Comparison of the crystal structures of two penta­dehydro­peptides containing ΔPhe residues, namely (Z,Z)-N-(tert-butoxy­carbonyl)­glycyl-α,β-phenyl­alanyl­glycyl-α,β-phenyl­alanyl­glycine (or Boc0–Gly1–ΔZPhe2–Gly3–ΔZPhe4–Gly5–OH) methanol solvate, C29H33N5O8·CH4O, (I), and (E,E)-N-(tert-butoxy­carbonyl)­glycyl-α,β-phenyl­alanyl­glycyl-α,β-phenyl­alanyl­glycine (or Boc0–Gly1–ΔEPhe2–Gly3–ΔEPhe4–Gly5–OH), C29H33N5O8, (II), indicates that the ΔZPhe residue is a more effective inducer of folded structures than the ΔEPhe residue. The values of the torsion angles ϕ and ψ show the presence of two type-III′ β-turns at the ΔZPhe residues and one type-II β-turn at the ΔEPhe residue. All amino acids are linked trans to each other in both peptides. β-Turns present in the peptides are stabilized by intra­molecular 4→1 hydrogen bonds. Mol­ecules in both structures form two-dimensional hydrogen-bond networks parallel to the (100) plane.

Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ70 (the housekeeping σ subunit) and σS (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ70, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the −35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the −10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the −10/+1 region could improve promoter recognition by EσS.

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.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.