I-TRAP: A method to identify transcriptional regulator activated promoters

To identify sigma factor-regulated genes, we developed a method, termed I-TRAP, for the identification of transcriptional regulator activated promoters. The I-TRAP method is based on the fact that some genes will be differentially expressed in the presence and absence of a transcriptional regulator. I-TRAP uses a DNA library in a promoter-trap vector that contains two reporter genes, one to allow the selection of active promoters in the presence of the transcriptional regulator and a second to allow screening for promoter activity in the absence of the transcriptional regulator.To illustrate the development and use of the I-TRAP approach, the construction of the vectors, host strains, and library necessary to identify SigmaE-regulated genes of Mycobacterium tuberculosis is described.The I-TRAP method should be a versatile and useful method for identifying and characterizing promoter activity under a variety of conditions and in response to various regulatory proteins. In our study, we isolated 360 clones that may contain plasmids carrying SigmaE-regulated promoters genes of M. tuberculosis.The ability of Mycobacterium tuberculosis bacteria to infect and replicate inside host mononuclear phagocytes is key to its ability to establish an infection and cause tuberculosis. To elucidate the mechanisms used by tubercle bacilli to survive in macrophages, researchers at several laboratories are studying mycobacterial genes specifically expressed during growth in macrophages [1-6]. Among the genes specifically expressed during intracellular growth are genes encoding two extracytoplasmic function sigma factors, SigE (σE) and SigH (σH) [3-5]. Sigma factors play critical roles in the recognition of promoters by RNA polymerase. In several systems, individual sigma factors are known to regulate the differential expression of sets of genes involved in specific processes, such as genes involved in the heat-shock response or those required for sporulation in Bacillus subtilis [7,8].I