Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete
Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete
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DOI：
10.1046/j.1365-2958.2002.02758.x
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发表时间：
2002-01-01
影响因子：
3.6
通讯作者：
Radolf, JD
中科院分区：
文献类型：
--
作者：
Eggers, CH;Caimano, MJ;Radolf, JD
伯氏疏螺旋体的32 kb环状质粒（cp32）家族一直是深入研究的主题，因为其成员编码许多差异表达的脂蛋白。多达9种不同的cp32似乎能够在单个螺旋体中稳定复制。在这里，我们证明了一个含有4 kb片段的构建体（pCE310），该片段来自伯氏疏螺旋体CA-11.2A cp32的假定维持区，能够在高传代伯氏疏螺旋体B31和强毒伯氏疏螺旋体297中自主复制。缺失分析表明，只有旁系家族57的成员和邻近的非编码片段是复制所必需的。pCE310插入片段编码的PF32同源物与B31和297 cp32-3质粒上编码的PF32同源物几乎相同。cp32-3在携带pCE310的B31和297转化子中被选择性删除的发现，证明了PF32蛋白对cp32相容性的重要性，并证实了表达相同PF32同源物的cp32质粒不相容的预测。利用含有CA-11.2A cp32质粒维持区的穿梭载体，将绿色、黄色和青色荧光蛋白报告基因导入伯氏疏螺旋体。流式细胞术显示，绿色荧光蛋白在几乎90%的无毒和感染性转化体中都能很好地表达。除了增强我们对伯氏疏螺旋体质粒生物学的理解外，我们的研究结果还进一步发展了用于剖析莱姆病致病机制的遗传系统。
The 32 kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA-11.2A cp32 was capable of autonomous replication in both high-passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non-coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32-3 plasmids. The finding that cp32-3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA-11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.