scholarly journals An In Vitro Enzyme System for the Production of myo-Inositol from Starch

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Tomoko Fujisawa ◽  
Shohei Fujinaga ◽  
Haruyuki Atomi
Keyword(s):  
Enzyme System ◽  
Scale Production ◽  
Inositol Monophosphatase ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Conversion Rates ◽  
Maltodextrin Phosphorylase ◽  
Phosphate Synthase

ABSTRACT We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima. The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD+, resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD+ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. IMPORTANCE myo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol.

scholarly journals AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

2015 ◽  
Vol 197 (24) ◽  
pp. 3788-3796 ◽  
Author(s):  
Takayuki Kuge ◽  
Haruhiko Teramoto ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Large Scale ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Primary Role ◽  
Promoter Regions ◽  
Independent Manner ◽  
Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

scholarly journals A Cytochrome b 5 -Containing Plastid-Located Fatty Acid Desaturase from Chlamydomonas reinhardtii

2012 ◽  
Vol 11 (7) ◽  
pp. 856-863 ◽  
Author(s):  
Simone Zäuner ◽  
Wibke Jochum ◽  
Tara Bigorowski ◽  
Christoph Benning
Keyword(s):  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
Fluorescent Protein ◽  
Acyl Chain ◽  
Transit Peptide ◽  
Primary Electron ◽  
Chloroplast Targeting ◽  
Content Type ◽  
Cell Extracts

ABSTRACT Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn- 2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b 5 domain, distinguishing it from other known plastid desaturases. Cytochrome b 5 is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b 5 domain of CrΔ4FAD showed that it is functional in vitro . Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas . Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.

scholarly journals The Antidepressant Sertraline Provides a Promising Therapeutic Option for Neurotropic Cryptococcal Infections

2012 ◽  
Vol 56 (7) ◽  
pp. 3758-3766 ◽  
Author(s):  
Bing Zhai ◽  
Cheng Wu ◽  
Linqi Wang ◽  
Matthew S. Sachs ◽  
Xiaorong Lin
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Fungal Infections ◽  
Therapeutic Option ◽  
Susceptibility Gene ◽  
Content Type ◽  
Fungal Burden ◽  
Cell Extracts ◽  
Cns Penetration

ABSTRACTTherapeutic treatment for systemic mycoses is severely hampered by the extremely limited number of antifungals. The difficulty of treatment of fungal infections in the central nervous system is further compounded by the poor central nervous system (CNS) penetration of most antifungals due to the blood-brain barrier. Only a few fungistatic azole drugs, such as fluconazole, show reasonable CNS penetration. Here we demonstrate that sertraline (Zoloft), the most frequently prescribed antidepressant, displays potent antifungal activity againstCryptococcus neoformans, the major causative agent of fungal meningitis. Inin vitroassays, this neurotropic drug is fungicidal to all naturalCryptococcusisolates tested at clinically relevant concentrations. Furthermore, sertraline interacts synergistically or additively with fluconazole againstCryptococcus. Importantly, consistent with ourin vitroobservations, sertraline used alone reduces the brain fungal burden at an efficacy comparable to that of fluconazole in a murine model of systemic cryptococcosis. It works synergistically with fluconazole in reducing the fungal burden in brain, kidney, and spleen. In contrast to its potency againstCryptococcus, sertraline is less effective against strains ofCandidaspecies and its interactions with fluconazole againstCandidastrains are often antagonistic. Therefore, our data suggest the unique application of sertraline against cryptococcosis. To understand the antifungal mechanisms of sertraline, we screened a whole-genome deletion collection ofSaccharomyces cerevisiaefor altered sertraline susceptibility. Gene ontology analyses of selected mutations suggest that sertraline perturbs translation.In vitrotranslation assays using fungal cell extracts show that sertraline inhibits protein synthesis. Taken together, our findings indicate the potential of adopting this antidepressant in treating cryptococcal meningitis.

scholarly journals Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

2012 ◽  
Vol 79 (5) ◽  
pp. 1619-1628 ◽  
Author(s):  
Michihiro Ito ◽  
Ikuo Sato ◽  
Masumi Ishizaka ◽  
Shin-ichiro Yoshida ◽  
Motoo Koitabashi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Genomic Library ◽  
Enzyme System ◽  
Head Blight ◽  
Wheat Seedlings ◽  
Content Type ◽  
Cytochrome P450 System ◽  
Redox Partner ◽  
Catabolic Genes

ABSTRACTDeoxynivalenol (DON) is a natural toxin of fungi that causeFusariumhead blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON inSphingomonassp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 geneddnAwas cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent toddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstitutedin vitroin an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM−1s−1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

scholarly journals Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Daniel Braga ◽  
Mahmudul Hasan ◽  
Tabea Kröber ◽  
Daniel Last ◽  
Gerald Lackner
Keyword(s):  
Large Scale ◽  
Reductase Activity ◽  
Protein A ◽  
Model Organisms ◽  
Gram Negative Bacteria ◽  
Scale Production ◽  
Metabolome Analysis ◽  
Gram Negative ◽  
Content Type

ABSTRACT Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420. Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum. These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future. IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

scholarly journals Mechanisms Involved in Fe(III) Respiration by the Hyperthermophilic Archaeon Ferroglobus placidus

2015 ◽  
Vol 81 (8) ◽  
pp. 2735-2744 ◽  
Author(s):  
Jessica A. Smith ◽  
Muktak Aklujkar ◽  
Carla Risso ◽  
Ching Leang ◽  
Ludovic Giloteaux ◽  
...  
Keyword(s):  
Electron Acceptor ◽  
Expression Patterns ◽  
Hyperthermophilic Archaea ◽  
Binding Motifs ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Type Iv ◽  
Cell Extracts ◽  
Sds Page ◽  
Type Iv Pilin

ABSTRACTThe hyperthermophilic archaeonFerroglobus placiduscan utilize a wide variety of electron donors, including hydrocarbons and aromatic compounds, with Fe(III) serving as an electron acceptor. In Fe(III)-reducing bacteria that have been studied to date, this process is mediated byc-type cytochromes and type IV pili. However, there currently is little information available about how this process is accomplished in archaea.In silicoanalysis of theF. placidusgenome revealed the presence of 30 genes coding for putativec-type cytochrome proteins (more than any other archaeon that has been sequenced to date), five of which contained 10 or more heme-binding motifs. When cell extracts were analyzed by SDS-PAGE followed by heme staining, multiple bands corresponding toc-type cytochromes were detected. Different protein expression patterns were observed inF. placiduscells grown on soluble and insoluble iron forms. In order to explore this result further, transcriptomic studies were performed. Eight genes corresponding to multihemec-type cytochromes were upregulated whenF. placiduswas grown with insoluble Fe(III) oxide compared to soluble Fe(III) citrate as an electron acceptor. Numerous archaella (archaeal flagella) also were observed on Fe(III)-grown cells, and genes coding for two type IV pilin-like domain proteins were differentially expressed in Fe(III) oxide-grown cells. This study provides insight into the mechanisms for dissimilatory Fe(III) respiration by hyperthermophilic archaea.

scholarly journals A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Jian-qiang Jin ◽  
Shin-ichi Hachisuka ◽  
Takaaki Sato ◽  
Tsuyoshi Fujiwara ◽  
Haruyuki Atomi
Keyword(s):  
Lipoic Acid ◽  
Oxidative Decarboxylation ◽  
Biotin Synthase ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Archaeal Species ◽  
Lipoyl Synthase ◽  
H Protein

ABSTRACT Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea. IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea. Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

scholarly journals Enantioselective Dechlorination of Polychlorinated Biphenyls inDehalococcoides mccartyiCG1

2018 ◽  
Vol 84 (21) ◽  
Author(s):  
Ling Yu ◽  
Qihong Lu ◽  
Lan Qiu ◽  
Guofang Xu ◽  
Yanhong Zeng ◽  
...  
Keyword(s):  
Polychlorinated Biphenyls ◽  
Critical Role ◽  
Enrichment Factors ◽  
Reductive Dehalogenation ◽  
Content Type ◽  
Whole Cells ◽  
Dehalococcoides Mccartyi ◽  
Cell Extracts

ABSTRACTReductive dehalogenation mediated by organohalide-respiring bacteria plays a critical role in the global cycling of organohalides. Nonetheless, information on the dehalogenation enantioselectivity of organohalide-respiring bacteria remains limited. In this study, we report the enantioselective dechlorination of chiral polychlorinated biphenyls (PCBs) byDehalococcoides mccartyiCG1. CG1 preferentially removed halogens from the (−)-enantiomers of the three major environmentally relevant chiral PCBs (PCB174, PCB149, and PCB132), and the enantiomer compositions of the dechlorination products depended on their parent organohalides. Thein vitroassays with crude cell extracts or concentrated whole cells and thein vivoexperiments with living cells showed similar enantioselectivities, in contrast with the distinct enantiomeric enrichment factors (εER) of the substrate chiral PCBs. Additionally, these results suggest that concentrated whole cells might be an alternative to crude cell extracts inin vitrotests of reductive dehalogenation activities. The enantioselective dechlorination of other chiral PCBs that we resolved via gas chromatography further confirmed the preference of CG1 for the (−)-enantiomers.IMPORTANCEA variety of agrochemicals and pharmaceuticals are chiral. Due to the enantioselectivity in biological processes, enantiomers of chiral compounds may have different environmental occurrences, fates, and ecotoxicologies. Many chiral organohalides exist in anaerobic or anoxic soils and sediments, and organohalide-respiring bacteria play a major role in the environmental attenuation and global cycling of these chiral organohalides. Therefore, it is important to investigate the dehalogenation enantioselectivity of organohalide-respiring bacteria. This study reports the discovery of enantioselective dechlorination of chiral PCBs byDehalococcoides mccartyiCG1, which provides insights into the dehalogenation enantioselectivity ofDehalococcoidesand may shed light on future PCB bioremediation efforts to prevent enantioselective biological side effects.

scholarly journals Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance

2012 ◽  
Vol 11 (7) ◽  
pp. 932-942 ◽  
Author(s):  
Min Hsu ◽  
Eun Young Yu ◽  
Ondrej Sprušanský ◽  
Michael J. McEachern ◽  
Neal F. Lue
Keyword(s):  
Functional Analysis ◽  
Budding Yeast ◽  
Kluyveromyces Lactis ◽  
Telomere Maintenance ◽  
Physical Interaction ◽  
Content Type ◽  
Terminal Domain ◽  
Cell Extracts

ABSTRACT Est1 and Ebs1 in Saccharomyces cerevisiae are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast Kluyveromyces lactis (named Kl Est1). We show that similar to other Est1s, Kl Est1 is required for normal telomere maintenance in vivo and full telomerase primer extension activity in vitro . Kl Est1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of Kl Est1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between Kl Est1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to S. cerevisiae Ebs1 ( Sc Ebs1) (but not Sc Est1), Kl Est1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of Kl Est1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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