ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC50 of 8 ìM. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugsFil: Kurth, Daniel German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de la Iglesia, Agustina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bazet Lyonnet, Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lin, Ting Wang. University of California; Estados UnidosFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Tsai, Shiou Chuan. University of California; Estados UnidosFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Fitness costs associated with evolved herbicide resistance alleles in plants

Predictions based on evolutionary theory suggest that the adaptive value of evolved herbicide resistance alleles may be compromised by the existence of fitness costs. There have been many studies quantifying the fitness costs associated with novel herbicide resistance alleles, reflecting the importance of fitness costs in determining the evolutionary dynamics of resistance. However, many of these studies have incorrectly defined resistance or used inappropriate plant material and methods to measure fitness. This review has two major objectives. First, to propose a methodological framework that establishes experimental criteria to unequivocally evaluate fitness costs. Second, to present a comprehensive analysis of the literature on fitness costs associated with herbicide resistance alleles. This analysis reveals unquestionable evidence that some herbicide resistance alleles are associated with pleiotropic effects that result in plant fitness costs. Observed costs are evident from herbicide resistance-endowing amino acid substitutions in proteins involved in amino acid, fatty acid, auxin and cellulose biosynthesis, as well as enzymes involved in herbicide metabolism. However, these resistance fitness costs are not universal and their expression depends on particular plant alleles and mutations. The findings of this review are discussed within the context of the plant defence trade-off theory and herbicide resistance evolution

Key aspects on the biology, ecology and impacts of johnsongrass [Sorghum halepense (L.) Pers] and the role of glyphosate and non-chemical alternative practices for the management of this weed in Europe

Sorghum halepense (L.) Pers is a common and noxious worldwide weed of increasing distribution in many European countries. In the present review, information on the biology, ecology, agricultural, economic and environmental impact of johnsongrass is given, and the current status of this weed in Europe is discussed. Furthermore, special attention is given to the important role of field trials using glyphosate to control weeds in arable and perennial crops in many European countries. Some of the factors which affect control efficacy and should be taken into account are also discussed. Finally, several non-chemical alternative methods (cultural, mechanical, thermal, biological, etc.) for johnsongrass management are also presented. The adoption of integrated weed management (IWM) techniques such as glyphosate use, crop rotation, and deep tillage is strongly recommended to control plant species that originate from both seed and rhizomes.This research was funded by Bayer Agriculture BVBA, grant number 140319

Discovery and characterization of regulatory mechanisms affecting the heteromeric acetyl-coenzyme a carboxylase in Arabidopsis

Fatty acid biosynthesis (FAS) is an essential metabolic pathway used by all organisms to generate fatty acids. A staple component of this pathway is the enzyme acetyl-CoA carboxylase (ACCase), which catalyzes the committed step by converting acetyl-CoA to malonyl-CoA. The heteromeric form of this enzyme requires four different subunits for activity: biotin carboxylase, biotin carboxyl carrier protein (BCCP), and alpha- and beta-carboxyltransferase (CT). Heteromeric ACCase is present in prokaryotes and the plastids of most plants, and has been a focus of biotechnology research due to its prominent role in FAS. Many different regulatory mechanisms have been identified in both plants and E. coli. However, it is still unknown how most of these regulatory mechanisms are mediated. For example, ACCase is known to be feedback inhibited by 18:1-acyl carrier protein in plants, yet it is unknown how this inhibition is exerted on the enzyme. Therefore it was posited that other unknown factors, such as proteins or post-translational modifications, might play a role in ACCase regulation. To identify suspected regulatory factors associated with ACCase, we performed in vivo co-immunoprecipitation (co-IP) using subunit-specific antibodies to isolate the ACCase complex from Arabidopsis thaliana leaves. Quantitative mass spectrometry of these co-IPs revealed all four known subunits to ACCase and two unknown proteins annotated as 'biotin/lipoyl attachment domain containing' (BADC) proteins. The BADC proteins are a family of three proteins in A. thaliana and resemble the BCCP subunit to ACCase, but lack the conserved biotinylation motif. All three BADC proteins interacted with the two A. thaliana BCCP isoforms and the biotin carboxylase subunit of ACCase based on yeast two-hybrid and heterologous co-expression analyses. None of the BADC proteins were biotinylated in planta or when expressed in Escherichia coli, unlike BCCP controls. Gene orthologs to BADC were found only in plant and green algae species that contain a heteromeric ACCase suggesting BADC genes co-evolved with this form of ACCase. Expression of BADC proteins in a temperature-sensitive E. coli BCCP mutant in minimal media strongly inhibited cell growth through interaction with the homologous, bacterial ACCase. Also, addition of recombinant BADC protein to in vitro ACCase activity assays significantly reduced enzyme activity. Finally, partial silencing of one of the BADC genes in A. thaliana seed led to a slight, yet significant, increase in seed oil content. We conclude the BADC proteins are ancient BCCPs that acquired a new function through mutation of the biotinylation motif. We propose a poisoned complex model whereby BADCs function as negative regulators of ACCase by competing with BCCP for access to the holo-ACCase complex. In addition, a study was performed to identify the role of phosphorylation of the alpha-CT subunit. Multiple studies had identified two phosphorylation sites on the C-terminal domain of alpha-CT in A. thaliana. This C-terminal domain is not found in all plant species and has an unknown function. To determine the potential regulatory effect of phosphorylation on this domain, phosphomimic and phospho-deficient alpha-CT mutants were made and expressed in wild type A. thaliana. Multiple independent transgenic lines containing at least two-fold alpha-CT protein compared to empty vector controls were screened for seed oil content. The resulting data showed no clear phenotype that could be attributed to expression of the mutants. This result could be explained by a number of factors such as the presence of endogenous alpha-CT, the complexity of the seed oil phenotype, or a large margin of technical error in some lines. However, in vitro ACCase activity assays showed that a transgenic line overexpressing native alpha-CT contained increased specific activity of the enzyme compared to controls. Furthermore, analysis of transgenic lines expressing phosphomimic or phospho-deficient alpha-CT mutants also showed increased ACCase specific activity which was indistinguishable from the native alpha-CT overexpression line, regardless of the mutation. Therefore it appears that increased alpha-CT expression can increase ACCase activity by allowing for the formation of more active complexes. This observation suggests that alpha-CT is the limiting subunit of the ACCase complex in the stroma

Evolved polygenic herbicide resistance in Lolium rigidum by low-dose herbicide selection within standing genetic variation

The interaction between environment and genetic traits under selection is the basis of evolution. In this study, we have investigated the genetic basis of herbicide resistance in a highly characterized initially herbicide-susceptible Lolium rigidum population recurrently selected with low (below recommended label) doses of the herbicide diclofop-methyl. We report the variability in herbicide resistance levels observed in F1 families and the segregation of resistance observed in F2 and back-cross (BC) families. The selected herbicide resistance phenotypic trait(s) appear to be under complex polygenic control. The estimation of the effective minimum number of genes (NE), depending on the herbicide dose used, reveals at least three resistance genes had been enriched. A joint scaling test indicates that an additive-dominance model best explains gene interactions in parental, F1, F2 and BC families. The Mendelian study of six F2 and two BC segregating families confirmed involvement of more than one resistance gene. Cross-pollinated L. rigidum under selection at low herbicide dose can rapidly evolve polygenic broad-spectrum herbicide resistance by quantitative accumulation of additive genes of small effect. This can be minimized by using herbicides at the recommended dose which causes high mortality acting outside the normal range of phenotypic variation for herbicide susceptibility

Transcriptome profiling of a spirodiclofen susceptible and resistant strain of the European red mite Panonychus ulmi using strand-specific RNA-seq

Background: The European red mite, Panonychus ulmi, is among the most important mite pests in fruit orchards, where it is controlled primarily by acaricide application. However, the species rapidly develops pesticide resistance, and the elucidation of resistance mechanisms for P. ulmi has not kept pace with insects or with the closely related spider mite Tetranychus urticae. The main reason for this lack of knowledge has been the absence of genomic resources needed to investigate the molecular biology of resistance mechanisms. Results: Here, we provide a comprehensive strand-specific RNA-seq based transcriptome resource for P. ulmi derived from strains susceptible and resistant to the widely used acaricide spirodiclofen. From a de novo assembly of the P. ulmi transcriptome, we manually annotated detoxification enzyme families, target-sites of commonly used acaricides, and horizontally transferred genes implicated in plant-mite interactions and pesticide resistance. In a comparative analysis that incorporated sequences available for Panonychus citri, T. urticae, and insects, we identified radiations for detoxification gene families following the divergence of Panonychus and Tetranychus genera. Finally, we used the replicated RNA-seq data from the spirodiclofen susceptible and resistant strains to describe gene expression changes associated with resistance. A cytochrome P450 monooxygenase, as well as multiple carboxylcholinesterases, were differentially expressed between the susceptible and resistant strains, and provide a molecular entry point for understanding resistance to spirodiclofen, widely used to control P. ulmi populations. Conclusions: The new genomic resources and data that we present in this study for P. ulmi will substantially facilitate molecular studies of underlying mechanisms involved in acaricide resistance

Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acc1

Acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase) plays a central role in carbon metabolism and has been the site of action for the development of therapeutics or herbicides, as its product, malonyl-CoA, is a precursor for production of fatty acids and other compounds. Control of Acc1 activity in the yeast Saccharomyces cerevisiae occurs mainly at two levels, i.e., regulation of transcription and repression by Snf1 protein kinase at the protein level. Here, we demonstrate a strategy for improving the activity of ACCase in S. cerevisiae by abolishing posttranslational regulation of Acc1 via site-directed mutagenesis. It was found that introduction of two site mutations in Acc1, Ser659 and Ser1157, resulted in an enhanced activity of Acc1 and increased total fatty acid content. As Snf1 regulation of Acc1 is particularly active under glucose-limited conditions, we evaluated the effect of the two site mutations in chemostat cultures. Finally, we showed that our modifications of Acc1 could enhance the supply of malonyl-CoA and therefore successfully increase the production of two industrially important products derived from malonyl-CoA, fatty acid ethyl esters and 3-hydroxypropionic acid. IMPORTANCE ACCase is responsible for carboxylation of acetyl-CoA to produce malonyl-CoA, which is a crucial step in the control of fatty acid metabolism. ACCase opened the door for pharmaceutical treatments of obesity and diabetes as well as the development of new herbicides. ACCase is also recognized as a promising target for developing cell factories, as its malonyl-CoA product serves as a universal precursor for a variety of high-value compounds in white biotechnology. Yeast ACCase is a good model in understanding the enzyme's catalysis, regulation, and inhibition. The present study describes the importance of protein phosphorylation in regulation of yeast ACCase and identifies potential regulation sites. This study led to the generation of a more efficient ACCase, which was applied in the production of two high-value compounds derived from malonyl-CoA, i.e., fatty acid ethyl esters that can be used as biodiesel and 3-hydroxypropionic acid that is considered an important platform chemical

Herbicide resistance in weeds (1996)

New 8/95, Reprinted 5/96/4M

Glyphosate resistance in annual ryegrass (Lolium rigidum Gaud.) with multiple resistance mechanisms.

Glyphosate (N-(phosphonomethyl)glycine) is a post-emergent, systemic and non-selective herbicide for the control of annual and perennial weeds. This herbicide has very low toxicity to the mammals. The target enzyme for glyphosate in plants is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate inhibits the biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan in the plant. The first case of glyphosate resistance was reported in Lolium rigidum in Australia after 15 years of persistence use of this herbicide and the number weeds reported resistant to glyphosate has increased around the world. So far, two mechanisms known to be involved in resistance to glyphosate are target-site mutation and reduced herbicide translocation. Recently, two populations of L. rigidum from Australia have been discovered with very high levels of resistance to glyphosate. This project aims to determine the levels of glyphosate resistance in these populations, investigate glyphosate resistance mechanisms in the populations and finally assess the mode of inheritance of resistance. In this project, four resistant (NLR70, SLR77, SLR80 and SLR88) and one susceptible (VLR1) L. rigidum populations were evaluated for their response to glyphosate. From the dose response experiments, the susceptible population of VLR1 was completely controlled with the recommended rate of glyphosate (450 g a.e ha⁻ ¹). In contrast, the resistant populations were not fully controlled by this herbicide rate. There was considerable variation between the populations in their resistance to glyphosate. In comparison to the susceptible population VLR1, SLR77 was 2.2 to 3.5 fold resistant to glyphosate, NLR70 was 3.7 to 8.4 fold resistant to glyphosate, SLR88 was 5.6 to 11.4 fold resistant to glyphosate and SLR80 was 8.2 to 76.7 fold resistant to glyphosate. The mechanism of glyphosate resistance in the populations was investigated. ¹⁴ C-glyphosate was used to determine the absorption and translocation of glyphosate among the populations. There was no significant difference on the absorption of ¹⁴ C-glyphosate 48 hours after treatment in the population. However, the accumulation of ¹⁴ C-glyphosate in the stem region was higher in the susceptible VLR1 population (25.9%) and in resistant SLR77 (25%) than the other three populations. The resistant populations NLR70, SLR88 and SLR80 had about half the amount of glyphosate accumulating in the stem region. These three resistant populations appear to be resistant to glyphosate as a result of reduced translocation of glyphosate to the shoot meristem. Part of the EPSP synthase gene of the susceptible and four resistant populations was amplified and sequenced to identify any changes in the nucleotide sequence. The predicted amino acid sequence from the susceptible population VLR1 was the same as the consensus sequence from other plant species in the conserved region sequenced. However, the resistant populations of NLR70, SLR77, SLR80 and SLR88 showed polymorphisms within the nucleotide sequence in this region. Single nucleotide substitutions of A for C at codon 106 were observed in the resistant populations SLR77 and SLR80. This nucleotide change is predicted to substitute threonine for proline at position 106. In the resistant population SLR88, a nucleotide substitution of T for C was observed at the same codon. This nucleotide substitution is predicted to change the amino acid from proline 106 to serine. Therefore, these three populations appear to be resistant to glyphosate as a result of a target-site mutation. An inheritance study was conducted by cross pollinating the susceptible VLR1 and resistant SLR88 population. From the dose response, the parent susceptible was completely killed with the recommended rate of glyphosate and higher rates of glyphosate were required to control parental resistant and both F₁ progenies (maternal susceptible and resistant). Both F₁ progenies showed an intermediate response to glyphosate compared with the parental populations. This indicated that the resistance to glyphosate in population SLR88 is inherited by nuclear gene(s) through the transfer of pollen during the cross pollination. It is suggested that SLR88 and SLR80 population contain both glyphosate resistant mechanisms due to the cross pollination between individuals with different resistant mechanisms. Having two resistant mechanisms results in populations being highly resistant to glyphosate compared to those with one resistance mechanism. The higher level of glyphosate resistance in these multiple glyphosate resistance populations will likely make them harder to manage.Thesis (M.Ag.Sc.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Herbicide resistance in the Canadian prairie provinces : Five years after the fact

La résistance aux herbicides a été reconnue comme un problème pour la première fois dans les Prairies canadiennes, en 1988, quand une sétaire verte (Setaria viridis) résistante à la trifluraline a été détectée au Manitoba, puis une stellaire moyenne (Stellaria média) et un kochia à balais (Kochia scoparia) résistants au chlorsulfuron ont été identifiés en Alberta et en Saskatchewan, respectivement. Depuis lors, le nombre de mauvaises herbes résistantes s'est accru pour inclure la folle avoine (Avena fatua) résistante aux triallates, ainsi qu'aux aryloxyphénoxypropionates et aux cyclohexanediones (herbicides du groupe 1), la sétaire verte aux herbicides du groupe 1, la soude roulante (Salsola pestifer) et la moutarde des champs (Sinapis arvensis) résistantes au sulfonylurées et aux imidazolinones (herbicides du groupe 2), et finalement la moutarde des champs résistante aux herbicides régulateurs de croissance (herbicides du groupe 4). Les niveaux et patrons de résistance croisée aux molécules des groupes 1 et 2 diffèrent énormément entre les différentes populations, avec des facteurs de résistance (rapport de résistant à sensible [R:S]), obtenus à l'aide de courbes de réponse aux doses, se classant de 150. La résistance de la sétaire verte au groupe 1 et la résistance de la stellaire moyenne et du kochia à la classe 2 sont dues à des sensibilités réduites des enzymes-cibles: l'acétyl coenzyme-A carboxylase (ACCase) et l'acétolactate synthase (ALS), respectivement. Les mécanismes de résistance pour les autres espèces, incluant la folle avoine résistante aux inhibiteurs de rACCase (groupe 1) et aux triallate/difenzoquat (groupe 8) sont obscurs. À présent, le seul cas de résistance multiple dans l'ouest canadien est la sétaire verte résistante aux éléments chimiques des groupes 1 et 3 (inhibiteurs de l'ACCase et dinitroanilines). Les préoccupations à venir concernent la sévérité accrue de la résistance aux groupes 1 et 8 dans les Prairies, et la possibilité de sélectionner pour la résistance multiple chez les mauvaises herbes du type de la sétaire verte, contre lesquelles il existe peu d'alternatives efficaces.Herbicide resistance was first recognized as a problem on the Canadian Prairies in 1988 when trifluralin-resistant green foxtail (Setaria viridis) was reported in Manitoba, and chlorsulfuron-resistant chickweed (Stellaria media) and koehia (Kochia scoparia) in Alberta and Saskatchewan, respectively. Since then, the number of resistant weeds has increased to include wild oats (Avena fatua) resistant to triallate and to aryloxyphenoxypropionate and cyclohexanedione (group 1) herbicides, green foxtail to group 1 herbicides, Russian thistle (Salsola pestifer) and wild mustard (Sinapis arvensis) to sulfonylurea and imidazolinone (group 2) herbicides, and wild mustard to growth regulator (group 4) herbicides. The levels and patterns of cross-resistance to chemicals in groups 1 and 2 vary widely among different populations, with resistance factors [resistant to susceptible (R:S) ratios] derived from dose response curves typically ranging from 150. Group 1 resistance in green foxtail and group 2 resistance in chickweed and kochia populations are due to reduced sensitivities of the target enzymes, acetyl coenzyme-A carboxylase (ACCase) and acetolactate synthase (ALS), respectively. The mechanisms of resistance in the other species including wild oats resistant to ACCase inhibitors (group 1 ) and to triallate/difenzoquat (group 8) are unclear. At present, the only instance of multiple resistance in western Canada is green foxtail resistant to chemicals in both groups 1 and 3 (ACCase inhibitors and dinitroanilines). Future concerns focus mainly on the increasing seriousness of group 1 and 8 resistance across the Prairies, and on the possibility of selecting for multiple resistance in weeds such as green foxtail for which there are few remaining effective control options