Smith C J
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick, Maryland 21701.
J Bacteriol. 1987 Oct;169(10):4589-96. doi: 10.1128/jb.169.10.4589-4596.1987.
The Bacteroides pBI136 clindamycin resistance (Ccr) determinant from the composite transposon Tn4551 was cloned onto the shuttle plasmid pFD160, and the regions necessary for expression in Bacteroides fragilis were determined. These results suggested that transcriptional regulatory signals required for Ccr were located in the Tn4551 direct repeat sequence (DRS) adjacent to the resistance determinant. Analysis of the nucleotide sequence of this region revealed that the Ccr structural gene, 798 base pairs (bp), was located 17 bp from the terminus of the DRS and that this gene (ermFS) differed from ermF (pBF4) by one amino acid. The DRS element was found to be 1,155 bp and appeared to contain the ermFS transcription start signals. The DRS structure was typical of insertion sequence elements isolated from other bacterial species, and its termini were characterized by 25-bp regions of imperfect dyad symmetry. The DRS was dominated by a 978-bp open reading frame, which terminated in the left inverted repeat 27 bp from the ermFS start codon, and weak amino acid sequence homology was observed with the putative transposase of IS3. Promoter activity of the DRS in B. fragilis was demonstrated by in vitro construction of operon fusions with a promoterless ermFS gene followed by transformation of the recombinant plasmids with selection for resistance to clindamycin. The location of one DRS promoter was identified by using the ermFS fusions and then verified by in vitro mutagenesis of the site with single-stranded linkers. Northern blot (RNA blot) analysis of total RNA from B. fragilis strains containing pBI136 or ermFS recombinant plasmids confirmed the location of this promoter and indicated that it was used in vivo by Tn4551. A second DRS promoter, which activated ermFS transcription by readthrough of the large DRS open reading frame, was also identified by the Northern blot analysis. The bicistronic ermFS message was not observed in strains containing a complete copy of Tn4551, and the possibility of transcriptional regulation is discussed.
来自复合转座子Tn4551的脆弱拟杆菌pBI136克林霉素抗性（Ccr）决定簇被克隆到穿梭质粒pFD160上，并确定了在脆弱拟杆菌中表达所需的区域。这些结果表明，Ccr所需的转录调控信号位于与抗性决定簇相邻的Tn4551直接重复序列（DRS）中。对该区域核苷酸序列的分析表明，Ccr结构基因长798个碱基对（bp），位于DRS末端17 bp处，该基因（ermFS）与ermF（pBF4）仅相差一个氨基酸。发现DRS元件长1155 bp，似乎包含ermFS转录起始信号。DRS结构是从其他细菌物种中分离出的插入序列元件的典型结构，其末端的特征是有25 bp的不完全二重对称区域。DRS主要由一个978 bp的开放阅读框组成，该阅读框在左反向重复序列中终止，距ermFS起始密码子27 bp，并且与IS3的推定转座酶存在弱氨基酸序列同源性。通过将无启动子的ermFS基因与操纵子进行体外融合构建，然后将重组质粒转化并选择对克林霉素的抗性，证明了DRS在脆弱拟杆菌中的启动子活性。通过使用ermFS融合体鉴定了一个DRS启动子的位置，然后通过用单链接头对该位点进行体外诱变进行了验证。对含有pBI136或ermFS重组质粒的脆弱拟杆菌菌株的总RNA进行Northern印迹（RNA印迹）分析，证实了该启动子的位置，并表明Tn4551在体内使用该启动子。Northern印迹分析还鉴定了第二个DRS启动子，该启动子通过大DRS开放阅读框的通读激活ermFS转录。在含有完整Tn4551拷贝的菌株中未观察到双顺反子ermFS信息，并讨论了转录调控的可能性。
