School of Biological Sciences, The University of Hong Kong, Kadoorie Biological Sciences Building, Pofulam Road, Hong Kong SAR, P. R. China.
J Microbiol Biotechnol. 2010 Aug;20(8):1163-78. doi: 10.4014/jmb.1003.03007.
Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of 7 Vibrio plasmid replicons. Comparative sequences analysis of the 7 Vibrio plasmid replicons obtained in this study together with 8 published Vibrionaceae plasmid sequences revealed replication participating elements involved in the ColE1-mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation and, as a result the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids has been demonstrated by direct sequencing analysis and southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNAI species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing basis for rapid characterization and classification of Vibrionaceae plasmids.
质粒在弧菌科细菌的水平基因转移中发挥着重要作用，但令人惊讶的是，人们对它们的复制和不相容性系统知之甚少。在本研究中，我们成功开发了一种基于生物信息学的实验鉴定 7 种弧菌质粒复制子的策略。对本研究获得的 7 种弧菌质粒复制子与 8 个已发表的弧菌科质粒序列的比较序列分析表明，参与 ColE1 复制起始和调控模式的复制参与元件。与质粒 ColE1 一样，这些弧菌科质粒编码两种 RNA 种类（引物 RNA 和反义 RNA）用于复制起始和调控，因此这 15 个弧菌科质粒被指定为 ColE1 样弧菌科（CLV）质粒。基于复制子组织的比较和复制区的系统发育分析，对这 15 个 CLV 质粒进行分组，得到了两个亚组。通过直接测序分析和 Southern 杂交证实了 CLV 质粒的共存，强烈表明 CLV 质粒的不相容性主要由类似于 ColE1 样质粒的 RNAI 种类决定。在 CLV 质粒上也鉴定出了类似于保守 Xer 重组位点的序列，表明 CLV 质粒可能像 ColE1 样质粒一样，利用宿主特异性重组系统进行多聚体分辨率。所有结果表明，这 15 个质粒形成了一个新的 ColE1 样群，为快速鉴定和分类弧菌科质粒提供了依据。
