Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The mce operons are crucial for Mycobacterium tuberculosis entry into host cells.
• The mce1 operon, comprising over 13 genes including fadD5 (Rv0166), has a unique non-coding region between Rv0166 and Rv0167.

Purpose of the Study:

• To investigate the function of the non-coding region between Rv0166 and Rv0167 in the mce1 operon.
• To determine the regulatory role of this intergenic sequence on mce1 operon transcription.

Main Methods:

• In silico prediction of promoter activity using MEME software.
• Reporter assays in a surrogate host (M. smegmatis) to assess promoter function and regulation.
• Mapping of transcription start sites in M. tuberculosis clinical isolates and wild-type strains.

Main Results:

• The intergenic region (IGPr) exhibits promoter activity and a negative regulatory function.
• This regulatory element represses both the native mce1 promoter and a heterologous promoter.
• A point mutation in the negative regulatory element of a clinical isolate (VPCI591) leads to enhanced promoter activity.
• Two transcription start sites for the mce1 operon were identified and are utilized in both wild-type and clinical isolates.

Conclusions:

• The mce1 operon can be transcribed from two promoters (P1 and P2), with P2 located in the intergenic region.
• The intergenic region contains a negative regulatory element that influences P2 activity.
• A mutation in this regulatory element acts as a gain-of-function, increasing P2 activity and potentially affecting M. tuberculosis pathogenesis.