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Background  The ESAT-6 (early secreted antigenic target, 6 kDa) family collects small mycobacterial proteins secreted by  Mycobacterium tuberculosis , particularly in the early phase of growth. There are 23 ESAT-6 family members in  M. tuberculosis  H37Rv. In a previous work, we identified the Zur- dependent regulation of five proteins of the ESAT-6/CFP-10 family ( esxG ,  esxH ,  esxQ ,  esxR , and  esxS ).  esxG  and  esxH  are part of ESAT-6 cluster 3, whose expression was already known to be induced by iron starvation.    Results  In this research, we performed EMSA experiments and transcriptional analysis of ESAT-6 cluster 3 in  Mycobacterium smegmatis  ( msmeg0615 - msmeg0625 ) and  M. tuberculosis . In contrast to what we had observed in  M. tuberculosis , we found that in  M. smegmatis  ESAT-6 cluster 3 responds only to iron and not to zinc. In both organisms we identified an internal promoter, a finding which suggests the presence of two transcriptional units and, by consequence, a differential expression of cluster 3 genes. We compared the expression of  msmeg0615  and  msmeg0620  in different growth and stress conditions by means of relative quantitative PCR. The expression of  msmeg0615  and  msmeg0620  genes was essentially similar; they appeared to be repressed in most of the tested conditions, with the exception of acid stress (pH 4.2) where  msmeg0615  was about 4-fold induced, while  msmeg0620  was repressed. Analysis revealed that in acid stress conditions  M. tuberculosis rv0282  gene was 3-fold induced too, while  rv0287  induction was almost insignificant.    Conclusion  In contrast with what has been reported for  M. tuberculosis , our results suggest that in  M. smegmatis  only IdeR-dependent regulation is retained, while zinc has no effect on gene expression. The role of cluster 3 in  M. tuberculosis  virulence is still to be defined; however, iron- and zinc-dependent expression strongly suggests that cluster 3 is highly expressed in the infective process, and that the cluster contributes to the antigenic profile during the course of infection. Moreover, cluster 3 induction in acid stress conditions strengthens the hypothesis that cluster 3 is expressed in the course of infection.  In  M. smegmatis , the expression of  msmeg0615  and  msmeg0620  genes is broadly similar in differing growth phases and in stress conditions, with the exception of acid stress (pH 4.2). Differences in expression between cluster 3 genes can be explained by the presence of internal promoters, both in  M. smegmatis  and  M. tuberculosis .

Transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis