Isolation and characterization of two novel plasmids from pathogenic Leptospira interrogans serogroup Canicola Serovar Canicola strain Gui44.
Isolation and characterization of two novel plasmids from pathogenic Leptospira interrogans serogroup Canicola Serovar Canicola strain Gui44.
复制标题
致病性问号钩端螺旋体血清群 Canicola Serovar Canicola 菌株 Gui44 中两种新型质粒的分离和表征。
DOI：
10.1371/journal.pntd.0003103
复制
发表时间：
2014-08
影响因子：
3.8
通讯作者：
Guo XK
中科院分区：
文献类型：
--
作者：
Zhu WN;Huang LL;Zeng LB;Zhuang XR;Chen CY;Wang YZ;Qin JH;Zhu YZ;Guo XK
先前的致病性钩端螺旋体基因组分析鉴定了两条环状染色体，但没有质粒。本研究的目的是研究审问l.s ananserovar Canicola Gui44菌株可能的染色体外成分。采用改良碱法从病原菌Gui44中分离到两个新的质粒pGui1和pGui2。采用Southern印迹法测定它们的存在和大小。然后利用454和Hiseq测序获得两个质粒的完整序列并进行分析。此外，利用实时定量PCR和下一代测序技术比较两个质粒与染色体的相对拷贝数。最后，在体外连续传代2年以上后，对菌株Gui44进行了重新测序，以评估两种质粒的稳定性。较大的质粒pGui1全长74,981碱基对（bp）， GC含量为34.63%，具有62个开放阅读框（orf）。较小的质粒pGui2长度为66,851 bp， GC含量为33.33%，含有63个orf。pGui1和pGui2编码的复制起始蛋白序列与LA1839（86%和88%）具有显著的相似性，LA1839是另一种致病性l .疑问菌血清Lai菌株Lai中已知的复制蛋白，表明其具有自主质粒复制的能力。经过2年多的体外连续传代，定量PCR和下一代测序证实了这两个质粒的单一拷贝和它们在菌株Gui44中的稳定存在。有趣的是，这两个质粒都含有大量的新基因（pGui1中有35个，pGui2中有52个）。本报告证实了犬科血清菌株Gui44中存在两个独立的环状质粒，为钩端螺旋体的基因组组织、适应、进化和发病机制提供了新的认识，这将有助于开发致病性钩端螺旋体的体内遗传操作系统。钩端螺旋体是钩端螺旋体病的病原体，钩端螺旋体病是最常见的动物向人类传播的疾病之一。先前对Lai和Copenhageni的基因组分析已经确定了大（4.33百万碱基）和小（350千碱基）圆形染色体的存在，但没有任何质粒的证据。由于缺乏可用的遗传工具，对钩端螺旋体及其致病性的详细了解被推迟。在本研究中，我们证实了在中国流行毒株桂44中存在两个新的质粒。在这两个质粒中发现的一些新基因可能在菌株的特征中起重要作用。这两个质粒将为了解钩端螺旋体基因组的多样性提供有用的信息，并显著提高我们对钩端螺旋体的进化和发病机制的认识。特别是，它将有助于致病性钩端螺旋体物种遗传操作系统的发展。
Previous genomic analysis of pathogenic Leptospira has identified two circular chromosomes but no plasmid. This study aims to investigate potential extrachromosomal elements of L.interrogans serovar Canicola strain Gui44. Two novel plasmids, pGui1 and pGui2, were isolated from the pathogenic strain Gui44, using a modified alkaline lysis method. Southern blotting was performed to determine the presence and size of them. Then, 454 and Hiseq sequencing were applied to obtain and analyze the complete sequences of the two plasmids. Furthermore, real-time quantitative PCR and next-generation sequencing were used to compare relative copy numbers of the two plasmids with that of the chromosomes. Finally, after serial passages in vitro for more than 2 years, the strain Gui44 was subsequently re-sequenced to estimate stability of the two plasmids. The larger plasmid, pGui1, 74,981 base pairs (bp) in length with GC content of 34.63%, possesses 62 open reading frames (ORFs). The smaller plasmid, pGui2, is 66,851 bp in length with GC content of 33.33%, and contains 63 ORFs. The replication initiation proteins encoded by pGui1 and pGui2 demonstrate significant sequence similarity with LA1839 (86% and 88%), a well-known replication protein in another pathogenic L.interrogans serovar Lai strain Lai, suggesting the ability for autonomous plasmid replication. Quantitative PCR and next-generation sequencing confirms a single copy of both plasmids and their stable presence in the strain Gui44 with in vitro serial passages after more than 2 years. Interestingly, the two plasmids both contain a significant number of novel genes (35 in pGui1 and 52 in pGui2). This report confirms the presence of two separate circular plasmids in serovar Canicola strain Gui44 and provides a new understanding of genomic organization, adaptation, evolution and pathogenesis of Leptospira, which will aid in the development of in vivo genetic manipulation systems in pathogenic Leptospira species. Leptospira species are the causative agent of leptospirosis, one of the most common animal to human transmitted diseases. Previous genomic analysis of L.interrogans serovar Lai and Copenhageni has identified the presence of large (4.33 mega base) and small (350 kilo base) circular chromosomes without evidence of any plasmids. Detailed understanding of Leptospira and its pathogenicity was delayed by the lack of available genetic tools. In this study we confirm the existence of two novel plasmids in L.interrogans serovar Canicola strain Gui44, an epidemic strain in China. Some novel genes identified in the two plasmids may play important roles in the characterization of the strain. The two plasmids will provide useful information in understanding the diversity of Leptospira genome and markedly improve our understanding of the evolution and pathogenesis of L.interrogans. In particular, it will contribute to the development of genetic manipulation systems in pathogenic Leptospira species.
登录
查看更多内容
腐生的瘦肉纤溶酶的基因组序列提供了对钩端螺旋体进化和钩端螺旋体病的发病机理的见解。
影响因子：
3.7
作者：
Picardeau, Mathieu;Bulach, Dieter M.;Bouchier, Christiane;Zuerner, Richard L.;Zidane, Nora;Wilson, Peter J.;Creno, Sophie;Kuczek, Elizabeth S.;Bommezzadri, Simona;Davis, John C.;McGrath, Annette;Johnson, Matthew J.;Boursaux-Eude, Caroline;Seemann, Torsten;Rouy, Zoe;Coppel, Ross L.;Rood, Julian I.;Lajus, Aurelie;Davies, John K.;Medigue, Claudine;Adler, Ben
通讯作者：
Adler, Ben
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
3.7
作者：
Lin YP;McDonough SP;Sharma Y;Chang YF
通讯作者：
Chang YF
DOI：
10.1073/pnas.1109285109
发表时间：
2012-01-24
影响因子：
11.1
作者：
Kung, Vanderlene L.;Khare, Sonal;Hauser, Alan R.
通讯作者：
Hauser, Alan R.
影响因子：
3.6
作者：
Matsunaga, J;Barocchi, MA;Albert, KI
通讯作者：
Albert, KI