Laboratório de Genética Molecular de Microrganismos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
J Antimicrob Chemother. 2011 Apr;66(4):797-801. doi: 10.1093/jac/dkr011. Epub 2011 Feb 7.
Integrons are considered expression systems due to the presence of Pc promoters that drive gene cassette transcription. The role and configurations of Pc are well known in class 1 integrons; however, this region has not yet been identified in class 2 integrons. This study aimed to characterize the Pc promoter from class 2 integrons and to determine the effect of gene cassette position on transcription driven by this promoter.
The class 2 cassette arrays from Klebsiella pneumoniae and Vibrio cholerae strains were determined by sequencing. Transcription analyses were performed by real-time RT-PCR and relative quantification was carried out by comparing the transcripts of each normalized gene inserted in the integron to each other. The resistance profile was determined by the disc diffusion method. The class 2 Pc promoter was characterized by 5' rapid amplification of cDNA ends and promoter prediction programs.
Sequence analysis revealed the presence of the dfrA1-sat2-aadA1-ybeA and sat2-aadA1-ybeA arrangements in K. pneumoniae and V. cholerae strains, respectively. Real-time RT-PCR showed that the transcription of the first cassettes was higher than that of distal ones in wild-type and recombinant strains. All strains were resistant, indicating cassette expression. The Pc promoter of class 2 integrons (-35 TTTAAT |16 bp| -10 TAAAAT) was determined based on in silico analyses and on the transcription start site sequence of the class 2 integron cassette array.
The Pc from class 2 integrons was characterized for the first time and the cassette position effect on transcription was demonstrated.
整合子被认为是表达系统，因为存在 Pc 启动子驱动基因盒转录。Pc 在 1 类整合子中的作用和构型是众所周知的；然而，在 2 类整合子中尚未发现该区域。本研究旨在表征 2 类整合子的 Pc 启动子，并确定基因盒位置对该启动子驱动转录的影响。
通过测序确定肺炎克雷伯菌和霍乱弧菌菌株的 2 类盒阵列。通过实时 RT-PCR 进行转录分析，并通过比较整合子中每个归一化基因的转录本彼此之间进行相对定量。通过圆盘扩散法确定耐药谱。通过 5'快速扩增 cDNA 末端和启动子预测程序对 2 类 Pc 启动子进行了表征。
序列分析显示，肺炎克雷伯菌和霍乱弧菌菌株中分别存在 dfrA1-sat2-aadA1-ybeA 和 sat2-aadA1-ybeA 排列。实时 RT-PCR 显示，野生型和重组菌株中，第一个盒的转录高于远端盒的转录。所有菌株均具有抗性，表明存在盒表达。基于计算机分析和 2 类整合子盒阵列的转录起始位点序列，确定了 2 类整合子 Pc 启动子（-35 TTTAAT |16 bp| -10 TAAAAT）。
首次对 2 类整合子的 Pc 进行了表征，并证明了盒位置对转录的影响。
