Involvement of transcription elongation factor GreA in Mycobacterium tuberculosis viability, antibiotic susceptibility, and intracellular fitness

To understand how GreA may affect gene expression at the transcriptional level, we performed whole-transcriptome RNA-seq analyses. To examine whether GreA binds to specific chromosomal regions, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and then sought to verify the binding mode of GreA. ChIP-seq experiments were performed by using M. smegmatis strains producing GreA proteins fused with two repeats of the FLAG epitope (FLAG2). GreA-FLAG2-DNA nucleoprotein complexes fixed with formaldehyde in the log phase of growth were immunoprecipitated using magnetic beads. To exclude unspecific interactions with magnetic beads, we used a WT strain lacking the FLAG epitope as a negative control for our ChIP-seq experiments. Enriched regions were determined by comparison to the background noise level, which was estimated versus the input DNA of each ChIP-Seq replicate. Disrupting greA resulted in the differential regulation of 195 genes in M. smegmatis during the exponential growth. Among these, 28 were positively regulated and 167 were negatively regulated. The identified GreA-FLAG2 binding sites included 1005 ChIP-seq peaks that were confirmed in two biological replicates and absent in WT strain. Overall design: For RNA-seq: Examination of 2 strains (Wild type VS ?greA). For ChIP-seq: Examination of 2 strains (Wild type VS greA-flag).