Previously, others and we demonstrated that small RNAs MTS1338 and MTS0997 of , the causative agent of tuberculosis (TB), are essential for persistence inside host macrophages. However, their role in mycobacterial transcriptional profile regulation was characterized incompletely, and potential influence on host immune responses and the outcome of infection remained obscure. To obtain new insights in biology and TB pathogenesis we established unmarked single-knockout (KO) strains with deleted genes for MTS0997, or MTS1338, as well as the double KO strain. These strains, their parental wild type progenitor and complemented double KO variant were explored in three experimental settings: (i) whole genome expression profiling during cultural growth, (ii) whole genome transcriptional analysis of murine bone marrow-derived macrophages activated or not with IFN-γ, and (iii) TB infection in genetically highly TB-susceptible mice. The results of our experiments indicate that deletion of MTS0997 sRNA results in a more pronounced impact on biology compared to MTS1338. The most prominent phenotypic shifts in -deleted strain were modulation of bacterial succinate/fumarate respiration, up-regulation of genes belonging to the ESX-1 secretion system, increase in pathogen virulence for the murine host and decreased production of a few pro-inflammatory cytokines in the lung tissue. In addition, differences in the sizes of B-cell and other MHC-II-expressing cell populations in lungs of mice infected with single- compared to double-KO strains provided evidence for transcriptional epistasis, a phenomenon that remains poorly understood in mycobacteria.