新種洋菜分解性海洋細菌之分離與特性探討

Isolation and characterization of novel marine agarolytic bacteria

劉東岩(Tung Yen Liu)

指導教授 ： 謝文陽

國立臺灣大學/理學院/海洋研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01354

摘要

洋菜是由海洋中的紅藻所萃取出的多醣複合物，主要成分為聚半乳糖 (polygalactan)，聚半乳糖則約由70%洋菜糖（agarose）和30%洋菜膠（agaropectin）所構成。洋菜糖是經由一連串3,6-脫水左旋半乳糖(3,6-anhydro-L-galactose)與右旋半乳糖(D-galactose)所形成的長鏈狀聚合物。只有一小部分種類細菌具洋菜分解能力，大多發現於海洋環境。本研究分別至台灣東北角與安平港共篩選出110株具洋菜分解能力的細菌，經初步的生理生化特性與序列分析，其中有三分離株可能為新種細菌，分別為TMA1、SA1、AW18。
此三株具有洋菜分解能力的分離株皆為革蘭氏陰性的筆直或彎曲桿菌，皆需有鈉鹽才能生長。同時，也都具有catalase與oxidase活性，也能進行硝酸還原反應，但都無法進行脫氮反應。除了以上的共通特性外，個別分離株的特性如下：
TMA1沒有鞭毛、不具運動性，最適合生長於25℃及3% NaCl的培養基中，此株僅能進行好氧生長，無法以發酵葡萄糖的方式在無氧條件下生長。其細胞脂肪酸以C16:0 (17.5%)、C17:1ω8 c (12.8%)、C17:0 (11.1%)、C15:0 2-OH/C16:1ω7c (8.6%)和C13:0(7.3%)等為主要組成。DNA的G+C 含量為41.0 mol%。16S rRNA基因序列分析顯示，TMA1與Talassomonas loyana有97.0%的序列相似度， Talassomonas ganghwensis有95.2%的序列相似度；Talassomonas viridans有94.7%的序列相似度，與其餘已知菌種的序列相似度均低於94.0%。
SA1細胞個體大多不具有鞭毛且無法運動。最適合生長於30℃及2% NaCl的培養基中。此株在無氧條件下，無法以發酵葡萄糖或其他醣類的方式生長，但能以還原硝酸的方式生長。細胞脂肪酸的主要組成有C15:0 2-OH/C16:1ω7c (28.6%)、C17:1ω8 c (22.8%)、C16:0 (14.5%)、C18:1ω7 c (11.0%)與C17:0 (6.4%)，DNA的G+C 含量為55.6 mol%。根據16S rRNA基因序列分析結果， Teredinibacter turnerae、Pseudomonas argentinensis與Cellvibrio japonicus為此株最相近的物種，其序列相似度依序為92.7%、92.3%、92.1%，沒有其他已知菌屬與SA1的序列相似度高於92.0%。
AW18具有鞭毛與運動性，最適合生長於20-25℃及3% NaCl的培養基中，此株能以發酵葡萄糖或半乳糖等多種醣類的方式在無氧條件下生長。該株的細胞脂肪酸組成大於5%者有C15:0 iso 2-OH/C16:1ω7c (35.62%)、C18:1ω7c (27.03%)、C16:0 (16.26%)、C14:0 3-OH/C16:1 iso I (6.90%)，DNA的G+C 含量為52.9 mol%。經由16S rRNA基因序列分析，與Agarivorans albus最為相近，其序列相似度為93.9%，而與其他已知菌種的序列相似度皆低於92.0％。
综合各項表型及遺傳特性分析結果顯示，分離株TMA1、SA1與AW18可歸類為新種海洋洋菜分解細菌。

關鍵字

洋菜分解 ； 海洋細菌

並列摘要

Agar, a complex polysacchrides extracted from red algae, consists of polygalactan. Polygalactan is composed of above 70% agarose and 30% agaropectin, and agarose consists of a linear chain of alternating residues of 3,6-anhydro-L-galactose and D-galactose. Only a relatively few bacterial species are capable of degrading this refractory material. Agarolytic bacteria are ubiquitous in coastal and estuarine habitats. In this study, there were 110 agarolytic marine bacteria isolated from northeast coast and An-Ping Harbor. Phenotypic and phylogenetic data of the isolates revealed that three isolates, TMA1, SA1, and AW18, could be novel species.
These three agarolytic isolates were Gram-negative and straight or curved rods. They required NaCl for growth and presented catalase. They could reduce nitrate to nitrite but not further to N2. Other characteristics of the three isolates are listed below.
TMA1 was non-motile and non-flagellated. It grew aerobically and was incapable of anaerobic growth by fermenting glucose or other carbohydrates. Cellular fatty acids were predominated by C16:0 (17.5%), C17:1ω8c (12.8%), C17:0 (11.1%), C15:0 iso 2-OH/C16:1ω7c (8.6%) and C13:0 (7.3%). The DNA G+C content was 41.0 mol%. The strain showed highest 16S rRNA gene sequence similarities to JAMB-A33 (98.2%), Thalassomonas ganghwensis (95.2%) and Thalassomonas viridans (94.7%). No other known bacteria shared more than 94% sequence similarity with strain TMA1.
SA1 was non-motile and non-flagellated, but some cells were flagellated. It grew aerobically and was capable of anaerobic growth by reducing nitrite to nitrate. Cellular fatty acids were predominated by C15:0 2-OH/C16:1ω7c (28.6%), C17:1ω8 c (22.8%), C16:0 (14.5%), C18:1ω7 c (11.0%), and C17:0 (6.4%). The DNA G+C content was 55.6 mol%. The 16S rRNA gene sequence was similar to Teredinibacter turnerae (92.7%), Pseudomonas argentinensis (92.3%), and Cellvibrio japonicus (92.1%), and no other known bacteria shared more than 92% sequence similarity with strain SA1.
AW18 could motion by a single polar flagellum. It was capable of anaerobic growth by fermenting glucose or other carbohydrates. The major fatty acids present at levels greater than 5% included C15:0 iso 2-OH/C16:1ω7c (35.62%), C18:1ω7c (27.03%), C16:0 (16.26%), C14:0 3-OH/C16:1 iso I (6.90%). The DNA G+C content was 52.9 mol%. The strain showed highest 16S rRNA gene sequence similarities to Agarivorans albus (93.9%), and no known bacteria shared more than 92% sequence similarity with strain AW18.
Phylogenetic and phenotypic characteristics accumulated in this study support that TMA1, SA1, and AW18 are novel species in

並列關鍵字

agarolytic ； marine bacteria

參考文獻

Akagawa-Matsushita, M., Matsuo, M., Koga, Y. & Yamasato, K. (1992). Alteromonas atlantica sp. nov. and Alteromonas carrageenovora sp. nov., bacteria that decompose algal polysaccharides. Int J Syst Bacteriol 42:621-627.

Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol. 50:1861-1868.

Distel, D.L., Morrill, W., MacLaren-Toussaint, N., Franks, D. & Waterbury, J. (2002). Teredinibacter turnerae gen. nov., sp. nov., a dinitrogen-fixing, cellulolytic, endosymbiotic gamma-proteobacterium isolated from the gills of wood-boring molluscs (Bivalvia: Teredinidae). Int J Syst Evol Microbiol. 52:2261-2269.

Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P. Jr. (1992) . How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol. 42, 166–170.

Gevers, D., Cohan, F. M., Lawrence, J.G., Spratt, B.G., Coenye, T., Feil, E. J., Stackebrandt, E., Van de Peer, Y., Vandamme, P., Thompson, F. L. & Swings, J. (2005). Re-evaluating prokaryotic species. Nat Rev Microbiol. 3:733-739.