) 呈現部分雙套 (merodiploid) 之情形，這些菌株也對生活環境的「溫度」和「鹽度」因子有差異性的表現，而發光強度上更出現有1～60倍的差異。根據這些基因變異及生理、生化特性的差別，本研究推測P. mandapamensis與劍尖槍鎖管建立共生關係的初始，是由多個已具變異特性的菌株由海水中進入發光器官內拓殖，而非僅由單一菌株進入拓殖的情形。此外，本研究也發現除了基因型的差異之外，某些生理和生化特性的差異也可被應用於區分親緣關係接近的P. mandapamensis和P. leiognathi二菌種，包括P. mandapamensis可於含7% NaCl的培養液中生長，但P. leiognathi則否；P. mandapamensis的離胺酸去羧酶 (lysine decarboxylase) 測試呈陰性反應，而P. leiognathi則呈陽性反應；P. mandapamensis具有同化蘋果酸 (malate) 的能力，而P. leiognathi則否。而本研究所建立之發光細菌的簡易、準確生長測量方法，更可成為其他發光細菌研究的重要研究方法參考。

The previous study (Fukasawa& Dunlap, 1986) identified the bioluminescent bacteria that symbiosed in the light organ of swordtip squids [Uroteuthis (Photololigo) edulis] is Photobacterium leiognathi. However, the current study proposes a contradictory identification. In our study, 25 strains of symbiotic bacteria were isolated from the light organs of five swordtip squids collected off the coast of north Taiwan. Among these 25 isolates, 24 were identified to be Photobacterium mandapamensis, while the rest one being P. leiognathi, based upon the results of comparative analyses of the 16S rDNA and the luxF gene. Therefore, the predominant bacterial symbiont of swordtip squid should be P. mandapamensis, and only in rare cases that both P. mandapamensis and P. leiognathi might be cosymbiosed within the same light organ. Since we have observed among the 24 P. mandapamensis isolates some featured differences of genetic, physiological, and biochemical characteristics, it is hypothesized that polyclonal bacteria may have entered the light organ from the sea water during its early stage of development. Moreover, the current studies unraveled some physiological and biochemical features that can be employed to distinguish the two close-related bacterial species of P. mandapamensis and P. leiognathi . The current studies have also developed a simple and accurate photometric method for measuring the growth of luminescent bacteria.