Mastropaolo Matthew D, Thorson Mary L, Stevens Ann M
Department of Biological Sciences, Virginia Tech, 219 Life Sciences 1, Washington Street, Blacksburg, VA 24061-0910, USA.
Microbiology (Reading). 2009 Aug;155(Pt 8):2683-2693. doi: 10.1099/mic.0.027748-0. Epub 2009 May 14.
There are barriers to cross-expression of genes between Bacteroides spp. and Escherichia coli. In this study, a lux-based reporter system was developed for Bacteroides and used to compare the promoter structure and function of a Bacteroides thetaiotaomicron 4001 (BT4001) 16S rRNA promoter with those of E. coli in vivo. Analysis of the BT4001 sequences upstream of the 16S rRNA gene revealed the same overall structure known for E. coli 16S rRNA promoters in that there were two promoters separated by approximately 150 bp. However, the BT4001 16S rRNA promoter contains the proposed Bacteroides -7 and -33 consensus sequences instead of the E. coli -10 and -35 consensus sequences. The biological activity of various configurations of the BT4001 16S rRNA promoter was analysed. Experiments pairing the BT4001 16S rRNA promoter with an E. coli RBS, and vice-versa, confirmed that gene expression between the two species is restricted at the level of transcription. In Bacteroides, a difference in translation initiation also appears to limit expression of foreign genes.
拟杆菌属和大肠杆菌之间存在基因交叉表达的障碍。在本研究中,开发了一种基于荧光素酶的拟杆菌报告系统,并用于在体内比较多形拟杆菌4001(BT4001)16S rRNA启动子与大肠杆菌启动子的结构和功能。对16S rRNA基因上游的BT4001序列分析显示,其总体结构与已知的大肠杆菌16S rRNA启动子相同,即有两个启动子,间隔约150 bp。然而,BT4001 16S rRNA启动子包含拟杆菌属提议的-7和-33共有序列,而非大肠杆菌的-10和-35共有序列。分析了BT4001 16S rRNA启动子各种构型的生物学活性。将BT4001 16S rRNA启动子与大肠杆菌核糖体结合位点(RBS)配对的实验,以及反之亦然的实验,证实了两个物种之间的基因表达在转录水平受到限制。在拟杆菌中,翻译起始的差异似乎也限制了外源基因的表达。