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標題: Characterization of novel pigmented-bacteria and mutagenic impact of ethyl methanesulfonate on polyphasic taxonomic markers
作者: Mariyam Shahina
瑪 麗 央
關鍵字: Robertkochia marina;myxol;Cryomorphaceae;mutagenesis;BOX-profile;pigment;16S rRNA gene;蝦紅素;Robertkochia marina;藍藻葉黃素醇;Cryomorphaceae;致突變作用;BOX-圖譜;色素;16S rRNA基因
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Bioactive natural products from the marine resources, including microorganisms encompass potential industrial and bio-medical applications. Irrespective of the enormous difficulty in isolating and harvesting marine bacteria, naturally occurring bioactive microbial metabolites are increasingly attractive due to their broad-ranging pharmacological activities, especially those with unique color pigments. However, characterization of bioactive compounds including pigments is quite complicated and thus given least importance so far in polyphasic taxonomy, which on the other hand integrates several kinds of data pertaining phenotypic, genotypic and phylogenetic characteristics during bacterial systematics. Similarly, the cumulative impact of any mutagenic chemical substance on pigment profile as well as other taxonomic markers is poorly studied. In this work, five novel, pigment-producing, Gram-negative, rod-shaped, strictly aerobic and non-spore-forming marine bacterial strains designated CC-AMO-30B, CC-AMZ-30M, CC-AMZ-30N, CC-AMO-30D and CC-AMWY-103B, isolated from coastal surface seawater in Taiwan were subjected to detailed pigment analysis and polyphasic taxonomy. Pigments were analyzed through UV–visible spectrophotometry, high-performance liquid chromatography-diode array detector and liquid chromatographic mass spectrometry. Finally, the impact of mutagenic ethyl methanesulfonate (EMS; doses 0.5% and 1%) on pigment profile and taxonomic markers was assessed with reference to red-pigmented Serratia. Strains CC-AMO-30B, CC-AMZ-30M and CC-AMZ-30N were identified to be Sphingomicrobium species of the family Sphingomonadaceae and named as S. astaxanthinifaciens, S. marinum and S. flavum, respectively. Strains CC-AMO-30D (Robertkochia marina) and CC-AMWY-103B (Luteibaculum oceani) are recognized to be novel genus of the family Flavobacteriaceae and Cryomorphaceae representatives, respectively. Strain CC-AMO-30B synthesized astaxanthin and its putative glycosyl derivatives, strains CC-AMZ-30M, CC-AMZ-30N and CC-AMWY-103B synthesized zeaxanthin and its isomers, whereas strain CC-AMO-30D produced both myxol and zeaxanthin. In addition, five closely related reference strains affiliated to a total of four different genera (Sphingomicrobium, Owenweeksia, Joostella and Galbibacter) were also characterized for their carotenoid profiles. Presently defined doses of EMS proved to be lethal to all novel strains, whereas at similar doses, Serratia strains (EMS-mutants CC-NPM-11>CC-LPM-20>CC-HPM-11) showed extensive heterogeneous alterations in the molecular, structural and biochemical features. The biosynthesis of prodigiosin was blocked completely in CC-NPM-11, where as other strains showed quantitative variation in terms of their prodigiosin profile. EMS-mutation hampered carbohydrate, protein, lipid and nucleic acid metabolism heterogeneously. Biosynthesis of saturated, unsaturated, hydroxyl and cyclo fatty acids were significantly affected. Unidentified aminolipids were most susceptible besides phosphatidylethanolamine, phosphatidylserine and an unidentified phospholipid. An unidentified polyamine, putrescine, cadaverine and spermidine showed significant quantitative variations. In summary, five novel strains and four reference strains of phylogenetically distant taxa were fully characterized for their bioactive pigments that could serve as model organisms for related analyses in the future. Furthermore, current study provided evidence for the lethal impact of EMS on pigmented marine bacterial isolates besides demonstrating susceptibility of pigments and evolutionarily conserved bacterial structural and molecular taxonomic markers for chemical mutation at similar doses in red-pigmented Serratia. While considering the hazardous impact of EMS, it is recommended that preference should be given to the screening and characterization of natural isolates for bioactive pigments instead of opting for strain improvement via chemical mutagenesis.

來自包含微生物的海洋資源的生物活性天然產物具有工業及生醫應用的潛力。既使分離海洋細菌有很大的困難度,天然的生物活性的微生物代謝物,尤其是獨特的色素物質由於其廣泛的臨床活性而受到矚目。無論如何,迄今在細菌系統學中整合表現型、基因型和親源性特性分析的多相分類學中,包括色素的生物活性化合物的特性分析受到最少重視。同樣地,致突變化學物質對於色素圖譜和其他分類標誌的累積效應的研究很少。在本研究中,對於自台灣沿海表層海水的五個新穎的菌株分別為 CC-AMO-30B、 CC-AMZ-30M、 CC-AMZ-30N、CC-AMO-30D和CC-AMWY-103B,其為產色素的、革蘭式陰性、桿形、絕對好氧、產鞭毛的和無形成孢子的海洋細菌菌株,進行色素分析及多相分類研究。藉由結合 UV–可見光分光光度計 和高效液體層析-光二極體陣列偵測器(HPLC-SAD)和液體層析質譜儀(LC-MS)分析色素物質。 最後,利用0.5%和1%劑量的乙基甲磺酸鹽(EMS) 對於紅色色素產生Serratia菌株其色素圖譜和分類標誌的影響。CC-AMO-30B、CC-AMZ-30M 和CC-AMZ-30N 等菌株經鑑定為Sphingomonadaceae科 的Sphingomicrobium 菌種,並命名分別為 S. astaxanthinifaciens、 S. marinum 和S. flavum。確認CC-AMO-30D (Robertkochia marina) 和CC-AMWY-103B (Luteibaculum oceani)分别為 Flavobacteriaceae 和 Cryomorphaceae科的新屬。CC-AMO-30B 菌株合成蝦紅素 (astaxanthin) 及其醣基衍生物,而CC-AMZ-30M、CC-AMZ-30N 和CC-AMWY-103B 菌株合成玉米黃素(zeaxanthin)及其異構物,而CC-AMO30D產生分別合成藍藻葉黃素醇(myxol)和玉米黃素。此外,我們亦同時調查屬於四個不同屬(Sphingomicrobium、Owenweeksia、Joostella 和 Galbibacter) 的5個緊密相關的標準菌株的類胡蘿蔔素圖譜。目前證實限定劑量的EMS 對所有新穎菌株有致死效果,然而對於Serratia 菌株(CC-NPM-11> CC-LPM-20> CC-HPM-11)在分子、結構和生化特性呈現異質性的改變。CC-NPM-11中靈菌紅素(prodigiosin)的合成受到完全抑制 ,然而其他菌株之靈菌紅素的圖譜呈現出量的變化。EMS-致突變異質性地傷害醣類、蛋白質、脂肪和核酸代謝。飽合、不飽和、羥基和環狀脂肪酸明顯受到影響。除了磷脂酰乙醇胺(PEA)、磷脂醯絲胺酸(PS) 和一個未知的磷脂類(PL),未知的胺脂類(AL)是最受影響的。一個未知的多胺、腐胺、屍胺和亞精胺的量呈現明顯改變。簡言之,五個新穎菌株和親緣性較遠的四個標準菌株的具生物活性的色素的特性分析可以在未來相關分析被當作生物模式。尤有甚者,本研究除了闡明在與產紅色素的Serratia菌株類似劑量的色素和演化性保留細菌結構和分子分類標誌的化學變異作用,並且提供EMS對於產色素海洋細菌對致死效應的證據。當考慮EMS的有害效應時,我們建議優先篩選和天然的生物活性色素的特性分析代替藉由化學突變挑選改良菌株。
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