謝文陽臺灣大學：海洋研究所邱秀慧Chiu, Hsiu-HuiHsiu-HuiChiu2007-11-272018-06-282007-11-272018-06-282006http://ntur.lib.ntu.edu.tw//handle/246246/56493海洋弧菌泛指異營性、好鹽性、兼性嫌氣性革蘭氏陰性桿菌中，具有發酵葡萄糖等醣類能力者。它們遍布於內灣、沿岸和大洋水域及海底沉積物，也附存於形形色色海洋生物體表和體內。根據16S rDNA序列訊息，八十種海洋弧菌有效種均隸屬γ-Proteobacteria，分別被歸類於Enterovibrio、Grimontia、Photobacterium、Salinovibrio和Vibrio五屬。本研究以此類海洋弧菌為研究素材，首先建立弧菌有效種標準株16S rDNA和recA基因之基因型資料庫，做為快速鑑種之參考，其次將本實驗室所收集的類海洋弧菌鑑定分群，最後針對具有發光、耐高鹽及發酵產氣特性的弧菌，詳細探討其各類形質，並進一步證實菌株FG1T和VR1T為新種細菌。 八十二種海洋弧菌標準株16S rDNA經DdeI、HhaI、RsaI和ScaI四種核酸限制酶模擬切割後，其限制型(或模擬限制型)數目依序為七、十三、十和四種，共可整合為四十六種基因型(或模擬基因型)。16S rDNA之第一和第四基因型所屬十八株標準株之recA資料，經DdeI、HhaI和RsaI模擬酶切後，其限制型分別為九、十二和六種，可整合為十八種基因型。綜合模擬結果，16S rDNA和recA限制酶切基因型分析可做為鑑定海洋弧菌之簡便工具。 本實驗室保存五百八十七株類海洋弧菌分離株，吾人挑出四十一株具鈉鹽需求和發酵葡萄糖特性之革蘭氏陰性桿菌為實驗對象，進行表現型和基因型檢測。依照發酵產氣、菌落擴散性、洋菜分解酶、氧化酶、賴氨酸去羧酶(arginine dihydrolase)、鳥氨酸去羧酶(lysine decarboxylase)、42 ℃和10% NaCl培養條件下生長、利用甘露糖作為生長單一碳源等菌株表型特性檢測結果，可將這些分離株歸類為二十三個表型群。利用HhaI、DdeI、RsaI和ScaI四種限制酶分析菌株16S rDNA限制型，則可獲得十四個16S rDNA基因型群，其中有十型相異於前述有效種標準菌株的44種基因型。recA-RFLP的分析，則可進一步將相同16S rDNA基因型的菌株區分開。 二十七株發光細菌都是屬於需鹽性、兼性嫌氣性革蘭氏陰性桿菌，都可歸類為弧菌科(Vibrionaceae)菌種。依據菌株在4°C、40°C與8%NaCl的增殖情形，及是否利用甘露醇或蔗糖為唯一碳源的特性，分離株可被分為五群。經16S rRNA基因序列分析比對發現，第一菌群代表株之最相似菌種為Photobacterium leiognathi subsp. mandapamensis (ATCC 25521T) (相似度99.4%)。其餘四群代表株與Vibrio herveyi (ATCC 14126T)或其近緣種具有98%以上序列相似度，而它們的表型特性也都與Vibrio herveyi吻合。利用HhaI、DdeI、RsaI和ScaI四種限制酶分析分離株16S rDNA限制型，可將五群菌株區分為二個16S rDNA基因型群，第一菌群菌株與其最相似菌種Photobacterium leiognathi都屬於相同基因型，第二至第五菌群菌株經由進一步的gyrB序列分析，可鑑定為Vibrio herveyi和Vibrio campbellii。研究結果顯示，台灣沿岸海水中發光菌的菌種組成不外Photobacterium leiognathi和Vibrio herveyi/ campbellii，而以後者較占優勢。 台灣北部沿海海水中耐高鹽海洋弧菌之生菌數估算值約介於0-1500 CFU/ml。25株耐高鹽分離株可初步分為十四個菌群，其分群標準係依據菌株能否生長於42 ℃、發酵產氣、菌落擴散性、有無洋菜分解酶、ornithine decarboxylase和lysine decarboxylase、能否利用β-hydroxybutyrate為單一碳源以及發酵L-arabinose能力。利用前述四種內切限制酶分析菌株16S rDNA限制型，可將十四群菌株區分為八個16S rDNA基因型群。解析可耐受至15% NaCl之三株分離株16S rDNA序列訊息，菌株L3AN-7與Vibrio natriegens (ATCC 14048T)最為近緣，相似度為99.7%，菌株H3AN-1與2HAN4-3則最類似Salinivibrio costicola (NCIMB 701T)，相似度分別為98.9%和98.4%。 二株兼性嫌氣性發酵產氣菌株FG1T和VR1T均為具鞭毛之海洋弧菌，在嫌氣的環境下皆可發酵葡萄糖或其他碳水化合物產生有機酸。前者與最近緣菌種Vibrio mytili之16S rDNA相似度僅96.2%，而與後者最相近的菌種為Vibrio gazogenes，此二菌株16S rDNA序列相似度為95.8%，DNA-DNA雜交結果相似度為55.9%。根據表型特性和遺傳特性，菌株FG1T與VR1T均是Vibrio屬新種細菌，吾人將其分別命名為V. aerogenes (ATCC 700797T & CCRC 17041T)和V. ruber (CCRC 17186T & JCM 11486T)。Marine vibrios are heterotrophic, halophilic, facultatively anaerobic, Gram-negative, rod-shaped bacteria capable of fermenting glucose and other carbohydrates. They are ubiquitous in estuarine, coastal and oceanic waters and in marine sediments. They are closely associated with many kinds of marine organisms from plankton to fish. According to 16S rRNA, marine vibrios belongs to γ-Proteobacteria. Eighty Vibrionaceae species are classified into 5 genii including Enterovibrio, Grimontia, Photobacterium, Salinovibrio and Vibrio. Enterovibrio and Grimontia. This paper described 16S rDNA- and recA-RFLP (restriction fragment length polymorphism) of known Vibrionaceae species and identification and classification of vibrio-like strains, luminous, high-NaCl tolerant vibrios. Two unidentified strains of glucose-fermentation and gas-producting bacteria, FG1T and VR1T, reported by us previously were proven to represent two novel Vibrio species, respectively. The 16S rDNA sequences of eighty-two Vibrionaceae species were simulated by four restriction endonuclease enzymes, DdeI, HhaI, RsaI and ScaI, and were classified into seven, thirteen, ten and four restriction types. These Vibrionaceae species were separated into forty-six genotypes. Eighteen species in first and fourth genotypes could be classified into eighteen types by the restriction patterns of recA. The RFLP analysis of 16S rDNA and recA was a simple method to classify vibrios. Forty-one strains were chosen by the Gram-negative reaction, NaCl-requirement and glucose-fermentation from 587 vibrio strains in our laboratory. The selected strains were characterized into twenty-three groups by the features of swarming, agarase, oxidase, fermentation with gas-production, arginine dihydrolase, lysine decarboxylase, 42˚C, growth in 10% NaCl and utilization of mannose as sole carbon source. These isolates were classified into 14 genotypes by 16S rRNA-RFLP digested with the four enzymes, DdeI, HhaI, RsaI and ScaI. Ten genotypes were new and different from standard strains in Vibrionaceae. The analysis of recA-RFLP had distinguished into different strains in the same 16S rRNA genotype. All of them were halophilic, facultatively anaerobic, Gram-negative rods, and could be identified as the family of Vibrionaceae. According to the growth at 4 and 40˚C, in 8% NaCl and utilization of mannitol and sucrose as sole carbon source, these isolates were divided into five groups. The first group had the highest similarity with Photobacterium leiognathi (99.4%). The other 4 groups had higher than 98% similarity with Vibrio harveyi or its closely related species. However, they could be clustered into only two genotypes according to the analysis of 16S rRNA-RFLP digested with various restriction enzymes (DdeI, HhaI, RsaI and ScaI). The characterization data together with a 16S rRNA gene-based phylogenetic analysis revealed that the isolates included in phenotype I (seven isolates) could be members of Photobacterium leiognathi subsp. mandapamensis (ATCC 25521T), while the strains included in phenotypes II-V (twenty isolates) might be classified as V. harveyi (ATCC 14126T). However, phylogeny based on gyrB sequences indicated that phenotypes II-V could be classified into two species, Vibrio harveyi and Vibrio campbellii. Culturable luminous bacteria in the shallow coastal waters of Taiwan during the sampling period are dominated by V. harveyi/campbellii and P. leiognathi, and the former species appeared to be more prevalent and numerous than the latter species in general. Besides Salinovibrio, we want to find out if another vibrios could tolerate more than 10 or 12% NaCl. The coastal water samples were collected along the northern Taiwan. The count of high-NaCl tolerate bacteria was between 0-1500 CFU/ml. From the counting plates, twenty-five bacterial strains were isolated. According to the swarming, agarase, fermentation with gas-production, ornithine decarboxylase, lysine decarboxylase, growth at 42˚C and utilization β-hydroxybutyrate as sole carbon source, the twenty-five isolates were classified into fourteen groups. The isolates were further classified into eight genotypes by 16S rRNA-RFLP digested with 4 enzymes described above. Three strains, L3AN-7, H3AN-1 and 2HAN4-3, could grow in PY broth with 15% NaCl. L3AN-7 was identified as Vibrio natriegens (ATCC 14048T) with similarity of 99.7%. H3AN-1 and 2HAN4-3 were classified as Salinivibrio costicola (NCIMB 701T) with similarities 98.9 and 98.4%. Two strains, FG1T and VR1T, could both ferment glucose with gas production and were isolated from a seagrass bed sediment sample collected from Nanwan Bay and a sea-water sample collected in the shallow coastal region of Keelung, Taiwan. FG1T and VR1T were motile by means of two flagella at on pole of the cell and one polar sheathed flagellum, respectively. Strain FG1T required Na+ for growth, and exhibited optimal growth at 30-35°C, pH 6-7 and about 4% NaCl. The strain VR1T grew optimally at 25-30°C and pH 6-7 and required the presence of NaCl, the optimal concentration being about 2%. Both strains, FG1T and VR1T, grew anaerobically by fermenting glucose and other carbohydrates and producing acids and gases. The DNA G+C content of FG1T and VR1T were 45.9 and 45.8 mol%. The major cellular fatty acids of FG1T and VR1T were 2-OH-16:0 & 3-OH-14:0 and C12:0. Characterization data, together with the results of a 16S rDNA-based phylogenetic analysis, indicate that strain FG1T represents a new species of the genus Vibrio. Strain VR1T is phenotypically similar to Vibrio gazogenes. However, the reduction of nitrate to nitrite, the ability to utilize D-arabinose, melibiose and L-glycine as sole carbon sources, the inability to utilize sorbitol as a sole carbon source, resistance to O/129 and susceptibility to erythromycin and novobiocin allow differentiation between V. gazogenes and strain VR1T. The names Vibrio aerogenes sp. nov. and Vibrio ruber sp. nov. are proposed for the novel species, with strains FG1T and VR1T as the type strains.頁次 摘要．．．．．．．．．．．．．．．．．．．．． i Abstract．．．．．．．．．．．．．．．．．．．iv 前言．．．．．．．．．．．．．．．．．．．．． 1 第一章 已知海洋弧菌之16S rRNA和recA序列分析 8 前言 8 材料與方法 10 結果與討論 13 表 16 圖 19 第二章 台灣北部海水中海洋弧菌之收集及分類．． 23 前言 23 材料與方法 24 結果與討論 30 表 33 圖 37 第三章 發光海洋弧菌之多樣性與分類地位．．．．． 39 前言 39 材料與方法 40 結果與討論 44 表 53 圖 57 第四章 台灣北部海水中耐高鹽之海洋弧菌計數、分離及特性檢測．．．．．．．．．．．．． 65 前言 65 材料與方法 67 結果與討論 68 表 72 圖 76 第五章 發酵產氣之兩新種海洋弧菌分類地位之探討 80 前言 80 材料與方法 81 結果與討論 84 表 90 圖 94 參考文獻．．．．．．．．．．．．．．．．．．． 100 附錄一．．．．．．．．．．．．．．．．．．．．． 125 附錄二．．．．．．．．．．．．．．．．．．．．． 135 附錄三．．．．．．．．．．．．．．．．．．．．． 136 附錄四．．．．．．．．．．．．．．．．．．．．． 137 附錄五．．．．．．．．．．．．．．．．．．．．． 139 附錄六．．．．．．．．．．．．．．．．．．．．． 140 附錄七．．．．．．．．．．．．．．．．．．．．． 142 附錄八．．．．．．．．．．．．．．．．．．．．． 143 附錄九．．．．．．．．．．．．．．．．．．．．． 144 附錄十．．．．．．．．．．．．．．．．．．．．． 153 附錄十一．．．．．．．．．．．．．．．．．．．．． 180 附錄十二．．．．．．．．．．．．．．．．．．．．． 190 附錄十三．．．．．．．．．．．．．．．．．．．．． 197en-US弧菌酶切限制型基因型發光菌VibrionaceaeGammaproteobacterialuminousRFLPgenotype[SDGs]SDG14thesis