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Journal of Bacteriology, March 2008, p. 2227-2230, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01642-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Critical Role of a Single Position in the –35 Element for Promoter Recognition by Mycobacterium tuberculosis SigE and SigH{triangledown}

Taeksun Song,1 Seung-Eun Song,1 Sahadevan Raman,2 Mauricio Anaya,2 and Robert N. Husson2*

The Genome Research Center for Respiratory Pathogens, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea,1 Division of Infectious Diseases, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts2

Received 10 October 2007/ Accepted 31 December 2007


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ABSTRACT
 
Mycobacterial SigE and SigH both initiate transcription from the sigB promoter, suggesting that they recognize similar sequences. Through mutational and primer extension analyses, we determined that SigE and SigH recognize nearly identical promoters, with differences at the 3' end of the –35 element distinguishing between SigE- and SigH-dependent promoters.


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TEXT
 
Mycobacterial SigE and SigH are extracytoplasmic function sigma factors that regulate the transcriptional response to several stresses; both have been shown to be essential for virulence (1, 4, 7-9, 12, 16). SigH regulates the thioredoxin redox buffer system and several heat shock proteins in response to oxidative and heat stresses (12). SigH activity is regulated transcriptionally at its autoregulated promoter and posttranslationally by a redox-sensitive anti-sigma factor, RshA (13). The SigE regulon is less well defined. Phenotype analysis of sigE mutants indicates that SigE plays a role in the responses to oxidative and cell envelope stresses (9). Sequence analysis suggests that SigE, like SigH, is also regulated by a zinc-associated anti-sigma factor (2, 3, 5). sigE and sigH are linked in a transcriptional network; sigH is transcribed from a single autoregulated promoter, whereas sigE has at least three independent promoters, one of which is regulated by SigH (12, 15, 16). Both SigE and SigH can activate transcription from the single promoter of sigB, a primary sigma subfamily member that also appears to play a role in stress adaptation (6, 12).

The consensus promoter elements recognized by SigH have been defined on the basis of primer extension mapping and in vitro transcription of several SigH-dependent promoters (12). This consensus, gGGAACA-N16-17-cGTT, is similar to the consensus promoter recognized by SigR, the Streptomyces coelicolor orthologue of SigH, but with significant differences (10). The consensus promoter sequences proposed to be recognized by SigE are less clearly defined (GGa/g-a/c-c-N17-c/gGTTg) and were suggested on the basis of sequences 5' of SigE-regulated genes from microarray analysis that were similar to the consensus promoter recognized by S. coelicolor SigR (9). However, only a small fraction of the candidate SigE-regulated genes suggested by the microarray data were shown to have this sequence 5' of their coding sequence, and none of the nine putative SigE-dependent promoters used to generate this consensus was confirmed experimentally.

The similarity of the proposed promoter sequences recognized by SigE and SigH, the overlap in the types of stresses to which they respond, and the initiation of transcription by SigE and SigH from the same sigB promoter in vivo led us to investigate in more depth the promoter sequences recognized by SigE and SigH. To do this, we undertook in vitro mutational analysis of the sigB promoter and, on the basis of the results of this analysis, performed primer extension experiments to identify in vivo promoters dependent on SigE for their transcription. These data indicate near identity in the core –35 and –10 elements of the optimal promoters recognized by these two sigma factors, with the exception of the 3' position of the –35 element. Different bases at this position appear to account for nearly all of the specificity for recognition by SigH or SigE.

To perform the mutational analysis, each position of the sigB promoter from –37 to –28 and from –14 to –10, encompassing the –35 and –10 regions of this promoter, was changed by using mutagenic primers so that the naturally occurring base at each position was replaced with each of the other three bases (Fig. 1). Individual promoters with the native sequence and with each possible single mutation were cloned into an Escherichia coli plasmid vector (pRH1517). Clones were verified by sequencing, and plasmid DNA was linearized by digestion with Bsu36I and used as a template for single-round in vitro transcription as previously described (12). Transcripts, with a predicted size of 248 nucleotides, were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. These experiments were performed at least three times for each mutation at each position with consistent results. As shown in Fig. 1, each of the first four bases, GGAA, in the "core" –35 hexamer is essential for efficient transcription initiation by both SigE and SigH. At the fifth position, while SigE fails to initiate transcription in the gel shown unless the consensus C is present, in some experiments, a weak signal is seen with T at this position. SigH is more permissive at this position, allowing at least some transcription when any base is present, and generating a strong signal when T is present. Consistent with these data, all known in vivo SigH-dependent promoters have C or T at this position (12).


Figure 1
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  		FIG. 1. In vitro transcription analysis of the native and mutated forms of the M. tuberculosis sigB promoter. (A) Individual mutations were made at each position of the –10 and –35 elements shown, and runoff in vitro transcription reactions, with these sequences as a template, were performed with RNA polymerase incorporating SigE or SigH. The sequence of the native sigB promoter at each position is shown in capital letters, and the three individual base substitutions are shown below the line for each position. The –35 core hexamer and the –10 core trimer are indicated by a line above these positions. The lower band in the SigH panels is a nonspecific transcript. (B) Quantification by densitometry of at least two independent replicates of SigE in vitro transcription experiments. WT and wt, wild type.

The sixth position of the –35 core hexamer appears to be the key position for distinguishing between promoters that are optimally recognized by SigE and SigH. Replacing the native T of the sigB promoter with A greatly enhances transcription by SigH, whereas G or C at this position allows only weak transcription by SigH. All three substitutions at this position result in moderately reduced transcription by SigE, with substitution of C resulting in the strongest transcription and substitution of G resulting in the weakest transcription. Of the –35 region effects seen outside of the core hexamer in Fig. 1, substitution of G at the position immediately 3' of the core hexamer appears to inhibit transcription by both SigE and SigH. Changes at the position immediately 5' of the core hexamer do not have strong effects, and substitutions farther from the core –35 element did not have consistent effects (data not shown).

In the –10 element, substitution of any base at any position of the core trimer GTT essentially eliminates transcription by SigE or SigH, consistent with the conservation of these bases in all known SigH-dependent promoters (12). The C preceding this trimer, recognized as a partially conserved element in previously defined SigH-dependent promoters, appears to be more critical for transcription initiation by SigE. Whereas any substitution at this position allows moderately decreased transcription by SigH, substitution of T or G at this position essentially eliminates transcription by SigE and substitution of A markedly decreases transcription. There appears to be no preference for specific bases at the positions 3' of the GTT trimer (data not shown).

On the basis of these data, to identify candidate in vivo SigE-dependent promoters, we searched the Mycobacterium tuberculosis H37Rv genome for sequences up to 200 bases 5' of structural genes that matched the predicted optimal SigE-dependent promoter GGAAC-T/C-N17-18-GTT. In addition to sigB, 13 sequences were identified that met these criteria. Of these, one (Rv1798) was not investigated because its start codon overlapped the stop codon of the 5' gene, indicating that Rv1798 was part of an operon transcribed from a different promoter. Primer extension analysis of the remaining 12 genes was performed as previously described (12), by using RNA from wild-type H37Rv, isogenic sigE and sigH mutant strains, and a sigE sigH double mutant. Each culture was stressed with 1 mM diamide for 10 min prior to harvesting for RNA isolation. As an inducer of both sigE and sigH, analysis of RNA from diamide-induced cells allowed us to identify SigE-regulated promoters, as well as those that might also be transcribed by SigH.

This primer extension analysis identified eight SigE-dependent promoters on the basis of the presence of a transcript in RNA isolated from wild-type H37Rv and its absence (or a much weaker signal) in the sigE mutant (Fig. 2). Two promoters, those 5' of dnaJ2 and Rv2694c, though predominantly SigE dependent, appear to be weakly transcribed by SigH, based on the presence of a faint transcript signal in the sigE mutant lane and its absence in the sigE sigH double mutant, a finding that was previously observed for the sigB promoter (12). On the basis of our previous observation of stronger induction of transcription by heat stress of SigH-dependent heat shock genes (12), RNA was isolated following 50°C heat stress and primer extensions were performed with this RNA for the three SigE-dependent heat shock genes identified in this analysis. For hsp20 and htpX, stronger transcript signals were seen with RNA isolated from heat-stressed cells than with RNA isolated from diamide-stressed cells (data not shown). In contrast, the dnaJ2 transcript gave a weak but consistent signal with RNA isolated from cells induced by either stress. Despite the presence of a consensus-matching sequence, we did not identify a SigE-dependent promoter 5' of four genes (Rv0339, Rv1927, pknJ, and fadE25) that were identified in the database search described above.


Figure 2
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  		FIG. 2. Primer extension analysis of candidate in vivo SigE-dependent promoters. (A) Total RNA was isolated from the wild-type (Wt) and sigE (E–), sigH (H–), and sigE sigH (EH–) mutant strains of M. tuberculosis, and primer extensions were performed with gene-specific antisense primers. Specific transcripts were separated on sequencing gels, and the transcription start point was determined by comparison to a sequencing ladder run in the same gel. The sigA transcript, which does not vary under most growth conditions and is not SigE or SigH regulated, serves as a control for RNA quality and reverse transcription. For the experiments shown, RNA was isolated from diamide-stressed cells, except for hsp, htpX, and dnaJ2, for which RNA was isolated from heat-stressed cells. (B) Promoter sequences of SigE-regulated genes. The –35 core hexamer and the –10 core trimer are boxed. Transcription start points are in bold. In the consensus, Y = C or T.

In addition to the core sequences used to identify these SigE-dependent promoters, G precedes the –35 core hexamer in six of these nine promoters, as it does in six of seven previously identified SigH-dependent promoters (Fig. 2B). G was not identified as a preferred base at this position in the in vitro analysis. As suggested by the in vitro data, none of these in vivo SigE-dependent promoters has G in the position immediately 3' of the –35 hexamer, whereas A is present in five of these promoters. In contrast, three of seven SigH-regulated promoters have G at this position and two have A. Preceding the GTT –10 core trimer, C is present in seven of nine SigE-dependent promoters and five of seven SigH-dependent promoters, consistent with the in vitro data and suggesting that the –10 element does not play a role in distinguishing between SigH- and SigE-regulated promoters.

Of the eight SigE-regulated promoters identified in this work, four are among those previously predicted to be SigE dependent on the basis of some similarity to the promoter sequences recognized by the S. coelicolor sigma factor SigR (9). This analysis also predicted four other promoters to be SigE dependent, which contain –35 sequences that our in vitro data indicate are unlikely to be transcribed by SigE. This prior bioinformatic analysis did not identify the sixth position of the core hexamer as a conserved part of the –35 element.

These data demonstrate the close similarity of the promoters recognized by SigE and SigH and the critical importance of the sixth position of the –35 hexamer in distinguishing between promoters recognized by these two sigma factors. Optimal SigH-dependent transcription requires A at this position, and our previous data indicate that SigE-dependent transcription does not occur in vivo when A is present (12). In contrast, our in vitro and in vivo data indicate that C or T at this position is required for optimal SigE-dependent transcription. In the promoters where both SigE- and SigH-dependent transcription occur in vivo (sigB, dnaJ, and Rv2694c), this position is occupied by a SigE-preferred base, i.e., T or C, and these promoters are weak SigH-dependent promoters in vivo.

In addition, SigH is more tolerant of variation at the fifth position of the –35 core hexamer. The additional preferences for G preceding the –35 hexamer and C preceding the –10 trimer apply to both SigE- and SigH-dependent promoters, whereas A is present immediately 3' of the –35 hexamer in the majority of SigE- but not SigH-regulated promoters. It should be noted that we identified these SigE-regulated promoters by searching the M. tuberculosis genome sequence with the predicted optimal promoter sequences defined by our in vitro data. It is possible that there are additional SigE-dependent promoters in vivo that vary from this optimal consensus.

These data provide new insight into the sequence requirements of SigE- and SigH-regulated promoters. Though recognition of specific promoter sequences is critical to transcription regulation by these sigma factors, other mechanisms can contribute to the regulation of their activity. Recent data, for example, implicate polyphosphates and the two-component system mprAB in the regulation of sigE transcription and activation of the SigE regulon (11, 14). Our data also clearly identify several new genes in the SigE regulon of M. tuberculosis, including heat stress/chaperone genes and genes encoding a putative transcription factor, a DNA repair enzyme, and membrane proteins of unknown function. Characterization of these genes may lead to additional understanding of the function of the SigE regulon in M. tuberculosis physiology and pathogenesis.


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ACKNOWLEDGMENTS
 
This work was supported by grant RO1AI37901 from the National Institute of Allergy and Infectious Diseases to R.N.H., by a grant from the Potts Memorial Foundation to R.N.H., and by a grant from the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A010381), to T.S.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 919-2900. Fax: (617) 730-0254. E-mail: robert.husson{at}childrens.harvard.edu Back

{triangledown} Published ahead of print on 11 January 2008. Back


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Journal of Bacteriology, March 2008, p. 2227-2230, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01642-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Dona, V., Rodrigue, S., Dainese, E., Palu, G., Gaudreau, L., Manganelli, R., Provvedi, R. (2008). Evidence of Complex Transcriptional, Translational, and Posttranslational Regulation of the Extracytoplasmic Function Sigma Factor {sigma}E in Mycobacterium tuberculosis. J. Bacteriol. 190: 5963-5971 [Abstract] [Full Text]  

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