Jeters Robert T, Wang Gui-Rong, Moon Kyung, Shoemaker Nadja B, Salyers Abigail A
Department of Microbiology, 601 S. Goodwin Ave., Rm. B103, University of Illinois, Urbana, IL 61801.
J Bacteriol. 2009 Oct;191(20):6374-82. doi: 10.1128/JB.00739-09. Epub 2009 Aug 21.
Many human colonic Bacteroides spp. harbor a conjugative transposon, CTnDOT, which carries two antibiotic resistance genes, tetQ and ermF. A distinctive feature of CTnDOT is that its excision and transfer are stimulated by tetracycline. Regulation of the genes responsible for excision has been described previously. We provide here the first characterization of the regulation of CTnDOT transfer (tra) genes. Reverse transcription-PCR analysis of the region containing the tra genes showed that these genes are regulated at the transcriptional level. Surprisingly, increased production of tra gene mRNA in tetracycline-stimulated cells was mediated by the proteins encoded by the excision genes. Previous studies have shown that expression of the excision gene operon is controlled by the regulatory protein RteC. Accordingly, it was possible that RteC was also regulating tra gene expression and that the excision proteins were only accessory proteins. However, placing the excision gene operon under the control of a heterologous promoter showed that the excision proteins alone could activate tra gene expression and that RteC was not directly involved. We also found a second level of tra gene control. The transfer of CTnDOT was inhibited by a DNA segment that included only a portion of the 3' end of one of the excision genes (exc). This segment contained a small open reading frame, rteR. By replacing the codons encoding the first two amino acids of the putative protein product of this open reading frame with stop codons, we showed that the rteR gene might encode a small regulatory RNA. RteR acted in trans to reduce the number of tra transcripts in a way that was independent of the excision proteins. The repressive effect of RteR was not the result of decreased stability of the tra mRNA. Instead, RteR appears to be modulating the level of tra gene expression in some more direct fashion. The complex regulatory system that controls and links the expression of CTnDOT excision and transfer genes may be designed to ensure stable maintenance of CTnDOT in nature by reducing the fitness toll it takes on the cell that harbors it.
许多人结肠拟杆菌属菌种都携带一种接合转座子CTnDOT，它带有两个抗生素抗性基因tetQ和ermF。CTnDOT的一个显著特征是其切除和转移受四环素刺激。此前已对负责切除的基因调控进行过描述。我们在此首次对CTnDOT转移（tra）基因的调控进行了表征。对包含tra基因区域的逆转录PCR分析表明，这些基因在转录水平受到调控。令人惊讶的是，四环素刺激细胞中tra基因mRNA产量的增加是由切除基因编码的蛋白质介导的。先前的研究表明，切除基因操纵子的表达受调控蛋白RteC控制。因此，有可能RteC也在调控tra基因表达，而切除蛋白只是辅助蛋白。然而，将切除基因操纵子置于异源启动子控制下表明，仅切除蛋白就能激活tra基因表达，且RteC不直接参与其中。我们还发现了tra基因控制的第二个层面。CTnDOT的转移受到一个DNA片段的抑制，该片段仅包含一个切除基因（exc）3'端的一部分。这个片段包含一个小的开放阅读框rteR。通过将该开放阅读框假定蛋白质产物前两个氨基酸的编码密码子替换为终止密码子，我们表明rteR基因可能编码一种小的调控RNA。RteR以反式作用减少tra转录本数量，其方式独立于切除蛋白。RteR的抑制作用不是tra mRNA稳定性降低的结果。相反，RteR似乎以某种更直接的方式调节tra基因表达水平。控制并连接CTnDOT切除和转移基因表达的复杂调控系统可能是为了通过降低其对宿主细胞适应性的损害，确保CTnDOT在自然界中的稳定维持。
