Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.
Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.
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DOI：
10.1186/s12864-016-2445-4
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发表时间：
2016-02-16
期刊：
影响因子：
4.4
通讯作者：
Lale R
中科院分区：
文献类型：
--
作者：
Moghadam MS;Albersmeier A;Winkler A;Cimmino L;Rise K;Hohmann-Marriott MF;Kalinowski J;Rückert C;Wentzel A;Lale R
海洋低温环境是新生物发现的嗜冷微生物生命的宝贵来源。建立了一个北极海洋细菌菌株收集库，由1448个来自挪威大陆北部巴伦支海不同深度的生物群、水和沉积物样品的单独分离株组成，全年海水温度为4 °C。整个收集进行高通量筛选检测胞外漆酶活性与愈创木酚作为底物。总共鉴定了13株漆酶阳性菌株，均属于嗜冷杆菌属。从最多样化的四个菌株中，基于16 S rRNA基因序列分析，所有菌株都源自相同的葡萄孢属种菌落海鞘被囊动物样品，分离基因组DNA，并使用全基因组鸟枪法和8 kb配对文库测序的组合方法在Illumina MiSeq平台上进行基因组测序。基因组进行组装，并显示基因组大小在3.29和3.52 Mbp之间，平均G + C含量约为42%，在四个菌株中存在一到七个质粒。进行基于生物信息学的基因组挖掘以描述这四种菌株的代谢潜力，并鉴定可能导致所观察到的漆酶阳性表型的候选基因。多达两个不同的漆酶样多铜氧化酶（LMCO）编码基因的候选人被确定在四个菌株中的每一个。P11 F6-LMCO和P11 G5-LMCO 2在大肠杆菌BL 21（DE 3）中的异源表达产生了具有2，2 '-连氮基-双-3-乙基苯并噻唑啉-6-磺酸（ABTS）和愈创木酚氧化活性的重组蛋白。从北极海洋细菌中分离到13种漆酶阳性的嗜冷菌。对其中4个分离株进行了基因组测序。除P11 G5含有7个质粒外，其整体基因组特征与其他公开可用的嗜冷杆菌基因组序列相似。然而，在途径水平上存在差异，因为仅在本研究中报道的嗜冷杆菌菌株中检测到与酚类化合物、尼古丁、苯丙氨酸、苯乙烯、苯丙氨酸和乙醇胺降解相关的基因，而在其他公开可用的嗜冷杆菌基因组中不存在这些基因。此外，通过基因组挖掘鉴定了6个候选基因，并显示具有T1、T2和T3铜结合位点作为三结构域漆酶的主要特征。P11 F6-LMCO和P11 G5-LMCO 2重组表达，并显示出活性时，ABTS和愈创木酚作为底物。本文的在线版本（doi：10.1186/s12864-016-2445-4）包含补充材料，可供授权用户使用。
Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42 %, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates. The online version of this article (doi:10.1186/s12864-016-2445-4) contains supplementary material, which is available to authorized users.