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Journal of Bacteriology, May 2007, p. 3655-3659, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.00040-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Riboswitch Regulates Expression of the Coenzyme B₁₂-Independent Methionine Synthase in Mycobacterium tuberculosis: Implications for Differential Methionine Synthase Function in Strains H37Rv and CDC1551 ^,

Digby F. Warner,^* Suzana Savvi, Valerie Mizrahi, and Stephanie S. Dawes^*

MRC/NHLS/WITS Molecular Mycobacteriology Research Unit and DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, School of Pathology, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa

Received 9 January 2007/ Accepted 12 February 2007

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We observed vitamin B₁₂-mediated growth inhibition of Mycobacterium tuberculosis strain CDC1551. The B₁₂ sensitivity was mapped to a polymorphism in metH, encoding a coenzyme B₁₂-dependent methionine synthase. Vitamin B₁₂-resistant suppressor mutants of CDC1551 containing mutations in a B₁₂ riboswitch upstream of the metE gene, which encodes a B₁₂-independent methionine synthase, were isolated. Expression analysis confirmed that the B₁₂ riboswitch is a transcriptional regulator of metE in M. tuberculosis.

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Comparative genomics has been profitably utilized to elucidate interstrain variability between Mycobacterium tuberculosis clinical and laboratory isolates (7, 8, 19, 20). Moreover, recent studies (11, 14, 16) have begun to address the next challenge: mapping phenotypic consequences to identified polymorphisms. A whole-genome comparison between H37Rv, a virulent laboratory strain, and the clinical isolate CDC1551 identified a large sequence polymorphism at the PPE37-metH locus in CDC1551 that eliminates 1,196 bp at the 3' terminus of the metH gene (7) (Fig. 1). MetH is one of two methionine synthases predicted to catalyze the final S-methyl transfer reaction in de novo methionine biosynthesis in M. tuberculosis (4)—the formation of methionine from homocysteine—and requires a vitamin B₁₂-derived cofactor for activity; the other, MetE, does not. Full-length MetH is a monomer comprising four distinct domains (Fig. 1). In CDC1551, 398 amino acids have been lost from the C terminus, partially disrupting the coenzyme B₁₂ (or cobalamin [CBL])-binding domain and eliminating completely the S-adenosyl-L-methionine (SAM)-binding domain, which is required for the reductive reactivation of the B₁₂ moiety in MetH (6).

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FIG. 1. Deletion polymorphism at the PPE37-metH locus of CDC1551. Full-length MetH comprises an N-terminal homocysteine-binding domain (HCY), an N-methyltetrahydrofolate-binding domain (MTH), a CBL-binding domain, and a C-terminal SAM-binding domain (6). A large sequence polymorphism eliminates the SAM-binding domain in CDC1551 MetH (shaded box). Genomic regions deleted in the respective H37Rv metH mutants are also shown (open boxes).

Little is known about the regulation of corresponding B₁₂-dependent and B₁₂-independent enzymes in M. tuberculosis or their contribution to viability and pathogenesis (5). However, any suggestion of redundant methionine synthase function must be reconciled with the predicted essentiality of metE for optimal growth of M. tuberculosis in vitro (18). In particular, the apparent inability of metH to compensate for the loss of metE raises fundamental questions about the functionality of metH in this organism. Alternatively, the predicted essentiality of metE may indicate an inability of M. tuberculosis to produce the B₁₂ cofactor necessary to enable MetH to function as the sole methionine synthase in vitro, despite encoding an apparently complete vitamin B₁₂ biosynthetic pathway (17).

In genomes containing corresponding B₁₂-dependent and B₁₂-independent enzymes, the activity of the B₁₂-independent enzyme is often subject to regulation by a B₁₂ riboswitch (23). Riboswitches are highly structured domains found within the mRNA of the regulated gene (1). Ligand-specific binding of a small molecule (such as B₁₂) to the riboswitch results in the formation of an alternative RNA structure that attenuates transcription or translation (12). The association of riboswitch-mediated regulation with many essential metabolite biosynthetic and transport pathways has prompted the development of novel antibacterials that target these genetic control elements (2). Two B₁₂ riboswitch motifs have been identified in the M. tuberculosis genome and are located immediately upstream of metE (Fig. 2) and PPE2 (17, 23). Recently, Borovok et al. (3) took advantage of a B₁₂ riboswitch to effect growth arrest in a Streptomyces coelicolor mutant lacking the coenzyme B₁₂-dependent ribonucleotide reductase (RNR) NrdJ. In that case, transcription of the corresponding B₁₂-independent enzyme, NrdAB, was repressed by exogenous B₁₂, effectively abrogating essential RNR function in Streptomyces.

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FIG. 2. Predicted M. tuberculosis metE B12 riboswitch. The secondary structure is drawn according to the scheme presented by Vitreschak et al. (23). Uppercase letters indicate residues identified by Vitreschak et al. (23) to be invariant across approximately 200 B12 riboswitches from 67 bacterial genomes; lowercase letters are M. tuberculosis specific. The conserved B12-box is highlighted. The square and circle denote sites of CT transition mutations identified in B12 suppressor mutants of CDC1551 and H37Rv metH(B), respectively. The triangles denote single-nucleotide polymorphisms identified in the B12 riboswitch upstream of the nrdABS operon in B12 suppressor mutants of S. coelicolor (3) (see the text for details).

We sought to address by genetic means whether metH is functional in M. tuberculosis H37Rv and to investigate the consequences of the deletion polymorphism on the function of metH in CDC1551. Furthermore, the location of a putative riboswitch in the metE promoter region of M. tuberculosis suggested the possibility of exploiting the CDC1551 metH polymorphism to determine whether B₁₂ regulates the activity of metE in M. tuberculosis. In this paper, we describe the results of these investigations.

A metE mutant of M. tuberculosis H37Rv is viable when supplemented with vitamin B₁₂. To establish the ability of metH to compensate for the loss of metE in M. tuberculosis, we attempted to generate a metE deletion mutant of H37Rv (metE) (Fig. 3A) by allelic replacement with a hyg-marked metE deletion allele carried on the suicide plasmid p2metE17 (see Table S1 in the supplemental material) by previously described methods (9, 15). Consistent with the predicted essentiality of metE (18), a metE mutant could not be obtained on normal solid medium (Middlebrook 7H10; Difco). However, supplementing the growth medium with vitamin B₁₂ (cyanocobalamin; Sigma) at a concentration of 10 µg/ml enabled the ready isolation of a metE mutant (Fig. 3B and C). The successful generation of an H37Rv metE mutant on B₁₂-supplemented medium was significant, as it established simultaneously the ability of M. tuberculosis to transport vitamin B₁₂ despite the absence of identifiable B₁₂-specific transporters in the M. tuberculosis genome (17) and the capacity of the organism to convert exogenous vitamin B₁₂ into the methylcobalamin cofactor required for MetH-catalyzed S-methyl transfer (10). It also strongly supported functionality of the metH-encoded gene, although the need to supplement the growth medium suggested that M. tuberculosis was unable to produce sufficient B₁₂ cofactor in vitro to allow MetH to compensate for the loss of function of MetE.

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FIG. 3. Construction and phenotypic characterization of M. tuberculosis methionine synthase mutants. (A) Construction and genotypic characterization of mutant strains. Genomic DNA was digested with the relevant restriction enzyme and probed with either a metE- or metH-specific probe. Restriction maps of the various strains are illustrated schematically in the line drawings adjacent to each Southern blot (not drawn to scale). For metE, Southern blots were probed with a 1,989-bp fragment (metEp, represented by a spotted box) containing 564 bp of 5'-terminal metE coding sequence obtained by PCR amplification using the primer pair metEF2/metER2 (see Table S2 in the supplemental material). The metE::hyg mutation eliminates 1,367 bp of metE coding sequence; however, an additional BamHI site is introduced by the hyg cassette (B, BamHI; WT, wild-type H37Rv). For metH, Southern blots were probed with the 644-bp MluI-BglII fragment of H37Rv metH (metHp, represented by a hatched box). The probe corresponds to the SAM-binding domain of H37Rv metH and so fails to hybridize to CDC1551 genomic DNA. The metH(B) mutation eliminates 391 bp of metH coding sequence, whereas the metH(BB) mutation eliminates 1,417 bp metH coding sequence, including an MluI site (M, MluI; WT, wild-type H37Rv). metHKin is a single-crossover homologous recombination mutant of CDC1551 containing both truncated CDC1551 and full-length H37Rv metH alleles. (B) Effect of exogenous vitamin B12 on growth of CDC1551 and H37Rv methionine synthase mutants. Cells were incubated on solid 7H10 medium containing 10 µg/ml vitamin B12 (black bars), and CFU were scored after 4 weeks. Gray bars indicate normal medium without supplement. Data represent mean CFU from two independent experiments performed in duplicate, and error bars indicate standard deviations. The H37Rv metE mutant requires B12 supplementation for growth on 7H10, ensuring an absence of CFU on unsupplemented medium; B12P2 is a representative CDC1551 B12 suppressor strain containing a mutated B12 riboswitch (see the text and Fig. 2). (C) Representative plates.

Growth of CDC1551 is inhibited on solid medium containing vitamin B₁₂. We reasoned that if the truncation in CDC1551 metH rendered this gene nonfunctional, then it should not be possible to construct a CDC1551 metE deletion mutant by the approach described above for H37Rv. Indeed, we were unable to recover a metE mutant of this strain using the knockout vector p2metE17 (see Table S1 in the supplemental material): 102 white, sucrose-resistant, hygromycin-resistant clones recovered from the two-step allelic exchange procedure (15) were screened by PCR, and all were found to retain the wild-type metE allele. The inability to disrupt metE in CDC1551 thus provided a further indication that its truncated metH gene was not functionally active.

During the course of these experiments, we noticed that wild-type CDC1551 exhibited a marked plating defect on solid medium supplemented with vitamin B₁₂ (Fig. 3C): at a B₁₂ concentration of 10 µg/ml, a 3 log reduction in CFU was evident (Fig. 3B). In stark contrast, no effect on H37Rv growth was observed (Fig. 3B and C). These observations suggested that differential methionine synthase function might determine the contrasting growth phenotypes displayed by CDC1551 and H37Rv on Middlebrook 7H10 agar supplemented with B₁₂. That is, an inactivated metH coupled with B₁₂-mediated suppression of metE effectively abrogated all methionine synthase activity in CDC1551, thus preventing growth.

Disruption of metH renders H37Rv sensitive to vitamin B₁₂. To confirm that the lack of MetH function was directly responsible for the B₁₂ sensitivity of CDC1551, we constructed two independent hyg-marked metH deletion mutants of H37Rv (Fig. 3A; also see Table S1 in the supplemental material). A 391-bp BglII deletion in H37Rv metH(B) eliminated 63 amino acids in the CBL domain and 71 amino acids in the SAM domain (Fig. 1), roughly approximating the CDC1551 metH genotype in which 102 amino acids are missing from the CBL domain together with the entire SAM domain. H37Rv metH(BB), on the other hand, contained a much larger deletion (1,417-bp region spanned by BglII and BclI sites), which eliminated the entire CBL domain and disrupted both the methyltetrahydrofolate and SAM domains (Fig. 1). The metH(BB) allele was therefore expected to abrogate metH activity completely. Plating both mutants on medium supplemented with vitamin B₁₂ at 10 µg/ml confirmed that disruption of metH indeed renders M. tuberculosis B₁₂ sensitive (Fig. 3B and C). Furthermore, metH(B) recapitulated precisely the CDC1551 phenotype, indicating that the combined disruption of the CBL and SAM domains is sufficient to render M. tuberculosis MetH nonfunctional. Conversely, by introducing a copy of the full-length H37Rv metH gene into the CDC1551 chromosome, we were able to reverse the B₁₂ sensitivity of CDC1551 (metHKin) (Fig. 3B and 3C). CDC1551 metHKin (Fig. 3A) is a single-crossover recombinant that carries the full-length H37Rv metH open reading frame plus flanking and vector sequences (p2metHKin) (see Table S1 in the supplemental material), as well as retaining its native truncated metH allele. Together, these observations provided further evidence that CDC1551 MetH was inactive.

Vitamin B₁₂-resistant CDC1551 mutants containing mutated B₁₂ riboswitches. Colonies of CDC1551 of normal size and morphology arose on B₁₂-containing plates at a frequency of 10^–3 (Fig. 3B). We postulated that a mutation in the B₁₂ riboswitch motif upstream of metE might relieve B₁₂-mediated suppression of metE and so allow growth of these "vitamin B₁₂ suppressor" mutants. To ascertain the heritability of the suppressor phenotype, selected colonies were passaged in liquid medium (Middlebrook 7H9; Difco) without B₁₂ supplement. Restreaking of these cultures onto solid medium supplemented with B₁₂ established that the ability to grow on B₁₂ was not lost after passage (a representative suppressor mutant, B12P2, is shown in Fig. 3B and C). To characterize these mutants genotypically, we sequenced the 500-bp genomic region upstream of metE, which contains the B₁₂ riboswitch in 10 CDC1551 B₁₂ suppressor mutants. We also sequenced 10 B₁₂ suppressor mutants derived from H37Rv metH(B). This analysis revealed that 2 of the 10 CDC1551 mutants contained a CT transition in a conserved region of the B₁₂ riboswitch termed the "B12-box" (23). In addition, one H37Rv metH(B) suppressor mutant contained a CT transition in an adjacent position within the B12-box (Fig. 2). These data supported the idea that the vitamin B₁₂ plating deficiency of CDC1551 results from B₁₂-mediated repression of metE. However, the mechanism(s) of resistance in the remaining CDC1551 and metH(B) B₁₂ suppressor isolates remains to be elucidated. Preliminary sequence analysis has failed to reveal any mutations in the only other identified M. tuberculosis B₁₂ riboswitch—located upstream of PPE2 (17)—in these strains. Instead, it is possible that they represent B₁₂ transport mutants with diminished capacity to take up B₁₂.

The B₁₂ riboswitch is a transcriptional regulator of metE. To determine whether the B₁₂ riboswitch functions as a transcriptional or posttranscriptional regulator of M. tuberculosis metE, we analyzed the expression of metE during growth in liquid medium supplemented with vitamin B₁₂. Expression of the housekeeping gene sigA was used to benchmark relative expression levels, as described previously (5, 13). In both H37Rv and CDC1551, metE transcript levels were significantly reduced in B₁₂-supplemented medium, whereas expression of sigA was unaffected (Fig. 4). In contrast to the repression of metE observed in the parental CDC1551 strain, the expression of metE in the B12P2 mutant was completely unaffected by exogenous B₁₂. This observation confirmed that vitamin B₁₂-mediated repression of metE activity in wild-type M. tuberculosis occurs at the level of transcription. Interestingly, constitutive transcription of metE in the B12P2 mutant was noticeably diminished relative to H37Rv and CDC1551 when standardized against sigA. The B12P2 mutant displayed no growth deficiency under standard conditions in vitro, however, suggesting either that reduced metE expression has no effect on growth or that B12P2 harbors a second-site mutation(s) that ameliorates any growth-inhibitory effect of reduced metE expression.

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FIG. 4. Exogenous vitamin B12 represses transcription of M. tuberculosis metE. Reverse transcription (RT)-PCR analysis of metE expression in strains H37Rv, CDC1551, and the B12 suppressor mutant B12P2 is shown. Cells were grown for 5 h in Sauton's minimal medium in the presence (+) or absence (–) of 10 µg/ml vitamin B12 before being harvested for mRNA isolation. RT-PCR methods are described in the supplemental material, and primer sequences are detailed in Table S2. The data shown are representative of two independent experiments.

Conclusions and implications. In this study, we took advantage of an identified deletion polymorphism in a clinical isolate to confirm fundamental aspects of M. tuberculosis biology. Specifically, we have presented genetic evidence that both metE and metH encode functional methionine synthases and that transcription of metE is controlled by a B₁₂ riboswitch. In addition, through the construction of a metE deletion mutant of strain H37Rv, we have demonstrated the ability of M. tuberculosis to transport vitamin B₁₂. A previous comparative genomic analysis had situated mycobacteria (including M. tuberculosis) in a rare category of B₁₂-utilizing organisms lacking an identifiable B₁₂-specific transport system (17). Further analysis of those B₁₂ suppressor isolates of CDC1551 and metH(B) that did not map to the metE-related B₁₂ riboswitch may yield some insight into potential nonorthologous B₁₂ transport systems in M. tuberculosis.

The M. tuberculosis genome contains two other B₁₂-dependent enzymes in addition to metH (4): a class II RNR encoded by nrdZ (5) and a methylmalonyl coenzyme A mutase encoded by mutAB. The proof of functionality of both a B₁₂-dependent enzyme (MetH) and a B₁₂-dependent regulatory mechanism (the B₁₂ riboswitch), therefore, has broader implications for M. tuberculosis pathogenesis. For example, does M. tuberculosis synthesize B₁₂ and, if so, under what conditions? Alternatively, does M. tuberculosis acquire vitamin B₁₂ from the host? In either case, whether host acquired or synthesized de novo, does B₁₂ availability signal a shift in metabolism to the utilization of all three B₁₂-dependent enzymes? Also, given the presence of only two B₁₂ riboswitches in the M. tuberculosis genome, how is flux through the remaining B₁₂-dependent pathways (NrdZ and MutAB) regulated? Moreover, why has CDC1551 retained an intact B₁₂ riboswitch motif upstream of metE despite the loss of metH function? This question, in particular, might inform the observation that only a small proportion (3 of 20) of sequenced vitamin B₁₂-resistant isolates of CDC1551 and H37Rv metH(B) possessed mutated B₁₂ riboswitch motifs. Through the selective application of the methionine synthase mutants described here, as well as others containing disruptions in B₁₂ biosynthetic pathways, we are actively addressing these issues.

Finally, the disruption of metH in a clinical strain suggests the dispensability of this enzyme for M. tuberculosis pathogenesis. However, the effect that loss of the alternative methionine synthase has on the pathogenesis of M. tuberculosis remains to be determined. The epidemiology of disease caused by strain CDC1551 may be instructive: the strain was originally isolated as the highly infectious agent of a number of tuberculosis outbreaks (21, 22). Is it significant, therefore, that deletion polymorphisms affecting two of the three B₁₂-dependent enzymes (NrdZ and MetH) have been identified in individual M. tuberculosis clinical isolates (7, 19) that, by definition, are in active circulation? That is, does loss of B₁₂-dependent enzymes favor transmission and disease? In this regard, it is intriguing to note that preliminary sequence data from two other clinical isolates, strain C (Mycobacterium tuberculosis Sequencing Project, Broad Institute of Harvard and the Massachusetts Institute of Technology [http://www.broad.mit.edu]) and strain 210 (http://www.tigr.org), have revealed separate sequence polymorphisms in metH. The consequences of these polymorphisms for metH functionality are unknown; however, given that adaptive evolution of M. tuberculosis is dependent entirely on chromosomal rearrangements and mutations, at the very least these observations suggest that the metH locus may be under active selective pressure.

 
This work was supported by grants from the Medical Research Council of South Africa, the National Research Foundation, and the NHLS Trust and by an International Research Scholar's grant from the Howard Hughes Medical Institute (to V.M.).

Preliminary sequence data were obtained from the Broad Institute of Harvard and the Massachusetts Institute of Technology and from the Institute for Genomic Research. We are grateful to Helena Boshoff for the kind gift of M. tuberculosis CDC1551 and for constructively reviewing the manuscript. We also thank Stewart Cole for generously providing the M. tuberculosis H37Rv bacterial artificial chromosome library.

 
* Corresponding author. Mailing address: Molecular Mycobacteriology Research Unit, National Health Laboratory Service, P.O. Box 1038, Johannesburg 2000, South Africa. Phone: 2711-4899370. Fax: 2711-4899397. E-mail for Digby F. Warner: digby.warner{at}nhls.ac.za. E-mail for Stephanie S. Dawes: stephanie.dawes{at}nhls.ac.za

Published ahead of print on 16 February 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, May 2007, p. 3655-3659, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.00040-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Mowa, M. B., Warner, D. F., Kaplan, G., Kana, B. D., Mizrahi, V. (2009). Function and Regulation of Class I Ribonucleotide Reductase-Encoding Genes in Mycobacteria. J. Bacteriol. 191: 985-995 [Abstract] [Full Text]  
• Savvi, S., Warner, D. F., Kana, B. D., McKinney, J. D., Mizrahi, V., Dawes, S. S. (2008). Functional Characterization of a Vitamin B12-Dependent Methylmalonyl Pathway in Mycobacterium tuberculosis: Implications for Propionate Metabolism during Growth on Fatty Acids. J. Bacteriol. 190: 3886-3895 [Abstract] [Full Text]  

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