F420H2 Is Required for Phthiocerol Dimycocerosate Synthesis in Mycobacteria.

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.

Characterization of the MSMEG_2631 gene (mmp) encoding a multidrug and toxic compound extrusion (MATE) family protein in Mycobacterium smegmatis and exploration of its polyspecific nature using biolog phenotype microarray.

In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions.

Glucose 6-phosphate accumulation in mycobacteria: implications for a novel F420-dependent anti-oxidant defense system.

Glucose 6-phosphate (G6P) is a metabolic intermediate with many possible cellular fates. In mycobacteria, G6P is a substrate for an enzyme, F(420)-dependent glucose-6-phosphate dehydrogenase (Fgd), found in few bacterial genera. Intracellular G6P levels in six Mycobacterium sp. were remarkably higher ( approximately 17-130-fold) than Escherichia coli and Bacillus megaterium. The high G6P level in Mycobacterium smegmatis may result from 10-25-fold higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase when grown on glucose, glycerol, or acetate compared with B. megaterium and E. coli. In M. smegmatis this coincided with up-regulation of the first gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, when acetate was the carbon source, suggesting a cellular program for maintaining high G6P levels. G6P was depleted in cells under oxidative stress induced by redox cycling agents plumbagin and menadione, whereas an fgd mutant of M. smegmatis used G6P less well under such conditions. The fgd mutant was more sensitive to these agents and, in contrast to wild type, was defective in its ability to reduce extracellular plumbagin and menadione. These data suggest that intracellular G6P in mycobacteria serves as a source of reducing power and, with the mycobacteria-specific Fgd-F(420) system, plays a protective role against oxidative stress.

Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid.

Aldehyde oxidoreductase (carboxylic acid reductase) catalyzes the Mg(2+), ATP and NADPH dependent reduction of carboxylic acids to their corresponding aldehydes. The identification of the gene from Nocardia sp. NRRL 5646 and its expression in E. coli BL21-CodonPlus(®)(DE3)-RP/pHAT305 provided an avenue to develop a biocatalyst for reduction of carboxylic acids. In addition to aromatic acids, the recombinant carboxylic acid reductase also accepts several aliphatic mono, di and tri carboxylic acids as substrates. A recently identified Nocardia sp., phosphopantetheinyl transferase gene (npt) enhanced the activity of carboxylic acid reductase. Coexpression of car and npt in E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.83 resulted in a purified recombinant carboxylic acid reductase with improved specific activity of 2.2U/mg protein. The utility of the recombinant carboxylic acid reductase as a biocatalyst has been demonstrated using vanillic acid as substrate. E. coli BL21-CodonPlus(®)(DE3)-RP/pHAT305 expressing Car reduced 50% of vanillic acid to vanillin in 10h. E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.83 resting cells expressing Car and Npt reduced 90% of vanillic acid to vanillin in 6h. Enhanced, in vivo cofactor NADPH regeneration by glucose dehydrogenase (gdh) was accomplished using E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.85, that carried car, npt, and gdh. Resting cell reactions using E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.85 with in situ product removal by XAD-2 resin efficiently reduced 5g/L of vanillic and benzoic acids within 2h.

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria.

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25 degrees C), stoppered bottles incubated and sampled at 37 to 85 degrees C showed more methane upon analysis than bottles incubated at 25 degrees C: values at 50, 63, and 85 degrees C were 109, 126, and 125%, respectively, of the 25 degrees C value. All variation between 4 and 50 degrees C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63 degrees C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.

Pharmacy cases in Second Life: an elective course.

Interactive pharmacy case studies are an essential component of the pharmacy curriculum. We recently developed an elective course at the Rangel College of Pharmacy in pharmacy case studies for second- and third-year Doctor of Pharmacy students using Second Life® (SL), an interactive three-dimensional virtual environment that simulates the real world. This course explored the use of SL for education and training in pharmacy, emphasizing a case-based approach. Virtual worlds such as SL promote inquiry-based learning and conceptual understanding, and can potentially develop problem-solving skills in pharmacy students. Students were presented ten case scenarios that primarily focused on drug safety and effective communication with patients. Avatars, representing instructors and students, reviewed case scenarios during sessions in a virtual classroom. Individually and in teams, students participated in active-learning activities modeling both the pharmacist's and patient's roles. Student performance and learning were assessed based on SL class participation, activities, assignments, and two formal, essay-type online exams in Blackboard 9. Student course-evaluation results indicated favorable perceptions of content and delivery. Student comments included an enhanced appreciation of practical issues in pharmacy practice, flexibility of attendance, and an increased ability to focus on course content. Excellent student participation and performance in weekly active-learning activities translated into positive performance on subsequent formal assessments. Students were actively engaged and exposed to topics pertinent to pharmacy practice that were not covered in the required pharmacy curriculum. The multiple active-learning assignments were successful in increasing students' knowledge, and provided additional practice in building the communication skills beneficial for students preparing for experiential clinical rotations.

Mycobacterium smegmatis mc2 155 fbiC and MSMEG_2392 are involved in triphenylmethane dye decolorization and coenzyme F420 biosynthesis.

Mycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis mc2 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme F420. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis mc2 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme F420 and FO, the MSMEG_2392 mutant contained FO but not coenzyme F420. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme F420.

Reduction of carboxylic acids by Nocardia aldehyde oxidoreductase requires a phosphopantetheinylated enzyme.

Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.

Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis.

PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.

Large-scale production of coenzyme F420-5,6 by using Mycobacterium smegmatis.

Production of coenzyme F420 and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F420-5,6. M. smegmatis produced 1 to 3 micromol of intracellular F420 per liter of culture, which was more than the 0.85 to 1.0 micromol of F420-2 per liter usually obtained with Methanobacterium thermoautotrophicum and approximately 10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F420 than with previous methods.

Demonstration that fbiC is required by Mycobacterium bovis BCG for coenzyme F(420) and FO biosynthesis.

Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated Mycobacterium bovis strain BCG (M. bovis) mutants that could not make coenzyme F(420) were identified. Four independent mutants that could not make F(420) or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the M. bovis homologue of the Mycobacterium tuberculosis gene Rv1173, which we have named fbiC. Complementation of an M. bovis FbiC(-) mutant with fbiC restored the F(420) phenotype. These data demonstrate that fbiC is essential for F(420) production and that FbiC participates in a portion of the F(420) biosynthetic pathway between pyrimidinedione and FO. Homologues of fbiC were found in all 11 microorganisms that have been fully sequenced and that are known to make F(420). Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the M. bovis FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the M. bovis FbiC. Histidine-tagged FbiC overexpressed in Escherichia coli produced a fusion protein of the molecular mass predicted from the M. bovis BCG sequence (approximately 95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.

Nocardia sp. carboxylic acid reductase: cloning, expression, and characterization of a new aldehyde oxidoreductase family.

We have cloned, sequenced, and expressed the gene for a unique ATP- and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes. Recombinant CAR containing an N-terminal histidine affinity tag had K(m) values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes. car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.

Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I.

The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.

Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates.

Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes. They differ in their choice of substrates and use either tRNAGlu or both tRNAGlu and tRNAGln. Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown. A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain. To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. pylori contains one tRNAGlu and one tRNAGln species, whereas A. ferrooxidans possesses two of each. We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities. The H. pylori GluRS1 acylated only tRNAGlu, whereas GluRS2 was specific solely for tRNAGln. The A. ferrooxidans GluRS2 preferentially charged tRNA(UUG)(Gln). Conversely, A. ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the tRNA(CUG)(Gln) species. These three tRNA species have two structural elements in common, the augmented D-helix and a deletion of nucleotide 47. It appears that the discriminating or nondiscriminating natures of different GluRS enzymes have been derived by the coevolution of protein and tRNA structure. The coexistence of the two GluRS enzymes in one organism may lay the groundwork for the acquisition of the canonical glutaminyl-tRNA synthetase by lateral gene transfer from eukaryotes.