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

Abstract

BackgroundThe mce operons play an important role in the entry of M. tuberculosis into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of mce mutants. Recently, mce1 operon was found to extend over 13 genes, fadD5 (Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to mce1 among the four mce operons of M.tuberculosis. We have examined the function of this region.ResultsWe predicted putative promoter activity of the 200 base pairs of non-coding, intergenic region between Rv0166 and Rv0167 in silico using MEME software and designate it as intergenic promoter, IGPr. We demonstrate both promoter activity and a putative negative regulatory function of this fragment by reporter assays carried out in the surrogate host M.smegmatis. We find that the repressive elements not only control the native promoter but also repress a heterologous promoter of M.smegmatis. The higher activity of the intergenic promoter in a clinical isolate in comparison with the wild type sequence from M.tuberculosis H37Rv could be correlated with a point mutation within the negative element. We have mapped two transcription start sites for mce1 operon both of which are utilized in M.tuberculosis H37Rv as well as the clinical isolate VPCI591. Our studies show that the promoter activity in the non-coding region is relevant not only in reporter gene expression but also in the expression of mce1 operon in M. tuberculosis cells grown in synthetic medium.ConclusionThe mce operon of M.tuberculosis H37Rv potentially can be transcribed from two promoters P1 and P2, former mapping upstream of Rv0166 and the latter in the non-coding intergenic region between Rv0166 and Rv0167. The transcription initiation from P1 results in a transcript with Rv0166 while that from P2 will be without it. The sequences between the translation start site of Rv0167 and the promoter P2 have a negative regulatory role, as point mutation within the sequence leads to enhanced activity of P2 as well as a heterologous promoter from M.smegmatis. The mutation detected in the clinical isolate VPCI591 therefore behaves like a gain-of-function mutation.

Correlation of drug resistance with Single Nucleotide Variations through genome analysis and experimental validation in a multi-drug resistant clinical isolate ofM.tuberculosis

“…There are several intergenic regions that are flanked by divergently transcribed genes. The presence of promoters or regulatory regions within such intergenic non-coding regions are known in M.tuberculosis (40). Thus intergenic SNVs can affect the transcription/regulation of the downstream gene.…”

“…A number of intergenic variations lie within the known promoter/regulatory regions. Intergenic variation in the cis-regulatory sequences of mce1 operon between Rv0166 (fadD5) and Rv0167 (yrbE1A) were identified(40). SNV was identified within IS 1560 elements and mutation hotspot for anti-tuberculosis drug ethambutol, between embC and embAB gene.…”

“…The directed polymorphism analysis of the mce 1 operons in VPCI591, led to the identification of gain of function SNV at 196800 (G>C) in the intergenic region between Rv0166-Rv0167 (40). In the light of this, the whole genome sequencing and analysis was taken up to map the non-synonymous SNVs in VPCI591.…”

Genome-Wide Identification of Mycobacterium tuberculosis Exported Proteins with Roles in Intracellular Growth

“…White triangles mark TnЈbla TEM-1 insertion mutants that were not attenuated. The mce1 operon has two possible start sites and spans from either fadD5 or yrbE1A to mas1D (12,20). The mce2 and mce4 operons are transcribed in a single transcript comprised of the genes shown (11,24).…”

Detecting Operons in Bacterial Genomes via Visual Representation Learning

“…Since operons are often formed by genes that are related functionally or metabolically, predicting operons could help us predict higher levels of gene organization and regulatory networks 1,2,[8][9][10][11]14 . Such predictions could help annotate gene functions 15 , aid in drug development 16 , and antibiotic resistance 17 . Different methods use different features of operons to make their predictions.…”