SummaryMycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two-component system, named senX3-regX3. Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae. These could be grouped into three major families, containing elements of 77-101 bp, 46-53 bp and 58-101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40-50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two-component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.

Environmental regulation of bacterial gene expression is often mediated by two-component signal transduction systems, which are themselves tightly regulated. The response regulator RegX3 and the cytoplasmic portion of the histidine kinase SenX3 from Mycobacterium bovis BCG were overproduced in Escherichia coli and purified as N-terminally (His) 6 -tagged proteins. Phosphorylation assays demonstrated autophosphorylation of the cytoplasmic portion of SenX3 and a phosphotransfer from SenX3 to RegX3, involving conserved histidine and aspartate residues, respectively. In addition, as shown by electrophoretic mobility shift assays, (His) 6 RegX3 was able to specifically bind to the promoter region of the senX3-regX3 operon. Furthermore, operon fusion analyses indicated that the overexpression of the senX3-regX3 operon increases the activity of the senX3 promoter in Mycobacterium smegmatis. Together, these results indicate that the mycobacterial SenX3-RegX3 twocomponent system is positively autoregulated.