Transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis

Abstract

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.

Figure 1

Genetic organization of ESAT-6 cluster 3 in M. tuberculosis (A) and M. smegmatis (B). The position of the pr1 and pr2 promoters are indicated. The distance between rv0286 and rv0287, and between msmeg0619 and smeg0620 is arbitrary.

Figure 2

Promoter regions and transcriptional start sites of M. smegmatis ESAT-6 cluster 3. Sequences upstream of the msmeg0615 (A) and msmeg0620 (B) genes: primer sequences utilized for the cloning of promoter regions are underlined; stop codons of the upstream gene are in bold; translational start codons (+1) are in bold capital letters; transcriptional start sites are in bold and indicated with an arrow; hypothetical -35 and -10 regions are boxed; IdeR binding site is double underlined.

Figure 3

EMSA experiments on M. smegmatis and M. tuberculosis pr1 promoter with M. smegmatis IdeR (A) and Zur (B) proteins. (A) Migration of different DNA fragments representing the upstream region of the following genes: mmpS5-mmpL5 (unrelated fragment) (lanes 1–2), rv0282 (lanes 3–4), msmeg0615 (lanes 5–6), in the absence (-) and in the presence (+) of M. smegmatis IdeR. (B) EMSA experiments on the promoter region of M. tuberculosis rv0282 (lanes 1–4) and msmeg0615 (lanes 5–8) with M. smegmatis Zur. Lanes 1 and 5, negative control (without protein); lanes 2 and 6 no metal; lanes 3 and 7 200 μM Zn; lanes 4 and 8 400 μM Zn.

Figure 4

msmeg0615 (pr1) promoter activity. β-galactosidase activity of cultures grown in Sauton medium in the presence of varying divalent metal ions. The values, expressed as nanomoles of o-nitrophenol-β-D-galactopyranoside converted to o-nitrophenol min^-1 mg^-1 of protein, represent the average and the standard deviation of three independent clones. * indicates that values are significantly different from the control value (p < 0.01).

Figure 5

msmeg0620 (pr2 MS) and rv0287 (pr2 MT) promoter activity. β-galactosidase activity of msmeg0620 and rv0287 (pr2) in M. smegmatis cultures grown in 7H9 medium at mid-log phase. The value represents the average and the standard deviation of three independent clones. * indicates that values are significantly different from the control value (p < 0.01).

Figure 6

Expression of msmeg0615 and msmeg0620 genes. Level of expression of msmeg0615 (A) and msmeg0620 (B) genes in differing growth and stress conditions relative to the expression of the same gene in 7H9 culture in mid-log phase (OD = 0.8) (taken as 1). The level of sigA transcript was used to normalize the amount of RNA. The value represents the average and the standard deviation of three independent reactions. * indicates that values are significantly different from the control value (p < 0.01).