scholarly journals Steryl Ester Formation and Accumulation in Steroid-Degrading Bacteria

2019 ◽  
Vol 86 (2) ◽  
Author(s):  
Johannes Holert ◽  
Kirstin Brown ◽  
Ameena Hashimi ◽  
Lindsay D. Eltis ◽  
William W. Mohn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lipid Droplets ◽  
Steryl Ester ◽  
Biotechnological Production ◽  
Fatty Acid Substrate ◽  
Content Type ◽  
Adverse Conditions ◽  
Steryl Esters ◽  
Ester Formation ◽  
Acid Substrate

ABSTRACT Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics. IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

scholarly journals Steryl ester formation and accumulation in steroid-degrading bacteria

2019 ◽  
Author(s):  
Johannes Holert ◽  
Kirstin Brown ◽  
Ameena Hashimi ◽  
Lindsay D. Eltis ◽  
William W. Mohn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Lipid Droplets ◽  
Steryl Ester ◽  
Biotechnological Production ◽  
Fatty Acid Substrate ◽  
Adverse Conditions ◽  
Steryl Esters ◽  
Ester Formation ◽  
Acid Substrate

ABSTRACTSteryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl- and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCEOleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

scholarly journals The genetic requirements of fatty acid import by Mycobacterium tuberculosis within macrophages

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Evgeniya V Nazarova ◽  
Christine R Montague ◽  
Lu Huang ◽  
Thuy La ◽  
David Russell ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
The Novel ◽  
Novel Genes ◽  
Fatty Acid Substrate ◽  
Human Macrophages ◽  
Fatty Acid Transporter ◽  
Acid Transporter ◽  
Acid Substrate

Mycobacterium tuberculosis (Mtb) imports and metabolizes fatty acids to maintain infection within human macrophages. Although this is a well-established paradigm, the bacterial factors required for fatty acid import are poorly understood. Previously, we found that LucA and Mce1 are required for fatty acid import in Mtb (Nazarova et al., 2017). Here, we identified additional Mtb mutants that have a reduced ability to import a fluorescent fatty acid substrate during infection within macrophages. This screen identified the novel genes as rv2799 and rv0966c as be necessary for fatty acid import and confirmed the central role for Rv3723/LucA and putative components of the Mce1 fatty acid transporter (Rv0200/OmamB, Rv0172/Mce1D, and Rv0655/MceG) in this process.

Adhesion and Growth of Anaerobic Biofilms on Ion Exchange Resins

1986 ◽  
Vol 21 (4) ◽  
pp. 486-495 ◽  
Author(s):  
R.F.G. Selle Sardi ◽  
W. Bulani ◽  
W.L. Cairns ◽  
N. Kosaric
Keyword(s):  
Metabolic Activity ◽  
Cation Binding ◽  
Fatty Acid Substrate ◽  
Anaerobic Reactors ◽  
Initial Adhesion ◽  
Rapid Changes ◽  
Exchange Resins ◽  
Biofilm Development ◽  
First Time ◽  
Acid Substrate

Abstract Ion exchanger beads are explored as aids in accelerating the development of anaerobic biofilms for use in advanced anaerobic reactors. Initial adhesion and subsequent changes in adhesion and growth of anaerobic biofilms (as reflected in total supported biomass and metabolic activity) were monitored on different ion exchangers (strong cation, strong anion and weak anion) over a period of 12 days. Metabolic activity was recorded for the first time in this type of study using ATP biolumininescence assays which allowed monitoring of rapid changes in the biofilm development. Results indicate that the strong cation exchanger is a better overall substratum for anaerobic biofilm development due to its favorable property of dialent cation binding and adsorption of volatile fatty acid substrate for methanogens.

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.

scholarly journals Esterifications with 2-(Trimethylsilyl)ethyl 2,2,2-Trichloroacetimidate

Organics ◽  
2021 ◽  
Vol 2 (1) ◽  
pp. 17-25
Author(s):  
Wenhong Lin ◽  
Shea T. Meyer ◽  
Shawn Dormann ◽  
John D. Chisholm
Keyword(s):  
Carboxylic Acid ◽  
High Yield ◽  
Deuterium Labeling ◽  
Ester Formation ◽  
Acid Substrate

2-(Trimethylsilyl)ethyl 2,2,2-trichloroacetimidate is readily synthesized from 2-trimethylsilylethanol in high yield. This imidate is an effective reagent for the formation of 2-trimethylsilylethyl esters without the need for an exogenous promoter or catalyst, as the carboxylic acid substrate is acidic enough to promote ester formation without an additive. A deuterium labeling study indicated that a β-silyl carbocation intermediate is involved in the transformation.

scholarly journals Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

2015 ◽  
Vol 81 (6) ◽  
pp. 2215-2225 ◽  
Author(s):  
Sabrina Witthoff ◽  
Katja Schmitz ◽  
Sebastian Niedenführ ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biotechnological Production ◽  
Methanol Metabolism ◽  
Initial Oxidation ◽  
Content Type ◽  
Oxidation Of Methanol ◽  
Bacillus Methanolicus ◽  
Production Processes ◽  
Starting Point ◽  
Limited Application

ABSTRACTMethanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacteriumCorynebacterium glutamicumtoward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase fromBacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway ofBacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinantC. glutamicumstrain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of aC. glutamicumΔaldΔadhEmutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineeredC. glutamicumstrains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

scholarly journals A Novel 6-Benzyl Ether Benzoxaborole Is Active against Mycobacterium tuberculosisIn Vitro

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Nipul Patel ◽  
Theresa O'Malley ◽  
Yong-Kang Zhang ◽  
Yi Xia ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Medium ◽  
Inhibitory Concentration ◽  
Solid Medium ◽  
Eukaryotic Cells ◽  
Intracellular Bacteria ◽  
Benzyl Ether ◽  
Content Type ◽  
Resistant Mutants

ABSTRACT We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis. The compound had an MIC of 2 μM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 μM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 μM) without cytotoxicity against eukaryotic cells (IC50 > 100 μM). We isolated resistant mutants (MIC ≥ 100 μM), which had mutations in Rv1683, Rv3068c, and Rv0047c.

scholarly journals Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Amy Ellis ◽  
Alexis Balgeman ◽  
Mark Rodgers ◽  
Cassaundra Updike ◽  
Jaime Tomko ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Mhc Class Ii ◽  
Nonhuman Primates ◽  
Content Type ◽  
Host Immune Responses ◽  
Cell Responses ◽  
Cynomolgus Macaques ◽  
Mhc Haplotype

ABSTRACT Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis. Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis. Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis. All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.

scholarly journals Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Zhaojing Zong ◽  
Wei Jing ◽  
Jin Shi ◽  
Shu'an Wen ◽  
Tingting Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Novel Mutation ◽  
Multidrug Resistant ◽  
23S Rrna ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Significant Difference ◽  
Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

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