Transcriptional analysis of the 5' terminus of the flp fimbrial gene cluster from Actinobacillus actinomycetemcomitans.
Transcriptional analysis of the 5' terminus of the flp fimbrial gene cluster from Actinobacillus actinomycetemcomitans.
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来自放线放线杆菌的 flp 菌毛基因簇 5 末端的转录分析。
DOI：
10.1099/mic.0.25786-0
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Scannapieco,FrankA
中科院分区：
文献类型：
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作者：
Haase,ElaineM;Stream,JoshuaO;Scannapieco,FrankA
口腔病原菌新鲜分离物放线放线杆菌伴放线菌放线菌伴放线菌放线菌伴放线菌有证据表明，菌毛亚基基因flp是一组14个基因（flptotadG）的一部分，这些基因被认为编码参与这些菌毛合成、组装和输出的蛋白质。为了确定该基因簇5′端的转录组织，从A.用RT-PCR方法对放线菌伴生菌株283进行了分析。设计引物扩增跨越基因连接点或多个基因的区域，在每个单独的基因连接点产生扩增子，从flptotadD的粗糙和光滑的变体。半定量RT-PCR的thercpAtotadZamplicon显示，显着更多的mRNA转录从粗糙比光滑的变体。也检测到较长的扩增子，包括flptotadZ（3.9 kb）和tadAtotadD（2.1 kb），但仅来自粗糙变体。 用cDNA末端快速扩增法（RACE）鉴定含有flp基因的mRNA的5′端。位于rcpC或fBandflp-2内的反义引物能够扩增RACE产物，随后分离并亚克隆到pGEM-T中。DNA测序表明，mRNA的5′端位于offlp上游的G或T核苷酸的−102至−101 nt。相应的σ 70共有序列位于转录起始位点的−10（TATAAT）和−35（TTGCAT）处。这些数据证实，flp基因簇是一个操纵子转录的多顺反子的信息开始从一个G或T核苷酸位于基因间区上游oflp。利用转化大肠杆菌的alacZ报告基因构建体证实了flpupstream区的启动子功能。RT-PCR分析进一步表明，尽管转录确实发生在粗糙和光滑变体中，但全长转录物在光滑变体中迅速降解或显著下调。
Fresh isolates of the oral bacterial pathogenActinobacillus actinomycetemcomitansexhibit a fimbriated, rough colony phenotype. Evidence suggests that the fimbrial subunit geneflpis part of a cluster of 14 genes (flptotadG) thought to encode proteins involved in the synthesis, assembly and export of these fimbriae. To determine the transcriptional organization of the 5′ terminus of this gene cluster, total RNA from rough and smooth phenotype variants ofA. actinomycetemcomitansstrain 283 were analysed by RT-PCR. Primers designed to amplify regions spanning gene junctions or multiple genes yielded amplicons at each individual gene junction fromflptotadDfor both the rough and smooth variants. Semi-quantitative RT-PCR of thercpAtotadZamplicon revealed that significantly more mRNA was transcribed from the rough than the smooth variant. Longer amplicons encompassingflptotadZ(3·9 kb) andtadAtotadD(2·1 kb) were also detected, but only from the rough variant. Rapid amplification of cDNA ends (RACE) was used to identify the 5′ end of the mRNA containingflp. Antisense primers located withinrcpC,orfBandflp-2enabled amplification of a RACE product that was subsequently isolated and subcloned into pGEM-T. DNA sequencing indicated that the 5′ end of the mRNA was located at a G or T nucleotide −102 to −101 nt upstream offlp. Corresponding σ70consensus sequences were located at −10 (TATAAT) and −35 (TTGCAT) relative to the transcription start site. These data confirm that theflpgene cluster is an operon transcribed as a polycistronic message commencing from a G or T nucleotide located in the intergenic region upstream offlp. Promoter function of theflpupstream region was confirmed using alacZreporter gene construct transformed intoEscherichia coli. RT-PCR analysis further suggests that although transcription does occur in both the rough and smooth variants, full-length transcripts are rapidly degraded or are significantly downregulated in the smooth variant.