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Eric E Allen - One of the best experts on this subject based on the ideXlab platform.
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Genetic Suppression of lethal mutations in fatty acid biosynthesis mediated by a secondary lipid synthase
Applied and Environmental Microbiology, 2021Co-Authors: Marco N Allemann, Eric E AllenAbstract:The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes is a unique feature of certain marine Gammaproteobacteria inhabiting high-pressure and/or low temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated Type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as EPA (20:5n-3) and DHA (22:6n-3) are produced by a hybrid polyketide/fatty acid synthase - encoded by the pfa genes - also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by several-fold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to Suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD, malonyl CoA-acyl carrier protein transacylase, coding sequence. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo The co-occurrence of independent, yet compensatory fatty acid biosynthetic pathways in select marine bacteria may provide Genetic redundancy to optimize fitness under extreme conditions.ImportanceA defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two Genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a Genetic approach, new insights have been gained into the interactions between these two biosynthetic pathways. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be non-essential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to Genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
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Genetic Suppression of lethal mutations in fatty acid biosynthesis mediated by a secondary lipid synthase
Applied and Environmental Microbiology, 2021Co-Authors: Marco N Allemann, Eric E AllenAbstract:The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to Suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide Genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two Genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a Genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to Genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
Scott M Landfear - One of the best experts on this subject based on the ideXlab platform.
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transient Genetic Suppression facilitates generation of hexose transporter null mutants in leishmania mexicana
Molecular Microbiology, 2013Co-Authors: Xiuhong Feng, Dayana Rodriguezcontreras, Tamsen Polley, Lonfye Lye, David W Scott, Richard Burchmore, Stephen M Beverley, Scott M LandfearAbstract:The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2 and LmxGT3. Functional and Genetic studies of a cluster null mutant (Δlmxgt1-3) have dissected the roles of these proteins in Leishmania metabolism and virulence. However, null mutants were recovered at very low frequency, and comparative genome hybridizations revealed that Δlmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplification of a region on chromosome 29 not amplified in wild type parasites. These data suggested a model where this 29-40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the presence of individual overexpressed open reading frames covering the 29-40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facilitate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 overexpression was not required for Δlmxgt1-3 viability. These studies provide the first evidence that Genetic Suppression can occur by providing critical biological functions transiently. This novel form of Genetic Suppression may extend to other genes, pathways and organisms.
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transient Genetic Suppression facilitates generation of hexose transporter null mutants in leishmania mexicana
Molecular Microbiology, 2013Co-Authors: Xiuhong Feng, Dayana Rodriguezcontreras, Tamsen Polley, David W Scott, Richard Burchmore, Stephen M Beverley, Scott M LandfearAbstract:Summary The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2 and LmxGT3. Functional and Genetic studies of a cluster null mutant (Dlmxgt1-3) have dissected the roles of these proteins in Leishma- nia metabolism and virulence. However, null mutants were recovered at very low frequency, and compara- tive genome hybridizations revealed that Dlmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplificationofaregiononchromosome29notampli- fied in wild type parasites. These data suggested a model where this 29-40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the pres- enceofindividualoverexpressedopenreadingframes covering the 29-40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facili- tate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 over- expression was not required for Dlmxgt1-3 viability. These studies provide the first evidence that Genetic Suppression can occur by providing critical biological
Marco N Allemann - One of the best experts on this subject based on the ideXlab platform.
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Genetic Suppression of lethal mutations in fatty acid biosynthesis mediated by a secondary lipid synthase
Applied and Environmental Microbiology, 2021Co-Authors: Marco N Allemann, Eric E AllenAbstract:The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes is a unique feature of certain marine Gammaproteobacteria inhabiting high-pressure and/or low temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated Type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as EPA (20:5n-3) and DHA (22:6n-3) are produced by a hybrid polyketide/fatty acid synthase - encoded by the pfa genes - also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by several-fold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to Suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD, malonyl CoA-acyl carrier protein transacylase, coding sequence. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo The co-occurrence of independent, yet compensatory fatty acid biosynthetic pathways in select marine bacteria may provide Genetic redundancy to optimize fitness under extreme conditions.ImportanceA defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two Genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a Genetic approach, new insights have been gained into the interactions between these two biosynthetic pathways. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be non-essential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to Genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
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Genetic Suppression of lethal mutations in fatty acid biosynthesis mediated by a secondary lipid synthase
Applied and Environmental Microbiology, 2021Co-Authors: Marco N Allemann, Eric E AllenAbstract:The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to Suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide Genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two Genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a Genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to Genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
Alexander Bauer - One of the best experts on this subject based on the ideXlab platform.
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biological heart rate reduction through Genetic Suppression of gαs protein in the sinoatrial node
Journal of the American Heart Association, 2012Co-Authors: Patrick Lugenbiel, Kamilla Kelemen, Patrick A Schweizer, Rudiger Becker, Hugo A Katus, Alexander Bauer, Dierk ThomasAbstract:Background Elevated heart rate represents an independent risk factor for cardiovascular outcome in patients with heart disease. In the sinoatrial node, rate increase is mediated by β1 adrenoceptor mediated activation of the Gαs pathway. We hypothesized that Genetic inactivation of the stimulatory Gαs protein in the sinoatrial node would provide sinus rate control and would prevent inappropriate heart rate acceleration during β-adrenergic activation. Methods and Results Domestic pigs ( n =10) were evenly assigned to receive either Ad-small interfering RNA (siRNA)-Gαs gene therapy to inactivate Gαs or adenovirus encoding for green fluorescent protein (Ad-GFP) as control. Adenoviruses were applied through virus injection into the sinoatrial node followed by epicardial electroporation, and heart rates were evaluated for 7 days. Genetic inhibition of Gαs protein significantly reduced mean heart rates on day 7 by 16.5% compared with control animals (110±8.8 vs 131±9.4 beats per minute; P <0.01). On β-adrenergic stimulation with isoproterenol, we observed a tendency toward diminished rate response in the Ad-siRNA-Gαs group (Ad-siRNA-Gαs, +79.3%; Ad-GFP, +61.7%; n =3 animals per group; P = 0.294). Adverse effects of gene transfer on left ventricular ejection fraction (LVEF) were not detected following treatment (LVEFAd-siRNA-Gαs, 66%; LVEFAd-GFP, 60%). Conclusions In this preclinical proof-of-concept study targeted Ad-siRNA-Gαs gene therapy reduced heart rates during normal sinus rhythm compared with Ad-GFP treatment and prevented inappropriate rate increase after β-adrenergic stimulation. Gene therapy may provide an additional therapeutic option for heart rate reduction in cardiac disease. ( J Am Heart Assoc . 2012;1:jah3-e000372 doi: 10.1161/JAHA.111.000372)
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Genetic Suppression of gαs protein provides rate control in atrial fibrillation
Basic Research in Cardiology, 2012Co-Authors: Patrick Lugenbiel, Dierk Thomas, Kamilla Kelemen, Kerstin Trappe, Olympia Bikou, Patrick A Schweizer, Frederik Voss, Rudiger Becker, Hugo A Katus, Alexander BauerAbstract:Gene therapy-based modulation of atrioventricular (AV) conduction by overexpression of a constitutively active inhibitory Gα i protein effectively reduced heart rates in atrial fibrillation (AF). However, catecholamine stimulation caused an excessive increase in ventricular rate. We hypothesized that modest Genetic Suppression of a stimulatory G protein in the AV node would allow persistent rate control in acute AF and would prevent undesired heart rate acceleration during β-adrenergic activation. Atrial fibrillation was induced in 12 pigs by atrial burst pacing via an implanted cardiac pacemaker. Study animals were then assigned to receive either Ad-siRNA-Gαs gene therapy to inactivate Gαs protein or Ad-β-gal as control. Gαs protein inactivation resulted in a 20 % heart rate reduction (P < 0.01). AH and HV intervals were prolonged by 37 ms (P < 0.001) and 28 ms (P < 0.001), respectively, demonstrating atrioventricular conduction delay. Impairment of left ventricular ejection fraction (LVEF) during AF was attenuated by Gαs Suppression (LVEF 49 %) compared with controls (LVEF 34 %; P = 0.03). Isoproterenol application accelerated ventricular heart rate from 233 to 281 bpm (P < 0.001) in control animals but did not significantly affect pigs treated with Ad-siRNA-Gαs (192 vs. 216 bpm; P = 0.19). In conclusion, Genetic inhibition of Gαs protein in the AV node reduced heart rate and prevented AF-associated reduction of cardiac function in a porcine model. Rate control by gene therapy may provide an alternative to current pharmacological treatment of AF.
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Genetic Suppression of atrial fibrillation using a dominant negative ether a go go related gene mutant
Heart Rhythm, 2012Co-Authors: Radim Soucek, Dierk Thomas, Kamilla Kelemen, Olympia Bikou, Frederik Voss, Rudiger Becker, Hugo A Katus, Claudia Seyler, Michael Koenen, Alexander BauerAbstract:Background Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Gene therapy–dependent modulation of atrial electrophysiology may provide a more specific alternative to pharmacological and ablative treatment strategies. Objective We hypothesized that Genetic inactivation of atrial repolarizing ether-a-go-go–related gene (ERG) K + currents using a dominant-negative mutant would provide rhythm control in AF. Methods Ten domestic swine underwent pacemaker implantation and were subjected to atrial burst pacing to induce persistent AF. Animals were then randomized to receive either AdCERG-G627S to suppress ERG/ I Kr currents or green fluorescent protein (AdGFP) as control. Adenoviruses were applied using a novel hybrid technique combining atrial virus injection and epicardial electroporation to increase transgene expression. Results In pigs treated with AdCERG-G627S, the onset of persistent AF was prevented (n = 2) or significantly delayed compared with AdGFP controls (12 ± 2.1 vs. 6.2 ± 1.3 days; P P CERG-G627S = 62.1% ± 4.0% vs. LVEF GFP = 30.3% ± 9.1%; P Conclusion Inhibition of ERG function using atrial AdCERG-G627S gene transfer suppresses or delays the onset of persistent AF by prolongation of atrial refractoriness in a porcine model. Targeted gene therapy represents an alternative to pharmacological or ablative treatment of AF.
Waqar Ahmad - One of the best experts on this subject based on the ideXlab platform.
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dihydrolipoamide dehydrogenase Suppression induces human tau phosphorylation by increasing whole body glucose levels in a c elegans model of alzheimer s disease
Experimental Brain Research, 2018Co-Authors: Waqar AhmadAbstract:The microtubule associated tau protein becomes hyperphosphorylated in Alzheimer's disease (AD). While hyperphosphorylation promotes neurodegeneration, the cause and consequences of this abnormal modification are poorly understood. As impaired energy metabolism is an important hallmark of AD progression, we tested whether it could trigger phosphorylation of human tau protein in a transgenic Caenorhabditis elegans model of AD. We found that inhibition of a mitochondrial enzyme of energy metabolism, dihydrolipoamide dehydrogenase (DLD) results in elevated whole-body glucose levels as well as increased phosphorylation of tau. Hyperglycemia and tau phosphorylation were induced by either RNAi Suppression of the dld-1 gene or by inhibition of the DLD enzyme by the inhibitor, 2-methoxyindole-2-carboxylic acid (MICA). Although the calcium ionophore A23187 could reduce tau phosphorylation induced by either chemical or Genetic Suppression of DLD, it was unable to reduce tau phosphorylation induced by hyperglycemia. While inhibition of the dld-1 gene or treatment with MICA partially reversed the inhibition of acetylcholine neurotransmission by tau, neither treatment affected tau inhibited mobility. Conclusively, any abnormalities in energy metabolism were found to significantly affect the AD disease pathology.
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dihydrolipoamide dehydrogenase Suppression induce human tau phosphorylation by increasing whole body glucose levels in a c elegans model of alzheimer s disease
bioRxiv, 2017Co-Authors: Waqar AhmadAbstract:The microtubule associated tau protein becomes hyperphosphorylated in Alzheimers disease (AD). While tau hyperphosphorylation promotes neurodegeneration, the cause and consequences of this abnormal modification are poorly understood. As impaired energy metabolism is an important hallmark of AD progression, we tested whether it could trigger phosphorylation of human tau protein in a transgenic C. elegans model of AD. We found that Suppression or inhibition of a mitochondrial enzyme of energy metabolism, dihydrolipoamide dehydrogenase (DLD) either by RNAi or 2-methoxyindole-2-carboxylic acid (MICA) respectively, resulted in elevated whole-body glucose levels as well as increased phosphorylation of tau. Although the calcium ionophore A23187 could reduce tau phosphorylation induced by either chemical inhibition or Genetic Suppression of DLD, it was unable to reduce tau phosphorylation induced by hyperglycemia. While inhibition of the dld-1 gene or treatment with MICA partially reversed the inhibition of acetylcholine neurotransmission by tau, neither treatment affected tau inhibited mobility.