北极高维度海域海冰嗜冷菌系统发育多样性及其低温水解酶分析

Phylogenetic diversity and cold-adaptive hydrolytic enzymes of culturable psychrophilic bacteria associated with sea ice from high latitude ocean,Arctic

应用样品直接稀释涂布平板、-1℃富集培养和-20℃冷冻24h后富集培养等3种方法,从北极加拿大海盆和格陵兰海的高纬度海域(77°30′N^81°12′N)海冰中分离到37株嗜冷菌。根据其16S rDNA全长序列所进行的系统发育分析表明,分离菌株分属于γ_变形细菌群(γ_Proteobacteria)的Colwellia、Marinobacter、Shewanella、Thalassomonas、Glaciecola、Marinomonas、Pseudoalteromonas和嗜纤维菌_曲挠杆菌_拟杆菌群(Cytophaga_Flexibacter_Bacteroide,CFB)的Flavobacterium、Psychroflexus等9个属。其中有9株菌的16S rDNA序列与已明确鉴定种的相似性在93.4%~96.9%,为潜在的新种。北极加拿大海盆海冰细菌BSi20002与南极威德尔海海冰细菌Marinobactersp.ANT8277的16S rDNA序列相似性为100%,表明在种水平上南、北两极也存在相同的细菌。分离的嗜冷菌在4℃条件下能产生多种大分子物质水解酶类,其中62.6%、51.4%和40.5%的菌株分别能水解Tween_80、明胶和淀粉。

The phylogenetic diversity of culturable psychrophilic bacteria associated with sea ice from high latitude sea （77°30＇N - 81° 12＇N）, Canadian Basin and Greenland sea Arctic, was investigated. A total of 37 psychrophilic strains were isolated using three different methods of （ Ⅰ ） spread plate method： 100μL of each dilution ice-melt sample was spreaded onto the surface of Marine 2216 agar （DIFCO laboratories, Detroit, MI） and incubated for 2 to 6 weeks at 4℃ ; （ Ⅱ） bath culture and spread plate method： 1mL of sample was added to 9mL of NSW （unamended natural seawater, 0.2μm prefiltered and autoclaved） and incubated for 1 months at - 1 ℃, then spread plate method was used to isolate bacterial strains from the pre-cultured samples; （ Ⅲ ） cold shock, bath culture and spread plate method： samples were exposed to -20℃ for 24h, then bacterial strains isolated by bath culture and spread plate method under aerobic conditions. Nearly half of psychrophilic strains are isolated by using method Ⅲ . 16S rDNA nearly full-length sequence analysis reveal that psychrophilic strains fall in two phylogenetic divisions, γ-proteobacteria （in the genera Colwellia, Marinobacter, Shewanella, Thalassomonas, Glaciecola, Marinomonas and Pseudoalteromonas） and Cytophaga-Flexibacter-Bacteroides （in the genera Flavobacterium and Psychroflexus）. Nine of bacterial isolates （ BSi20007, BSi20497, BSi20517, BSi20537, BSi20170, BSi20001, BSi20002, BSi20675 and BSi20101） quite likely represent novel species （16S rDNA sequence similarity below 97%）. One of strains （BSi20002） from Canadian Basin shows 100% sequence similarity to the Antarctic Weddell sea ice isolate Marinobacter sp. ANT8277, suggesting bacteria may have a bipolar distribution at the species level. AF283859 sequences were submitted to the BLAST search program of the National Center for Biotechnology Information website （ NCBI, http,//www, ncbi. nlm.nih, gov）. Twenty sequences showing 100% similarity each other are retrieved from the database, eleven from Antarctic seawater bacteria, three from Antarctic sea-ice bacteria, one from Spitzbergen sea-ice bacteria, two from Chukchi Sea sea-ice bacteria, two from Canadian Basin sea-ice bacteria （in this study） and one from uncultured bacterium clone PDA-OTU11 associated with the coral Pocillopora damicornis from the Great Barrier Reef. These may indicate that the physiological and geographic barriers appear to be permeable and some bacterial species can survive in different environment. The majority of the bacterial strains are able to secrete diversity cold-adaptive hydrolytic enzymes into the medium at 4℃. The isolates that are able to degrade Tween-80, glutin, and starch account for, respectively, 62.6%, 51.4% and 40.5%,