Sae Woong Park

Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier

Recent advances in liquid chromatography and mass spectrometry have enabled the highly parallel, quantitative measurement of metabolites within a cell and the ability to trace their biochemical fates. In Mycobacterium tuberculosis (Mtb), these advances have highlighted major gaps in our understanding of central carbon metabolism (CCM) that have prompted fresh interpretations of the composition and structure of its metabolic pathways and the phenotypes of Mtb strains in which CCM genes have been deleted. High-throughput screens have demonstrated that small chemical compounds can selectively inhibit some enzymes of Mtbs CCM while sparing homologs in the host. Mtbs CCM has thus emerged as a frontier for both fundamental and translational research.

Evaluating the sensitivity of mycobacterium tuberculosis to biotin deprivation using regulated gene expression

In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens.

Growth of Mycobacteria on Carbon Monoxide and Methanol

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.

Pseudonocardia carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete, and an emended description of the genus Pseudonocardia

A bacterial strain, Y8(T), capable of oxidizing carbon monoxide, was isolated from a soil sample collected from a roadside in Seoul, Korea. On the basis of 16S rRNA gene sequence similarity analyses, strain Y8(T) was shown to belong to the genus Pseudonocardia and was related most closely to the type strain of Pseudonocardia alni (99.6 % similarity). The cells were aerobic and stained Gram-positive, with white aerial mycelium and brown substrate mycelium. The predominant fatty acids were 16 : 0 iso and 16 : 1 iso. The cell-wall peptidoglycan of strain Y8(T) contained meso-diaminopimelic acid. The DNA G+C content was 77 mol%. Strain Y8(T) contained MK-9 as the major menaquinone, which is different from the major menaquinone reported previously in the genus Pseudonocardia, MK-8(H(4)). DNA-DNA relatedness between strain Y8(T) and the type strains of P. alni and Pseudonocardia antarctica was respectively 10 and 63 %. Based on phylogenetic, morphological and chemotaxonomic evidence, it is proposed that strain Y8(T) (=KCCM 42678(T) =JCM 14827(T)) be classified as the type strain of a novel species, Pseudonocardia carboxydivorans sp. nov. An emended description of the genus Pseudonocardia is also presented.

O2- and NO-Sensing Mechanism through the DevSR Two-Component System in Mycobacterium smegmatis

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.

Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features. Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.

Perturbation of Cytochrome c Maturation Reveals Adaptability of the Respiratory Chain in Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis. IMPORTANCE Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis. Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis.

Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.

Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.

Tsukamurella carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete

A Gram-positive, slightly acid-alcohol-fast, carbon monoxide-oxidizing bacterium, strain Y2(T), was isolated from a soil sample collected from a roadside in Seoul, Korea. On the basis of 16S rRNA gene sequence comparative analyses, strain Y2(T) was shown to belong to the genus Tsukamurella and was most closely related to Tsukamurella tyrosinosolvens DSM 44234(T) (GenBank accession no. AY238514; 99.8 %). The predominant fatty acids were C(18 : 1)omega9c and C(16 : 0). The cell-wall peptidoglycan of strain Y2(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. Strain Y2(T) contained galactose and arabinose as the whole cell sugars. The DNA G+C content was 77 mol%. The DNA-DNA relatedness value between strain Y2(T) and T. tyrosinosolvens DSM 44234(T) was 62.7 %. Based on the combination of the carbon source utilization pattern, fatty acid profile, cell-wall chemotype, DNA G+C content and DNA-DNA hybridization experiments, it is proposed that strain Y2(T) (=KCCM 42885(T)=JCM 15482(T)) represents the type strain of a novel species, Tsukamurella carboxydivorans sp. nov.

Cloning and expression analysis of the duplicated genes for carbon monoxide dehydrogenase of Mycobacterium sp. strain JC1 DSM 3803

Carbon monoxide dehydrogenase (CO-DH) is an enzyme catalysing the oxidation of CO to carbon dioxide in Mycobacterium sp. strain JC1 DSM 3803. Cloning of the genes encoding CO-DH from the bacterium and sequencing of overlapping clones revealed the presence of duplicated sets of genes for three subunits of the enzyme, cutB1C1A1 and cutB2C2A2, in operons, and a cluster of genes encoding proteins that may be involved in CO metabolism, including a possible transcriptional regulator. Phylogenetic analysis based on the amino acid sequences of large subunits of CO-DH suggested that the CO-DHs of Mycobacterium sp. JC1 and other mycobacteria are distinct from those of other types of bacteria. The growth phenotype of mutant strains lacking cutA genes and of a corresponding complemented strain showed that both of the duplicated sets of CO-DH genes were functional in this bacterium. Transcriptional fusions of the cutB genes with lacZ revealed that the cutBCA operons were expressed regardless of the presence of CO and were further inducible by CO. Primer extension analysis indicated two promoters, one expressed in the absence of CO and the other induced in the presence of CO. This is believed to be the first report to show the presence of multiple copies of CO-DH genes with identical sequences and in close proximity in carboxydobacteria, and to present the genetic evidence for the function of the genes in mycobacteria.