徐 叢,張文燕,陳一然,張 蕊,董 逸,杜海艦,潘紅苗,肖 天,*
1 中國科學院海洋研究所,海洋生態(tài)與環(huán)境科學重點實驗室, 青島 266071 2 中國科學院大學, 北京 100049
?
青島太平灣潮間帶趨磁細菌多樣性
徐叢1,2,張文燕1,陳一然1,張蕊1,董逸1,杜海艦1,2,潘紅苗1,肖天1,*
1 中國科學院海洋研究所,海洋生態(tài)與環(huán)境科學重點實驗室, 青島2660712 中國科學院大學, 北京100049
在青島太平灣潮間帶沉積物中發(fā)現(xiàn)了一定量的海洋趨磁細菌,最大豐度可達350 個/cm3。透射電鏡觀察發(fā)現(xiàn)該區(qū)域趨磁細菌均為趨磁球菌。磁小體個體形狀單一,皆是立方體狀;磁小體排列方式多樣,以鏈狀排列為主,包括單鏈、雙鏈與多鏈,也有少數(shù)成簇排列。EDS結果表明,磁小體成分為四氧化三鐵。據估算,趨磁細菌的鐵元素含量(干重)范圍在0.40%—6.91%之間,平均為2.19%。通過16S rRNA基因文庫的構建與測序得到了47個趨磁細菌序列,分屬13個OTU。系統(tǒng)發(fā)育分析結果表明,它們都屬于α-變形菌綱,其中9個OTU與已知最相似序列的相似性低于97%,有5個OTU與已知最相似序列的相似性低于93%,可能代表了趨磁細菌的9個新種、5個新屬,說明該區(qū)域潛在的微生物新種質資源十分可觀。
潮間帶;趨磁細菌;多樣性;磁小體;16S rRNA 基因
趨磁細菌(magnetotactic bacteria)是一類能夠沿磁力線運動的特殊細菌[1],其形態(tài)多樣,有單細胞的球菌、桿菌、弧菌、螺旋菌以及多細胞趨磁原核生物[2]。趨磁細菌的趨磁性,源于體內含有的起導向作用的磁小體(magnetosome)與提供運動動力的鞭毛[3]。磁小體通常呈鏈狀排列,有單鏈、雙鏈以及多鏈,少數(shù)趨磁細菌的磁小體不成鏈排列[4-5]。磁小體的大小、形狀以及成分都具有種屬特異性[4-6];磁小體個體大小為35—120 nm,處于穩(wěn)定的單磁疇范圍內[5-7];形狀有立方八面體、六面體棱柱、子彈頭形、淚滴狀、薄片狀、球狀以及不規(guī)則狀等形態(tài)[7-8];成分有四氧化三鐵或/與四硫化三鐵[2]。自1975年美國學者Blakemore在Science上報道了趨磁細菌之后[9],科學家陸續(xù)從海洋、湖泊以及土壤等不同水陸生境中發(fā)現(xiàn)了趨磁細菌[2,10]。這些趨磁細菌屬于變形菌門下的α-變形菌綱、γ-變形菌綱與δ-變形菌綱、硝化螺旋菌門以及Candidate division OP3[2];在GeneBank中收錄的趨磁細菌16S rRNA基因序列約有1200條(截至2014年6月)。在中國,趨磁細菌在多種生境被發(fā)現(xiàn),這些生境主要分布在4個地區(qū):北京的湖泊水庫(北海、密云水庫等)、西安湖泊河流(曲江池、護城河等)、山東半島沿岸的海灣潟湖(膠州灣、匯泉灣、月湖等)以及海南島八門灣[10-15]。在海洋環(huán)境中發(fā)現(xiàn)了形態(tài)相異、生理特點不同、分屬不同分類單元的趨磁細菌[11-15]。本文收集了青島太平灣潮間帶沉積物中的海洋趨磁細菌,對趨磁細菌的形態(tài)、大小,磁小體大小、數(shù)目、排列方式以及16S rRNA基因系統(tǒng)發(fā)育做了研究,并對其與不同地域趨磁細菌群落構成作了比較。
1.1樣品采集
采樣點位于青島黃海沿岸(36°03′03″N,120°21′09″E)太平灣潮間帶的中潮帶,每天都有時間裸露出水或浸沒入水下。2014年3月份在采樣點3次采集表層沉積物(0—10 cm),分別裝在500 mL的塑料采樣瓶中,每瓶沉積物約100—150 g,之后向瓶中注入原位海水(沉積物與原位海水的比例約為1∶1),帶回實驗室進行后續(xù)實驗。
1.2趨磁細菌的收集
將磁鐵粘在采樣瓶的外壁,S極朝向收集瓶。30 min后,用巴斯德管吸取磁鐵附近的樣品約500 μL,注入100 mL螺口玻璃瓶中進行二次磁收,瓶中菌液約40 mL。使用相同方法對二次磁收瓶中的樣品進行磁富集。之后采用“T-T”法進一步富集趨磁細菌[16]:將5 mL移液槍頭的頂端插入無菌離心管中,離心管與移液槍頭的頂端都注滿0.22 μm過濾的無菌原位海水,將二次磁收集所得的菌液由移液槍頭的粗端注入移液槍頭的無菌原位海水中,置于勻強磁場(磁場強度約0.35 mT),待離心管底端出現(xiàn)明顯的灰色的菌斑,則停止收集。取離心管底端菌液進行顯微觀察與種類鑒定。
1.3計數(shù)、形態(tài)觀察與磁小體成分測定
使用OLYMPUS BX51顯微鏡的微分干涉(Differential Interference Contrast,DIC)模式,懸滴法觀察趨磁細菌并計數(shù)。將收集的菌液,滴在銅網上(北京中鏡科儀技術有限公司),用透射電鏡(HITACHI H8100,中國海洋大學電鏡室)對菌體與磁小體進行觀察,包括趨磁細菌菌體形態(tài)、大小,磁小體形態(tài)、大小、數(shù)目及排列方式。用高分辨率透射電子顯微鏡(JEM2100,山東大學化學與化工學院結構成分測試中心)對磁小體成分進行分析。
1.4趨磁細菌遺傳多樣性分析
1.4.1制備DNA模板
取T-T方法收集的菌液,用DEPC水洗滌3次,以去除海水中的離子。而后用液氮與80 ℃水浴反復凍融3次,使細胞壁破碎、細胞內DNA溶出。
1.4.2對16S rRNA基因進行擴增
PCR參照Bosshard等的方法進行[17]。引物:27f(5′-AGA GTY TGA TCC TGG CTC AG-3′)與1492r(5′-GGT TAC CTI GTI AGG ACT T-3′)。PCR的反應條件是:94 ℃變性10 min后,94 ℃ 1 min,50 ℃ 45 s,72 ℃ 1 min,循環(huán)25次,72 ℃延伸10 min。PCR擴增的產物利用濃度為0.1g/L的瓊脂糖凝膠進行電泳,并用瓊脂糖凝膠DNA回收試劑盒(北京康為世紀生物科技有限公司)回收PCR產物。
1.4.3連接、轉化與克隆及DNA序列測定
將瓊脂糖凝膠中回收的PCR產物連接到載體pMD18-T(Takara,Japan)上,轉化入E.coliTop10感受態(tài)細胞(北京全式金生物技術有限公司)中,在含有X-gal、IPTG與氨芐青霉素的SOB固體培養(yǎng)基上培養(yǎng)感受態(tài)細胞。選擇具有氨芐青霉素抗性的菌落,挑出少量菌體作為模板,用T載體的通用引物M13-47與RV-M進行菌落PCR。擴增產物通過濃度為1%(w/v)的瓊脂糖凝膠電泳,檢驗其是否為陽性。將陽性克隆交予南京金斯瑞生物科技有限公司進行測序,從而獲得16S rRNA基因序列。
1.4.416S rRNA基因序列分析
將16S rRNA基因序列進行BLAST比對(http://www.ncbi.hlm.nih.gov),得到與實驗所得的各條序列相似性最高的已知序列。通過BioEdit軟件進行相似性分析,以MEGA3.1軟件的鄰位相接法進行系統(tǒng)進化分析。
2.1趨磁細菌的豐度
通過光學顯微鏡鏡檢計數(shù),經計算沉積物中趨磁細菌豐度最高可達350 個/cm3。
2.2趨磁細菌的形態(tài)
通過透射電鏡觀察,發(fā)現(xiàn)太平灣的趨磁細菌為球形趨磁細菌與卵圓形趨磁細菌(圖1),菌體大小為(2.23±0.69) μm ×(1.84±0.47) μm(n=21),寬長比為0.87±0.10(n=21)。
圖1 透射電子顯微鏡下趨磁細菌的形態(tài)Fig.1 Morphology of magnetotactic bacteria based on transmission electron microscopyA:聚集在一起的多個趨磁球菌 Magnetotactic bacteria togethered in a cluster; B,D:具有四條磁小體鏈的趨磁球菌 Magnetotactic bacteria with four chains of magnetosomes; C:具有一條磁小體鏈的趨磁球菌 Magnetotactic bacteria with only one chain of magnetosomes; E,F:具有兩條磁小體鏈的趨磁球菌 Magnetotactic bacteria with two chain of magnetosomes
2.3磁小體的多樣性與成分
通過透射電鏡鏡觀察,可以發(fā)現(xiàn)太平灣趨磁細菌的磁小體鏈排列方式多樣,磁小體個數(shù)、大小差別很大。圖2顯示了數(shù)量占優(yōu)的趨磁細菌中磁小體的各種排列方式。54%的趨磁細菌含有兩條磁小體鏈,平行或成角度排列;僅具有一條磁小體鏈的趨磁細菌占18%,具有多條磁小體鏈與磁小體成簇排列的趨磁細菌各占14%??梢园l(fā)現(xiàn),具有多條磁小體鏈的趨磁細菌,其磁小體鏈大都是兩兩平行排列。透射電鏡觀察結果顯示,每個趨磁細菌所含磁小體個數(shù)不等,數(shù)量介于7—43個。磁小體平均體積介于2.71×105—1.63×106nm3(假設磁小體為長方體)。EDS分析結果顯示,磁小體化學成分為四氧化三鐵,假設趨磁細菌含水量為85%,則本實驗中趨磁細菌的含鐵元素量(干重)范圍在0.40%—6.91%之間,平均為2.19%(總鐵元素質量/總細菌干重)。
圖2 透射電子顯微鏡下趨磁細菌的不同磁小體排列方式Fig.2 Magneosomes organizing in different modes based on transmission electron microscopyA:單條磁小體鏈 One chain of magnetosomes; B:平行的兩條磁小體鏈 Two parallel chains of magnetosomes; C:成簇的磁小體 Magnetosomes in a cluster; D:成角度的兩條磁小體鏈 Two unparallel chains of magnetosomes; E:四條磁小體鏈 Four chains of magnetosomes
2.4趨磁細菌遺傳多樣性分析
2.4.1趨磁細菌16S rRNA基因序列測定
將擴增片段以pMD18-T作為載體連接到E.coliTOP10感受態(tài)細胞中,用T載體通用引物進行PCR擴增,得到135個陽性克隆。對這135個陽性克隆進行測序與比對分析,發(fā)現(xiàn)其中屬于趨磁細菌的序列有47條,分屬13個OTU(相似性<97%),分別為:XCQD1-18、XCQD1-2、XCQD1-19、XCQD51、XCQD81、XCQD6、XCQD2-2、XCQD4-20、XCQD34、XCQD2-23、XCQD53、XCQD130、XCQD1-21。根據遺傳多樣性分析,有23條序列所代表的細菌屬于OTU(XCQD1-18),優(yōu)勢度達48.93%。屬于OTU(XCQD2-2)、OTU(XCQD1-21)、OTU(XCQD1-2)、OTU(XCQD2-23)、OTU(XCQD130)的序列分別有6條、5條、2條、2條、2條,其余各OTU都只有一條。香農威納群落多樣性指數(shù)為2.64 nit,均勻性指數(shù)為0.71。
2.4.2趨磁細菌16S rRNA基因的系統(tǒng)發(fā)育分析
根據所得序列在NCBI數(shù)據庫中Blast的比對結果,結合在BioEdit軟件中進行比對得到的相似性,將得到的13個OTU序列與其相似性最高的序列以及已獲得純培養(yǎng)的趨磁細菌序列進行系統(tǒng)發(fā)育分析。
結果顯示(圖3),本實驗所發(fā)現(xiàn)的趨磁細菌OTU分別與Uncultured Magnetococcus sp. clone MRT130(EF371494)、Uncultured Magnetococcus sp. clone MRT97(EF371493)、Uncultured Magnetococcus sp.clone M-67(EF371491)、Uncultured Magnetococcus sp.clone M-52(EF371485),Uncultured Magnetococcus sp.clone XSE-42(EF379385)、Uncultured Magnetococcus sp.clone 37(EU780681)、ncultured Magnetococcus sp.clone M-40(EF371486),最為相似。相似情況見表1。
表1 GeneBank中與本實驗所發(fā)現(xiàn)的OTU最相似的序列
圖3 趨磁細菌16S rRNA基因序列的系統(tǒng)發(fā)生樹(加粗的是本實驗所獲得的序列)Fig.3 Phylogenetic tree based on 16S rRNA gene sequence analysis (The sequences determined in this study is shown in bold)
在系統(tǒng)進化樹上,13個OTU皆屬于α-變形菌綱。根據Stackebrandt等[18]認為16S rRNA基因相似性大于97%屬于一個種,小于93%—95%屬于不同的屬。對13個OTU相似性分析表明,其中有9個OTU(XCQD 1-18、XCQD 1-2、XCQD 1-19、XCQD 81、XCQD 6、XCQD 4-20、XCQD 34、XCQD 2-23、XCQD 53)與已知最接近的序列的相似性小于97%,可能是新的種;其中,又有5個OTU(XCQD 81、XCQD 4-20、XCQD 34、XCQD 2-23、XCQD 53)與已知最接近序列的相似性小于93%,可能是新的屬。
青島太平灣為南向半封閉海灣,海灣內無明顯排污口,水質良好,底質為泥沙與礫石混合。該環(huán)境沉積物中趨磁細菌的豐度可達350 個/cm3,較已報道的一般環(huán)境中趨磁細菌的數(shù)量(103—104個/ cm3)低[19],遠低于Files等[20]報道的淡水環(huán)境沉積物中的趨磁細菌豐度(104個/cm3)。與緊鄰的匯泉灣趨磁細菌的豐度(105個/cm3)[21]相比,僅相當于其1/300。
在多次富集的樣品中,始終只能觀察到一種形態(tài)類型的趨磁細菌——趨磁球菌。已有研究表明,無論是在海洋還是淡水生境中,趨磁球菌在數(shù)量上都是占優(yōu)勢的[22-27]。本調查發(fā)現(xiàn)趨磁球菌占絕對優(yōu)勢(>99%)。透射電鏡下可以觀察到磁小體的排列方式與數(shù)目各異。鐵是地殼中含量第四大的元素,對幾乎所有已知生物都是必需的。同時,鐵循環(huán)也是生物地化循環(huán)的關鍵過程。趨磁細菌群落在鐵循環(huán)與鐵元素沉積過程中扮演重要角色[28-29]:趨磁細菌主動地吸收自然環(huán)境中的鐵離子與亞鐵離子,積累在鐵硫化物或鐵氧化物質的磁小體中[30];當趨磁細菌死亡,磁小體中的鐵元素一部分以離子的形式回到環(huán)境中,一部分沉淀到沉積物中去[31];還有的鐵元素被捕食者攝取,從而進入食物鏈[32]。趨磁細菌是地質微生物學和生物礦化作用研究的模式微生物[2]。一般地,鐵元素占趨磁細菌干重的2%—3%,這個比例比其他生物高數(shù)個數(shù)量級[33]。林巍等[31]假設,50%湖泊、池塘、水庫環(huán)境分布有趨磁細菌,且位于沉積物的最上層10 cm,每立方厘米的沉積物含有1000個趨磁細菌,每個趨磁細菌含20個磁小體,每個磁小體體積為1.25×10-4μm3,趨磁細菌的代時為12 h,則每平方千米湖泊趨磁細菌磁鐵礦年產量為0.95 kg。本實驗每立方厘米的沉積物含有350個趨磁細菌,每個趨磁細菌平均有磁小體18個,每個磁小體平均體積為8.02×10-4μm3,則每平方千米潮間帶趨磁細菌可產生磁鐵礦1.92 kg,在太平灣潮間帶的鐵元素循環(huán)可能起到重要作用。趨磁細菌豐度與每個細菌的磁小體個數(shù)都不及淡水,但由于磁小體平均體積較大,因此海洋潮間帶單位面積年平均磁鐵礦產量是淡水的估算值的2.02倍。本實驗中,將磁小體質量以四氧化三鐵計,以細胞含水85%計,估算出太平灣潮間帶趨磁細菌平均含鐵元素質量比為2.19%,與文獻報道一致。此外,磁小體質量與趨磁細菌的菌體質量成明顯的相關性(r=0.637,P=0.002<0.05)(圖4)。推測在自然狀態(tài)趨磁細菌菌體越大,就需要越多的磁性物質來幫助其感知地磁場以調節(jié)其運動。
圖4 磁小體質量與細菌質量的關系(r=0.637,P=0.002<0.05) Fig.4 The relationship between the mass of bacteria and magnetosome (r=0.637,P=0.002<0.05)
圖5 太平灣趨磁細菌與匯泉灣趨磁細菌的系統(tǒng)發(fā)生樹(加粗的是本實驗所獲得的序列)Fig.5 Phylogenetic tree of magnetotactic bacteria in Taiping Bay and Huiquan Bay based on 16S rRNA gene sequence analysis (The sequences determined in this study is shown in bold)
與邢素娥等[21]對青島匯泉灣趨磁細菌的調查結果比較(圖5,圖6),匯泉灣趨磁細菌分為8個OTU(6個OTU在2006年屬于新發(fā)現(xiàn)),其中有兩個與太平灣趨磁細菌相同。兩地趨磁細菌群落的Jaccard群落相似度指數(shù)為0.1053。Jaccard群落相似度指數(shù)取值范圍為0—1,越接近于0,則群落相似性越低。太平灣與匯泉灣群落相似性指數(shù)僅為0.1053,說明兩海灣雖然緊鄰,且采樣地點同屬潮間帶,但是趨磁細菌群落的種類構成差別顯著。這可能與這兩個海灣的環(huán)境緊密相關。首先,匯泉灣采樣地處在青島市第一海水浴場,人類活動比較頻繁,對潮間帶環(huán)境影響較大;太平灣的采樣地不在海水浴場范圍內,人類活動對該地影響較小。第二,匯泉灣有一城市雨水下水道排放口,對匯泉灣潮間帶存在潛在的淡水輸入與有機物補充。第三,匯泉灣海岸線平緩,而太平灣多有礁石與海岬分布,兩地水文地質條件相異[34-36]。第四,對匯泉灣潮間帶趨磁細菌的采集是在2006年秋季的11月,而對太平灣趨磁細菌樣品的采集是在2014年春季的3月,群落演替與季節(jié)差異也可能是造成兩地趨磁細菌群落組成差異巨大的重要原因。
圖6 太平灣趨磁細菌與匯泉灣趨磁細菌群落組成相似性比較Fig.6 Comparison of magnetotactic bacteria communites in Taiping Bay and Huiquan Bay
與大西洋法國地中海沿岸兩個潮間帶的趨磁細菌群落的研究結果比較[37-38]。在Six-Fours-les-Plages潮間帶(43°06′03″N, 5°49′20″E,受到聯(lián)合國環(huán)境規(guī)劃署的特殊保護的一個海灘,沒有污染,環(huán)境良好)發(fā)現(xiàn)了球形、弧狀、桿狀、螺旋形的趨磁細菌,磁小體的形狀有立方體形、子彈頭形、棱柱形、八面體形,發(fā)現(xiàn)了12個趨磁細菌OTU,其中11個是新OTU(相似性<97%)。太平灣趨磁細菌菌體的形態(tài)與磁小體的形狀較為單一,發(fā)現(xiàn)13個趨磁細菌的OTU,其中9個是新OTU(相似性<97%)。相比較,Six-Fours-les-Plages的趨磁細菌多樣性更豐富。另一個法國地中海Gulf of Fos的潮間帶(43°26′ N, 4°49′E)受石油精煉與鋼鐵冶煉活動的影響,在這里發(fā)現(xiàn)了球形、弧狀、桿狀的趨磁細菌,磁小體的形狀僅有立方體形,僅發(fā)現(xiàn)4個趨磁細菌新OTU(相似性<97%),說明環(huán)境的污染可能會影響趨磁細菌的多樣性。
以上所述,不同環(huán)境和時間季節(jié)差異可能是造成不同潮間帶地區(qū)趨磁細菌種類組成差異的原因,這表明趨磁細菌的多樣性非常豐富,有必要對其進行更多的調查研究和資源收集,豐富海洋微生物資源庫。
致謝:中國科學院海洋研究所夏青同學幫助統(tǒng)計;中國海洋大學姜明老師與山東大學馬希騁老師幫助使用電子顯微鏡;徐劍虹老師幫助采樣。
[1]Frankel R B. The discovery of magnetotactic/magnetosensitive bacteria. Chinese Journal of Oceanology and Limnology, 2009, 27(1): 1-2.
[2]Bazylinski D A, Lefèvre C T, Schüler D. Magnetotactic bacteria // Rosenberg E, DeLong E F, Lory S, Stackebrandt E, Thompson F, eds. The Prokaryotes. Heidelberg: Springer, 2013: 453-494.
[3]Frankel R B, Bazylinski D A, Johnson M S, Taylor B L. Magneto-aerotaxis in marine coccoid bacteria. Biophysical Journal, 1997, 73(2): 994-1000.
[4]Benzerara K, Menguy N. Looking for traces of life in minerals. Comptes Rendus Palevol, 2009, 8(7): 617-628.
[5]Farina M, Lin de Barros H G P, Esquivel D M S, Dannon J. Ultrastructure of a magnetotactic microorganism. Biology of the Cell, 1983, 48(1): 85-88.
[6]Bazylinski D A, Frankel R B. Magnetosome formation in prokaryotes. Nature Reviews Microbiology, 2004, 2(3): 217-230.
[7]Bazylinski D A, Moskowitz B M. Microbial biomineralization of magnetic iron minerals: microbiology, magnetism and environmental significance. Reviews in Mineralogy and Geochemistry, 1997, 35: 181-223.
[8]Schüler D. Formation of magnetosomes in magnetotactic bacteria. Journal of Molecular Microbiology and Biotechnology, 1999, 1(1): 79-86.
[9]Blakemore R P. Magnetotactic bacteria. Science, 1975, 190(4212): 377-379.
[10]Lin W, Wang Y Z, Gorby Y, Nealson K, Pan Y X. Integrating niche-based process and spatial process in biogeography of magnetotactic bacteria. Scientific Reports, 2013, 3: 1643-1643.
[11]Chen Y R, Zhang R, Du H J, Pan H M, Zhang W Y, Zhou K, Li J H, Xiao T, Wu L F. A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China. Environmental Microbiology, 2014, 17(3): 637-647, doi: 10.1111/1462-2920.12480.
[12]Zhou K, Pan H M, Zhang S D, Yue H D, Xiao T, Wu L F. Occurrence and microscopic analyses of multicellular magnetotactic prokaryotes from coastal sediments in the Yellow Sea. Chinese Journal of Oceanology and Limnology, 2011, 29(2): 246-251.
[13]Zhang R, Chen Y R, Du H J, Zhang W Y, Pan H M, Xiao T, Wu L F. Characterization and phylogenetic identification of a species of spherical multicellular magnetotactic prokaryotes that produces both magnetite and greigite crystals. Research in Microbiology, 2014, 165(7): 481-489.
[14]Zhang W Y, Zhou K, Pan H M, Du H J, Chen Y R, Zhang R, Ye W N, Lu C J, Xiao T, Wu L F. Novel rod-Shaped magnetotactic bacteria belonging to the classAlphaproteobacteria. Applied and Environmental Microbiology, 2013, 79(9): 3137-3140.
[15]Zhang W Y, Zhou K, Pan H M, Yue H D, Jiang M, Xiao T, Wu L F. Two genera of magnetococci with bean-like morphology from intertidal sediments of the Yellow Sea, China. Applied and Environmental Microbiology, 2012, 78(16): 5606-5611.
[16]周克. 黃海沉積物多細胞趨磁原核生物的特性研究[D]. 北京: 中國科學院研究生院, 2010.
[17]Bosshard P P, Santini Y, Grüter D, Stettler R, Bachofen R. Bacterial diversity and community composition in the chemocline of the meromictic alpine Lake Cadagno as revealed by 16S rDNA analysis. FEMS Microbiology Ecology, 2000, 31(2): 173-182.
[18]Stackebrandt E, Goebel B M. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International Journal of Systematic Bacteriology, 1994, 44(4): 846-849.
[19]Blakemore R P. Magnetotactic bacteria. Annual Review of Microbiology, 1982, 36(1): 217-238.
[20]Flies C B, Jonkers H M, Beer D, Bosselmann K, B?ttcher M E, Schüler D. Diversity and vertical distribution of magnetotactic bacteria along chemical gradients in freshwater microcosms. FEMS Microbiology Ecology, 2005, 52(2): 185-195.
[21]邢素娥, 潘紅苗, 朱開玲, 肖天, 吳龍飛. 青島匯泉灣海洋趨磁細菌多樣性研究. 高技術通訊, 2008, 18(3): 312-317.
[22]Lin W, Pan Y X. Uncultivated magnetotactic cocci from Yuandadu park in Beijing, China. Applied and Environmental Microbiology, 2009, 75(12): 4046-4052.
[23]Lin W, Li J H, Schüler D, Jogler C, Pan Y X. Diversity analysis of magnetotactic bacteria in Lake Miyun, northern China, by restriction fragment length polymorphism. Systematic and Applied Microbiology, 2009, 32(5): 342-350.
[24]Spring S, Amann R, Ludwig W, Schleifer K H, Gemerden H V, Petersen N. Dominating role of an unusual magnetotactic bacterium in the microaerobic zone of a freshwater sediment. Applied and Environmental Microbiology, 1993, 59(8): 2397-2403.
[25]Spring S, Lins U, Amann R, Schleifer K H, Ferreira L C, Esquivel D M, Farina M. Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes. Archives of Microbiology, 1998, 169(2): 136-147.
[26]Pan H M, Zhu K L, Song T, Yu-Zhang K, Lefèvre C, Xing S E, Liu M, Zhao S J, Xiao T, Wu L F. Characterization of a homogeneous taxonomic group of marine magnetotactic cocci within a low tide zone in the China Sea. Environmental Microbiology, 2008, 10(5): 1158-1164.
[27]Flies C B, Peplies J, Schüler D. Combined approach for characterization of uncultivated magnetotactic bacteria from various aquatic environments. Applied and Environmental Microbiology, 2005, 71(5): 2723-2731.
[28]Westbroek P, de Jong E W. Biomineralization and Biological Metal Accumulation: Biological and Geological Perspectives Papers Presented at the Fourth International Symposium on Biomineralization, Renesse, The Netherlands, June 2-5, 1982. Berlin: Springer, 1983.
[29]Winklhofer M. Magnetoreception. Journal of the Royal Society Interface, 2010, 7(Suppl 2): S131-S134.
[30]Schüler D. Genetics and cell biology of magnetosome formation in magnetotactic bacteria. FEMS Microbiology Reviews, 2008, 32(4): 654-672.
[31]Lin W, Bazylinski D A, Xiao T, Wu L F, Pan Y X. Life with compass: diversity and biogeography of magnetotactic bacteria. Environmental Microbiology, 2013, 16(9): 2646-2658, doi: 10.1111/1462-2920.12313.
[32]Martins J L, Silveira T S, Abreu F, Silva K T, Silva-Neto D, Inácio D, Lins U. Grazing protozoa and magnetosome dissolution in magnetotactic bacteria. Environmental Microbiology, 2007, 9(11): 2775-2781.
[33]Heyen U, Schüler D. Growth and magnetosome formation by microaerophilicMagnetospirillumstrains in an oxygen-controlled fermentor. Applied Microbiology and Biotechnology, 2003, 61(5/6): 536-544.
[34]張蕊, 陳一然, 周克, 張文燕, 肖天, 吳龍飛. 青島潮間帶趨磁細菌的垂直分布特征及與環(huán)境因子的關系. 海洋科學, 2013, 37(10): 24-31.
[35]趙鐵虎, 李春, 叢鴻文, 褚宏憲. 青島近岸海區(qū)海底地貌類型及聲學特征. 海洋測繪, 2005, 25(1): 40-43.
[36]孫靜. 青島市海灘沉積地貌及質量評價[D]. 青島: 中國海洋大學, 2012.
[37]Fuduche M, Postec A, Davidson S, Chauvin J P, Galès G, Hirschler-Réa A, Olivier B, Wu L F, Pradel N. Diversity of Magnetotactic Bacteria from a French Pristine Mediterranean Area. Current Microbiology, 2014, 70(4): 499-505.
[38]Postec A, Tapia N, Bernadac A, Joseph M, Davidson S, Wu L F, Olivier B, Pradel N. Magnetotactic bacteria in microcosms originating from the French Mediterranean coast subjected to oil industry activities. Microbial Ecology, 2012, 63(1): 1-11.
Diversity of magnetotactic bacteria in the intertidal zone of Taiping Bay, Qingdao
XU Cong1,2,ZHANG Wenyan1,CHEN Yiran1,ZHANG Rui1,DONG Yi1,DU Haijian1,2,PAN Hongmiao1,XIAO Tian1,*
1KeyLaboratoryofMarineEcologyandEnvironmentalSciences,InstituteofOceanology,ChineseAcademyofSciences,Qingdao266071,China2UniversityofChineseAcademyofSciences,Beijing100049,China
Magnetotactic bacteria (MTB) are gram-negative motile prokaryotes that produce magnetosomes and can orient and migrate along the geomagnetic lines of force. They are ubiquitous in sediments and stratified water columns, distributed predominantly in the oxic-anoxic transition zone (OATZ). MTB comprise several morphological types, including cocci, rods, vibrios, spirilla, and multicellular magnetotactic prokaryotes. Usually, cocci are the dominant morphology. Variable phylogenetic relatedness of MTB has been confirmed on the basis of 16S rRNA genes. MTB can biomineralize iron oxide and/or iron sulfide magnetosomes. In most MTB, magnetosomes are organized in chain(s). In this study, we found a certain amount of MTB in the intertidal zone of Taiping Bay, Qingdao City, where the maximum abundance reaches up to 350 ind./cm3. Transmission electron microscopy revealed that all the MTB were magnetotactic cocci, with a size of (2.23 ± 0.69) μm × (1.84±0.47) μm and the width/length ratio of 0.87±0.10 (n=21). Fifty-four percent of the MTB contained two chains of magnetosomes, eighteen percent with one chain, fourteen percent with more than two chains and fourteen percent with cluster. All of these magnetosomes were prismatic mineral crystals. There were 7—43 (mean=18,n=21) magnetosomes in a cell and the volume of magnetosomes varied between 2.71 × 105nm3and 1.63 × 106nm3. Assuming that all the magnetosomes were magnetite, the percentage of Fe in MTB was 0.40%—6.91% (average 2.19%) and per square kilometer of intertidal zone produced 1.92 kg magnetite every year. This suggests that MTB may play an important role in the iron biogeochemical cycle in this area. Additionally, according to the statistics for each magnetotactic bacterium, we observed that the mass of magnetosome increased with the increasing mass of MTB (r=0.637,P=0.002 < 0.05). Phylogenetic analysis based on 16S rRNA gene sequences revealed that 47 sequences of MTB belonged to 13 OTUs (XCQD1-18, 1-2, 1-19, 51, 81, 6, 2-2, 4-20, 34, 2-23, 53, 130, 1-21) and affiliated toAlphaproteobacteria. OTU XCQD1-18 containing 23 sequences had the highest dominance index (48.93%). In addition, 6, 5, 2, 2, 2 sequences belonged to OTU XCQD2-2, XCQD1-21, XCQD1-2, XCQD2-23, XCQD130, respectively. The other OTUs had only one sequence. Shannon′s diversity indexH′ of MTB in Taiping Bay was 2.64nit, and Species Evenness J′ was 0.71. Nine OTUs (XCQD 1-18, 1-2, 1-19, 81, 6, 4-20, 34, 2-23, 53) shared less than 97% 16S rRNA gene sequence similarity with the nearest known sequences, in which, five OTUs (XCQD 81, 4-20, 4, 2-23, 53) shared lower than 93%. It suggested that they represented 9 new species and 5 novel genera. Our results indicate that there were substantial potential microorganism resources in Taiping Bay. Compared to the MTB community in Huiquan Bay, a bay adjacent to Taiping Bay, here were two MTB OTUs discovered both in Taiping Bay and Huiquan Bay. Two MTB OTUs were shared. Jaccard similarity coefficient was 0.1053, indicating that although the two bays were close to each other, the MTB communities showed great differences. Comparison of the features of MTB in Taiping Bay with two French Mediterranean coasts, Six-Fours-les-Plages and Gulf of Fos, revealed that environmental factors may have a great influence on the diversity of MTB. It is assumed that community succession, seasonal variation, and environmental distinction may contribute to the low similarity between the MTB communities and features of different intertidal areas. Our results imply that further investigation on MTB in terms of their diversity is required.
intertidal; magnetotactic bacteria; diversity; magnetosome; 16S rRNA gene
國家自然科學基金項目(41276170, 41206150, 41330962);國際海域資源調查與開發(fā)“十二五”項目(深海(微)生物資源勘探與資源潛力評價)(任務書編號:DY125-15-R-03)
2014-12-01; 網絡出版日期:2015-10-30
Corresponding author.E-mail: txiao@qdio.ac.cn
10.5846/stxb201412012380
徐叢,張文燕,陳一然,張蕊,董逸,杜海艦,潘紅苗,肖天.青島太平灣潮間帶趨磁細菌多樣性.生態(tài)學報,2016,36(14):4346-4354.
Xu C,Zhang W Y,Chen Y R,Zhang R,Dong Y,Du H J,Pan H M,Xiao T.Diversity of magnetotactic bacteria in the intertidal zone of Taiping Bay, Qingdao.Acta Ecologica Sinica,2016,36(14):4346-4354.