Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, Virginia, USA.
Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani, Thailand.
J Bacteriol. 2014 Aug;196(15):2762-74. doi: 10.1128/JB.01693-14. Epub 2014 May 16.
Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5' endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.
铁是淋病奈瑟菌生存和感染建立所必需的营养物质。奈瑟菌转铁蛋白结合蛋白（Tbps）构成了从人转铁蛋白中获取铁的双组分系统。TbpA 是完成铁内化的 TonB 依赖性转运体。TbpB 是一种表面暴露的脂蛋白，可使铁摄取过程更有效。先前的研究表明，编码这些蛋白质的基因排列在双顺反子操纵子中，tbpB 基因位于 tbpA 的上游，由反向重复序列分隔。该操纵子受铁摄取调节因子（Fur）控制；然而，对于基因的调节表达所需的启动子元件尚未通过实验来定义。在这项研究中，通过诱变鉴定并证实了假定的调控基序。对这些启动子/操纵子基序上游序列的进一步检查导致了几个新的重复序列的鉴定。我们假设这些重复序列参与了操纵子的额外调控。在已鉴定的启动子区域上游的区域进行插入诱变，导致 tbpB 和 tbpA 转录本水平降低，但 TbpA 和 TbpB 的蛋白水平升高。使用 RNA 测序（RNA-Seq）技术，我们确定从 tbpB 上游区域产生了长 RNA。通过定性 RT-PCR，我们将该转录本的 5'末端定位在两个上游插入之间。我们提出，该上游 RNA 的表达导致 tbpBA 操纵子内基因产物的优化表达。
