scholarly journals Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

2006 ◽  
Vol 72 (4) ◽  
pp. 2783-2792 ◽  
Author(s):  
T. Ledger ◽  
D. H. Pieper ◽  
B. González
Keyword(s):  
Toxic Effect ◽  
Degradation Pathway ◽  
Phenoxyacetic Acid ◽  
Hydroxylase Activity ◽  
Acid Degradation ◽  
Burkholderia Xenovorans ◽  
Mutant Strains ◽  
Negative Effect ◽  
Chlorophenoxyacetic Acid ◽  
Specific Contribution

ABSTRACT Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB I and tfdB II genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB I and tfdB II genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdBI contributes to a significantly higher extent than TfdBII. Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.

Plasmids of the Chlorophenoxyacetic-Acid Degradation of Bacteria of the Genus Raoultella

2021 ◽  
Vol 57 (3) ◽  
pp. 335-343
Author(s):  
N. V. Zharikova ◽  
T. R. Iasakov ◽  
E. I. Zhurenko ◽  
V. V. Korobov ◽  
T. V. Markusheva
Keyword(s):  
Acid Degradation ◽  
Chlorophenoxyacetic Acid

Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants

2005 ◽  
Vol 51 (7) ◽  
pp. 599-604 ◽  
Author(s):  
P Aneja ◽  
A Zachertowska ◽  
T C Charles
Keyword(s):  
Sinorhizobium Meliloti ◽  
Degradation Pathway ◽  
Nodule Occupancy ◽  
Wild Type ◽  
Term Survival ◽  
Mutant Strains ◽  
Key Factor ◽  
Carbon Excess ◽  
Synthesis And Degradation

The competitive abilities of Sinorhizobium meliloti mutant strains containing lesions in the PHB synthesis (phbC) and degradation (bdhA) pathways were compared. While the bdhA mutant showed no noticeable symbiotic defects on alfalfa host plants when inoculated alone, in mixed inoculation experiments it was found to be less competitive than the wild type for nodule occupancy. Long-term survival of the bdhA mutant on a carbon-limiting medium was not affected. However, when subjected to competition with the wild-type strain in periodic subculturing through alternating carbon-limiting and carbon-excess conditions, the bdhA mutant performed poorly. A more severe defect in competition for growth and nodule occupancy was observed with a mutant unable to synthesize PHB (phbC). These results indicate that the ability to efficiently deposit cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability, whereas the ability to use these stores is less important.Key words: Sinorhizobium meliloti, PHB metabolism, competition.

scholarly journals Enzymes Involved in a Novel Anaerobic Cyclohexane Carboxylic Acid Degradation Pathway

2014 ◽  
Vol 196 (20) ◽  
pp. 3667-3674 ◽  
Author(s):  
J. W. Kung ◽  
A.-K. Meier ◽  
M. Mergelsberg ◽  
M. Boll
Keyword(s):  
Carboxylic Acid ◽  
Degradation Pathway ◽  
Acid Degradation ◽  
Cyclohexane Carboxylic Acid

scholarly journals Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway

2014 ◽  
Vol 80 (11) ◽  
pp. 3341-3349 ◽  
Author(s):  
Tony Campillo ◽  
Sébastien Renoud ◽  
Isabelle Kerzaon ◽  
Ludovic Vial ◽  
Jessica Baude ◽  
...  
Keyword(s):  
Ferulic Acid ◽  
Protocatechuic Acid ◽  
Coenzyme A ◽  
Degradation Pathway ◽  
Hydroxycinnamic Acid ◽  
Plant Materials ◽  
Toxic Compounds ◽  
Content Type ◽  
Acid Degradation ◽  
Species Specific

ABSTRACTThe soil- and rhizosphere-inhabiting bacteriumAgrobacterium fabrum(genomospecies G8 of theAgrobacterium tumefaciensspecies complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genesatu1416,atu1417, andatu1420have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genesatu1415andatu1421have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)–CoA β-keto-thiolase, respectively. We thus demonstrated that theA. fabrumhydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transformp-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

scholarly journals Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by Burkholderia xenovorans LB400

2007 ◽  
Vol 190 (5) ◽  
pp. 1575-1583 ◽  
Author(s):  
Daryl J. Smith ◽  
Marianna A. Patrauchan ◽  
Christine Florizone ◽  
Lindsay D. Eltis ◽  
William W. Mohn
Keyword(s):  
Cytochromes P450 ◽  
Degradation Pathway ◽  
Dehydroabietic Acid ◽  
Burkholderia Xenovorans ◽  
Substrate Removal ◽  
Burkholderia Xenovorans Lb400 ◽  
Abietane Diterpenoids ◽  
Insertion Mutants

ABSTRACT The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

scholarly journals P0072GENERATION OF A C. ELEGANS MODEL TO STUDY THE INTERALLELIC INTERACTIONS OF PODOCIN

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Regina Légrádi ◽  
Magdolna Tália Keszthelyi ◽  
Tímea Köles ◽  
Kálmán Tory
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Chromosomal Integration ◽  
Rescue Effect ◽  
C Elegans ◽  
Filtration Barrier ◽  
Mutant Strains ◽  
Negative Effect ◽  
Touch Sensation ◽  
Fluorescent Tag

Abstract Background and Aims NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. NPHS2 encodes podocin, a key component of the glomerular filtration barrier. Podocin is a 42 kDa integral membrane protein, which accumulates in lipid raft microdomains at the podocyte slit diaphragm and is known to form oligomers. The c.686G>A, p.R229Q is the most common non-silent variant of NPHS2. We formerly described that R229Q is pathogenic only when trans-associated to specific 3’ missense mutations on the other parental allele. These C-terminal podocin mutants exert a dominant negative effect on R229Q podocin and retain it in intracellular compartments. As such, R229Q is the first variant in human genetics with a mutation-dependent pathogenicity. Based on FRET analysis and structural modeling, we formerly showed that the dominant negative effect is mediated by an altered oligomerization. However, the pathogenicity of several [mutation];[R229Q] associations is still in question and the identification of other rare NPHS2 variants with a mutation-dependent pathogenicity require an in vivo model. We therefore aimed to generate a Caenorhabditis elegans model, deficient for the homologous gene of NPHS2 (mec-2) and coexpressing differently (GFP- and mCherry-) tagged human podocin pairs. MEC-2 shares 45% identity and 83% similarity over 275aa (72%) of podocin (383aa). Expressed in six neurons, it is responsible for gentle-touch mechanosensation. The mec-2 mutants are insensitive to gentle touch in the center part of the body. Method Vectors encoding C-terminal GFP- or mCherry-tagged MEC-2 or human podocin with C. elegans codon optimization under mec-2 promoter and a selection marker (unc-119) were generated. Double (mec-2 and unc-119) mutant strains were established. Mutant strains were transformed by microparticle bombardment. The gentle-touch mechanosensation was examined by cat’s whiskers in a blinded experiment. Results The expression pattern of GFP and mCherry under mec-2 promoter corresponded to the six neurons responsible for gentle-touch sensation, indicating the proper functioning of the promoter. Strains with extrachromosomal MEC-2 or podocin coding vectors were successfully established (GFP-tagged MEC-2: n= 55 strains, GFP-tagged podocin: n= 81, mCherry-tagged podocin: n= 18). However, we found no rescue of the gentle touch sensation in any of them in blinded experiments. We hypothesized that either the fluorescent tag or the lack of chromosomal integration prevents the rescue effect of MEC-2. We therefore aimed to achieve self-cleaving of MEC-2 and the fluorescent tag, and inserted a T2A self-cleaving peptide-encoding sequence between them. To achieve chromosomal integration, we are implementing the MosSCI (Mos1-mediated Single Copy Insertion) technique. Conclusion Once the rescue with MEC-2 is achieved, the rescue effect of wild type and next different human podocin variant(s) will be aimed to analyze. The generation of the first animal model to study human interallelic interactions is challenging.

Phytic Acid Degradation as a Means of Improving Iron Absorption

2004 ◽  
Vol 74 (6) ◽  
pp. 445-452 ◽  
Author(s):  
Hurrell
Keyword(s):  
Ascorbic Acid ◽  
Phytic Acid ◽  
Iron Absorption ◽  
Low Cost ◽  
Molar Ratio ◽  
Infant Formulas ◽  
Complementary Foods ◽  
Acid Degradation ◽  
Negative Effect ◽  
Single Meal

Phytic acid is a potent inhibitor of native and fortification iron absorption and low absorption of iron from cereal- and/or legume-based complementary foods is a major factor in the etiology of iron deficiency in infants. Dephytinization of complementary foods or soy-based infant formulas is technically possible but, as phytic acid is strongly inhibitory at low concentrations, complete enzymatic degradation is recommended. If this is not possible, the phytic acid to iron molar ratio should be decreased to below 1:1 and preferably below 0.4:1. Complete dephytinization of cereal- and legume-based complementary foods has been shown to increase the percentage of iron absorption by as much as 12-fold (0.99% to 11.54%) in a single-meal study when the foods were reconstituted with water. The addition of milk, however, inhibits iron absorption and overcomes the enhancing effect of phytic acid degradation. Dephytinization can therefore be strongly recommended only for cereal/legume mixtures reconstituted with water, especially low-cost complementary foods destined for infants in developing countries. In countries where infant cereals are consumed with milk, ascorbic acid addition can more easily be used to overcome the negative effect of phytic acid on iron absorption. Similarly with soy-based infant formulas, especially if manufactured from low-phytate isolates, ascorbic acid can be used to ensure adequate iron absorption.

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