Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd-and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C 3 ) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B 12 -derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B 12 is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Mycobacterium tuberculosis is an obligate human pathogen that is expected to adapt metabolically to conditions that are often hostile and nutrient poor during successive cycles of infection, replication, persistence, and transmission. In particular, glucose deficiency and an abundance of fatty acids are thought to dictate mycobacterial metabolism during infection (3, 35), consistent with the complex repertoire of genes involved in lipid metabolism in the M. tuberculosis genome (10). Subsistence on fatty acids requires the sequential action of the catabolic ␤-oxidation cycle and, where glycolytic substrates are limiting, the anaplerotic glyoxylate cycle, which enables the assimilation of derivative two-carbon (C 2 ) acetyl-coenzyme A (CoA) subunits (37). In addition to producing acetyl-CoA, ␤-oxidation of odd-and branched-chain fatty acids yields the C 3 subunit propionyl-CoA. This metabolite can also be generated by the catabolism of branched-chain amino acids (24) and cholesterol. Recently, a cassette of genes involved in the catabolism of the A and B rings of cholesterol to propionyl-CoA, pyruvate, and other metabolites was identified in actinomycetes, including members of the M. tuberculosis complex (27,52). Although the relevance of cholesterol as a carbon source for M. tuberculosis in vivo has yet to be established, the likely action of this catabolic pathway during intracellular growth and survival of M. tuberculosis (52) suggests that it may constitute an additional, and potentially significant, source of propionylCoA in this pathogen.Propionyl-CoA is a key precursor in several lipid biosynthetic pathways in M. tuberculosis (28); however, while providing a high-energy metabolite, the accumulation of propionate is toxic to the cell, and as such, efficient mechanisms are required for its disposal (5). This dual nature implies a central role for propionate metabolism in the growth and persistence of M. tuberculosis in vivo (18,37). Evi...

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance. (1), a Cfamily DNA polymerase implicated in error-prone bypass of DNA lesions. Loss of DnaE2 activity renders Mtb hypersensitive to DNA damage and eliminates induced mutagenesis. Moreover, dnaE2 deletion attenuates virulence and reduces the frequency of drug resistance in vivo. Mtb contains two DnaE-type polymerases; the other, DnaE1, provides essential, high-fidelity replicative polymerase function (1). However, the basis for the functional specialization of the DnaE subunits remains unclear (2, 3). Although structural determinants such as active-site architecture contribute significantly to inherent fidelity, it is possible that differential interactions with other DNA metabolic proteins modulate polymerase function.Bacterial genomes containing a DnaE2-type DNA polymerase almost invariably encode a homolog of ImuB (4-6), a putative Yfamily polymerase that is usually present in a LexA-regulated imuA-imuB-dnaE2 gene cassette (5). In Caulobacter crescentus, both ImuB and ImuA are required for induced mutagenesis and damage tolerance (6) whereas plasmid-encoded DnaE2 and ImuB mediate UV-induced mutagenesis in Deinococcus deserti (7). Although distributed widely across the bacterial domain, the imuA-imuB-dnaE2 cassette is not found in organisms possessing umuDC homologs (5). This suggests that the encoded proteins perform an analogous function to DNA polymerase V (8), the Y-family member required for damage-induced mutagenesis in Escherichia coli (9).Mtb contains a putative SOS-inducible operon, Rv3395c-Rv3394c (1, 10), locat...

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

The emergence of drug-resistant pathogens poses a major threat to public health. Although influenced by multiple factors, high-level resistance is often associated with mutations in target-encoding or related genes. The fitness cost of these mutations is, in turn, a key determinant of the spread of drug-resistant strains. Rifampicin (RIF) is a frontline anti-tuberculosis agent that targets the rpoB-encoded β subunit of the DNA-dependent RNA polymerase (RNAP). In Mycobacterium tuberculosis (Mtb), RIF resistance (RIFR) maps to mutations in rpoB that are likely to impact RNAP function and, therefore, the ability of the organism to cause disease. However, while numerous studies have assessed the impact of RIFR on key Mtb fitness indicators in vitro, the consequences of rpoB mutations for pathogenesis remain poorly understood. Here, we examine evidence from diverse bacterial systems indicating very specific effects of rpoB polymorphisms on cellular physiology, and consider these observations in the context of Mtb. In addition, we discuss the implications of these findings for the propagation of clinically relevant RIFR mutations. While our focus is on RIF, we also highlight results which suggest that drug-independent effects might apply to a broad range of resistance-associated mutations, especially in an obligate pathogen increasingly linked with multidrug resistance.

We observed vitamin B 12 -mediated growth inhibition of Mycobacterium tuberculosis strain CDC1551. The B 12 sensitivity was mapped to a polymorphism in metH, encoding a coenzyme B 12 -dependent methionine synthase. Vitamin B 12 -resistant suppressor mutants of CDC1551 containing mutations in a B 12 riboswitch upstream of the metE gene, which encodes a B 12 -independent methionine synthase, were isolated. Expression analysis confirmed that the B 12 riboswitch is a transcriptional regulator of metE in M. tuberculosis.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 3Ј 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 B 12 -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 B 12 (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 B 12 moiety in MetH (6).Little is known about the regulation of corresponding B 12 -dependent and B 12 -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 B 12 cofactor necessary to enable MetH to function as the sole methionine synthase in vitro, despite encoding an apparently complete vitamin B 12 biosynthetic pathway (17).In genomes containing corresponding B 12 -dependent and B 12 -independent enzymes, the activity of the B 12 -independent enzyme is often subject to regulation by a B 12 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 B 12 ) to the riboswitch results in the formation of an alternative RNA structure that attenuates transcription or translation (12). The association of riboswitchmediated regulation with many essent...

SUMMARY The global tuberculosis (TB) control effort is focused on interrupting transmission of the causative agent, Mycobacterium tuberculosis, through chemotherapeutic intervention in active infectious disease. The insufficiency of this approach is manifest in the inexorable annual increase in TB infection and mortality rates and the emergence of multidrug-resistant isolates. Critically, the limited efficacy of the current frontline anti-TB drug combination suggests that heterogeneity of host and bacillary physiologies might impair drug activity. This review explores the possibility that strategies enabling adaptation of M. tuberculosis to hostile in vivo conditions might contribute to the subversion of anti-TB chemotherapy. In particular, evidence that infecting bacilli are exposed to environmental and host immune-mediated DNA-damaging insults suggests a role for error-prone DNA repair synthesis in the generation of chromosomally encoded antibiotic resistance mutations. The failure of frontline anti-TB drugs to sterilize a population of susceptible bacilli is independent of genetic resistance, however, and instead implies the operation of alternative tolerance mechanisms. Specifically, it is proposed that the emergence of persister subpopulations might depend on the switch to an altered metabolic state mediated by the stringent response alarmone, (p)ppGpp, possibly involving some or all of the many toxin-antitoxin modules identified in the M. tuberculosis genome.

The emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis remains a major concern of tuberculosis control programmes worldwide, as treatment depends on low-efficacy, toxic compounds that often lead to poor outcomes. M. tuberculosis develops drug resistance exclusively through chromosomal mutations, in particular single-nucleotide polymorphisms. Moreover, in laboratory assays the organism exhibits a spontaneous mutation rate that is at the lower end of the bacterial spectrum. Despite this, whole-genome sequencing technology has identified unexpected genetic diversity among clinical M. tuberculosis populations. This suggests that the mycobacterial mutation rate may be modulated within the host and, in turn, implies a potential role for constitutive and/or transient mutator strains in adaptive evolution. It also raises the possibility that environmental factors might act as key mutagens during M. tuberculosis infection. Here we consider the elements that might influence the mycobacterial mutation rate in vivo and evaluate the potential roles of constitutive and transient mutator states in the generation of drug resistance mutations. In addition, we identify key research questions that will influence future efforts to develop novel therapeutic strategies for a disease that continues to impose a significant global health burden.

Vitamin B12-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B12 in vitro, it is uncertain whether the organism is able to scavenge B12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B12. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B12 and related corrinoids in vitro. Our results establish an alternative to the canonical BtuCD-type system for B12 uptake in M. tuberculosis, and elucidate a role in B12 metabolism for an ABC protein implicated in chronic mycobacterial infection.