The complex architecture of mycobacterial promoters

Newton-Foot, Mae; Pittius, Nicolaas C. Gey van

doi:10.1016/j.tube.2012.08.003

Cited by 65 publications

(93 citation statements)

References 216 publications

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“…This was done in order to determine if dosR itself has any role to play in activating transcription in the presence of the C601T mutation. Plasmid pAA6 is a derivative of pDC6 (contain- 54 Glu mutation (pAA6) is also indicated. A schematic representation of the inserts included in the plasmids used to transform dosR KO mutants of H37Rv and HN878 is shown.…”

Section: Resultsmentioning

confidence: 99%

“…Rose et al also suggest that the C3500149T SNP generates a novel Ϫ10 TANNNT consensus motif, thereby effectively creating a new TSS within the dosR promoter region from which transcription is activated via the housekeeping SigA factor (29,(53)(54)(55). Although this seems a reasonable assumption, it has not been confirmed experimentally.…”

Section: Fig 6 the Dost Defect In Beijing Isolates Has No Impact On Tmentioning

confidence: 99%

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Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

et al. 2017

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The DosR regulon, a set of 48 genes normally expressed in Mycobacterium tuberculosis under conditions that inhibit aerobic respiration, is controlled via the DosR-DosS/DosT two-component system. While the regulon requires induction in most M. tuberculosis isolates, for members of the Beijing lineage, its expression is uncoupled from the need for signaling. In our attempts to understand the mechanistic basis for this uncoupling in the Beijing background, we previously reported the identification of two synonymous single-nucleotide polymorphisms (SNPs) within the adjacent Rv3134c gene. In the present study, we have interrogated the impact of these SNPs on dosR expression in wild-type strains, as well as a range of dosRdosS-dosT mutants, for both Beijing and non-Beijing M. tuberculosis backgrounds. In this manner, we have unequivocally determined that the C601T dosR promoter SNP is the sole requirement for the dramatic shift in the pattern of DosR regulon expression seen in this globally important lineage. Interestingly, we also show that DosT is completely nonfunctional within these strains. Thus, a complex series of evolutionary steps has led to the present-day Beijing DosR phenotype that, in turn, potentially confers a fitness advantage in the face of some form of host-associated selective pressure.IMPORTANCE Mycobacterium tuberculosis strains of the Beijing lineage have been described as being of enhanced virulence compared to other lineages, and in certain regions, they are associated with the dramatic spread of multidrug-resistant tuberculosis (TB). In terms of trying to understand the functional basis for these broad epidemiological phenomena, it is interesting that, in contrast to the other major lineages, the Beijing strains all constitutively overexpress members of the DosR regulon. Here, we identify the mutational events that led to the evolution of this unique phenotype. In addition, our work highlights the fact that important phenotypic differences exist between distinct M. tuberculosis lineages, with the potential to impact the efficacy of diagnosis, vaccination, and treatment programs.KEYWORDS Tuberculosis, DosR regulon, strain variation, evolution, Mycobacterium tuberculosis D espite being an ancient disease, human tuberculosis (TB) resulting from infection with Mycobacterium tuberculosis still remains a leading cause of death worldwide as a consequence of multiple contributing factors, including drug resistance, HIV coinfection, and inadequate access to high-quality health care (1). Furthermore, recent evidence suggests that the level of genetic diversity that exists among distinct M. tuberculosis isolates was previously underestimated and has the potential to impact pathogenicity, as well as to limit the effectiveness of current diagnostic and therapeutic interventions (2-4).

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Section: Resultsmentioning

confidence: 99%

Section: Fig 6 the Dost Defect In Beijing Isolates Has No Impact On Tmentioning

confidence: 99%

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

et al. 2017

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“…1). Mycobacterial promoters usually have well‐conserved ‐10 regions, while ‐35 are less conserved and hardly recognizable, probably because of the high number of sigma factors operating in these bacteria (Bashyam et al ., 1996; Newton‐Foot and Gey van Pittius, 2013; Manganelli, 2014). Moreover, several mycobacterial promoters present extended ‐10 regions (containing a TGn sequence at the 5′‐end) which increase their potency, and it was proposed that these promoters do not require the presence of a ‐35 sequence (Kenney and Churchward, 1996).…”

Section: Discussionmentioning

confidence: 99%

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Boldrin

Degiacomi

Serafini

et al. 2017

Microbial Biotechnology

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SummaryA range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip‐OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr, a strong Pip‐repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild‐type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip‐OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole‐cell screening assay to identify inhibitors of this enzyme.

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“…T ranscriptional regulation in mycobacteria is complex, with many different sigma factors and promoter sequences (1)(2)(3)(4)(5). Mycobacterium smegmatis and Mycobacterium tuberculosis encode 28 and 13 different sigma factors, respectively, but SigA, the sole group 1 member in each species, is essential and is presumably responsible for expression of all or most housekeeping genes (4).…”

mentioning

confidence: 99%

“…Multiple types of promoter specificities have also been reported, and although the correspondence with specific sigma factors is incomplete, the SigA promoters have similarity to the Sig-70 promoters of Escherichia coli. A consensus sequence derived from promoters with mapped transcription start sites (TSSs) identified a 5=-TTGACW Ϫ35 motif and a 5=-TATAMT Ϫ10 motif, spaced 16 to 21 bp apart (3,(6)(7)(8)(9). Mutational analysis of the Ϫ35 motif in a synthetic promoter indicated that optimal activity was provided by mutations in the sequence 5=-TTGCGA (10).…”

mentioning

confidence: 99%

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

Oldfield

Hatfull

2014

Journal of Bacteriology

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The P R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P R showed that it uses a canonical ؊10 hexamer recognized by SigA, and mutants with mutations to the sequence 5=-TATAMT had the greatest activities. It does not contain a 5=-TGN-extended ؊10 sequence, although mutants with mutations creating an extended ؊10 sequence had substantially increased promoter activity. Mutations in the ؊35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the ؊35 hexamer differentially affected promoter activity, depending on the ؊10 and extended ؊10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.

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The complex architecture of mycobacterial promoters

Cited by 65 publications

References 216 publications

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

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