大西洋洋中脊深海多环芳烃降解菌群的优势菌分析

摘要：【目的】为了分析大西洋洋中脊深海海水及表层沉积物中多环芳烃(PAHs)降解菌群中的优势菌。【方法】采用富集培养法和平板涂布法从深海样品中分离可培养细菌及PAHs降解菌。通过16S rRNA基因测序完成系统发育分析。采用变性梯度凝胶电泳（DGGE）及DNA测序分析降解菌群中的优势菌。【结果】总共分离到16株细菌,包括一株PAHs降解菌Novosphingobium sp. 4D。系统发育分析发现,可培养细菌中两个最大的类群分别与Alcanivorax dieselolei NO1A（5/16）和Tistrella mobilis TISTR 1108T（5/16）亲缘关系最近。DGGE结果表明,在菌群MC2D中菌株4L（以及4M、4N, Alcanivorax dieselolei NO1A, 99.21％）、4D（Novosphingobium pentaromativorans US6-1T,97.07％）和4B（以及4E、4H、4K,Tistrella mobilis TISTR 1108T,＞99％）是降解菌群中的优势菌。而降解菌群MC3CO中的优势菌是菌株5C（以及5H,Alcanivorax dieselolei NO1A,＞99％）、条带5-8代表的未培养菌株（Novosphingobium aromaticivorans DSM 12444T,99.41%）、5J（Tistrella mobilis TISTR 1108T,99.52％）和5F（以及5G,Thalassospira lucentensis DSM 14000T,＜97％）。【结论】本研究发现在大西洋洋中脊深海海水及表层沉积物中Alcanivorax、Novosphingobium、Thalassospira、Tistrella属的细菌是PAHs降解菌群中的优势菌,其中的主要降解菌是Novosphingobium属的细菌。     

几株新鞘氨醇杆菌的趋化性及其相关基因组成特点

【目的】选择了几株隶属于新鞘氨醇杆菌属(Novosphingobium)的有和没有多环芳烃(PAHs)降解能力的细菌,在基因组水平比较分析它们的趋化通路,并探究其中一些菌株对芳香化合物和三羧酸(TCA)循环中间代谢产物的趋化性。【方法】通过基因组的比较分析,研究了几株新鞘氨醇杆菌的趋化蛋白组成和趋化基因分布,并采用滴定法和游动平板法检测了相关菌对芳香族化合物和TCA循环中间代谢产物的趋化性。【结果】N.pentaromativorans US6-1、N.pentaromativorans F2、Novosphingobium indicum K13、Novosphingobium stygium DSM 12445、Novosphingobium sp.C2AC等5株新鞘氨醇杆菌对芳香族化合物和TCA循环中间代谢物具有不同程度的趋化性;所选的7株已完成基因组测序的新鞘氨醇杆菌N.pentaromativorans F2、N.pentaromativorans US6-1、Novosphingobium sp.PP1Y、Novosphingobium sp.AP12、Novosphingobium sp.Rr 2-17,Novosphingobium sp.B-7和Novosphingobium sp.DSM 19370均含有趋化蛋白MCP、CheW、CheA、CheB、CheR和CheY,且亲缘关系最近的US6-1、F2和PP1Y 3株菌的che基因簇中的基因排列一致,为che W-Y-D-B-R-A-(X);新鞘氨醇杆菌的趋化系统属Fla类型。【结论】几株新鞘氨醇杆菌都具有较为完整的趋化通路,且对多种芳烃及其代谢物的趋化性各不相同,其中菌株US6-1趋化现象最明显。     

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern

Background

Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome.

Results

PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y’s phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism.

Conclusions

In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-384) contains supplementary material, which is available to authorized users.     

一株深海热液环境来源的PAHs降解菌TVG9-Ⅶ的系统发育与降解基因

【目的】对一株深海热液环境来源的多环芳烃(PAHs)降解菌进行系统发育分析并对其降解特性和降解机制进行研究。【方法】对16S rRNA基因进行扩增和测序,进行基于16S rRNA基因序列的系统发育分析;利用GC-MS测定其对PAHs的降解率;通过构建基因组Fosmid文库,克隆PAHs降解基因簇;并利用RT-PCR和qPCR研究关键降解酶基因在不同PAHs诱导下的表达情况。【结果】从西南太平洋劳盆地热液沉积物中分离到一株PAHs降解菌株TVG9-Ⅶ,系统发育分析结果表明,该菌株属于新鞘氨醇杆菌属(Novosphingobium),与该属的Novosphingobium indicum H25T系统发育关系最为密切,它们的16S rRNA基因序列相似性高达99.7%。该菌株在21 d内对菲、荧蒽和芘的降解率分别为95.2%,57.3%和69.6%。从Fosmid文库中筛选得到一个负责PAHs降解的上游基因簇,包含了PAHs起始降解双加氧酶大小亚基(pheA1a/b)基因和一个脱氢酶基因;RT-PCR和qPCR实验表明,双加氧酶大亚基基因pheA1a在菲的诱导下上调表达4.2倍,而在萘及高环荧蒽和芘的诱导下无上调。【结论】菌株TVG9-Ⅶ是Novosphingobium属深海热液来源的PAHs降解菌,具有良好的降解特性,特别是对高环PAHs的降解效果较好。     

深海多环芳烃降解菌新鞘氨醇杆菌H25的降解特性及降解基因

[目的]为了从深海环境中筛选新的多环芳烃降解菌,了解其降解基因及降解特性.[方法]以原油作为碳源从印度洋深海海水样品中富集筛选出降解能力较强的多环芳烃降解菌,并根据已报道的相关菌属的多环芳烃起始双加氧酶大亚基序列及侧翼序列设计兼并引物进行扩增.[结果]获得了1株能够高效降解原油、柴油及多种多环芳烃的菌株H25.经16S rDNA序列系统发育分析表明它属于新鞘氨醇杆菌属(Novosphingobium)(96%).并从该菌株中扩增获得2条相似度为91.0%双加氧酶基因片段.2条序列在NCBI上Blastn分析表明均与菌株N.aromaticivorans DSM12444T的降解质粒pNL1上的双加氧酶大亚基具有最高相似度,分别为99.6%和91.0%.根据pNL1上的双加氧酶序列设计引物获得了包含H25双加氧酶大亚基及上下游序列的2个基因片段H25 Ⅰ(2.9kb)和H25Ⅱ(4.5kb).另外,单碳降解实验表明H25对联苯、2-甲基萘、2,6-二甲基萘、菲、二苯并噻吩、二苯并呋喃等均有较好的降解能力.[结论]H25菌株是Novosphingobium属可能的新种.深海细菌在大洋环境多环芳烃污染的自然净化中起到一定作用,并在环境生物修复中有较大的应用前景.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Characterization of Two Compatible Small Plasmids from Sphingobium yanoikuyae

We isolated and characterized two small cryptic indigenous plasmids, pYAN-1 (4,896 bp) and pYAN-2 (4,687 bp), from Sphingobium yanoikuyae, and developed a versatile system that permitted genetic manipulation of the genus Sphingomonas. Nucleotide sequencing of both plasmids revealed that they contained mobA, mobs, and repA genes, which are predicted to encode proteins associated with mobilization and replication, in common. Transformation with each plasmid harboring the antibiotic resistance gene by electroporation was fully successful, using Novosphingobium capsulatum as a host.     

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (k_cat = 39–91 s⁻¹) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity.