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Wolfgang Babel - One of the best experts on this subject based on the ideXlab platform.
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localization and characterization of two novel genes encoding stereospecific dioxygenases catalyzing 2 2 4 dichlorophenoxy propionate cleavage in Delftia acidovorans mc1
Applied and Environmental Microbiology, 2004Co-Authors: Kathleen M Schleinitz, Sabine Kleinsteuber, Tatiana Vallaeys, Wolfgang BabelAbstract:Two novel genes, rdpA and sdpA, encoding the enantiospecific α-ketoglutarate dependent dioxygenases catalyzing R,S-dichlorprop cleavage in Delftia acidovorans MC1 were identified. Significant similarities to other known genes were not detected, but their deduced amino acid sequences were similar to those of other α-ketoglutarate dioxygenases. RdpA showed 35% identity with TauD of Pseudomonas aeruginosa, and SdpA showed 37% identity with TfdA of Ralstonia eutropha JMP134. The functionally important amino acid sequence motif HX(D/E)X23-26(T/S)X114-183HX10-13R/K, which is highly conserved in group II α-ketoglutarate-dependent dioxygenases, was present in both dichlorprop-cleaving enzymes. Transposon mutagenesis of rdpA inactivated R-dichlorprop cleavage, indicating that it was a single-copy gene. Both rdpA and sdpA were located on the plasmid pMC1 that also carries the lower pathway genes. Sequencing of a 25.8-kb fragment showed that the dioxygenase genes were separated by a 13.6-kb region mainly comprising a Tn501-like transposon. Furthermore, two copies of a sequence similar to IS91-like elements were identified. Hybridization studies comparing the wild-type plasmid and that of the mutant unable to cleave dichlorprop showed that rdpA and sdpA were deleted, whereas the lower pathway genes were unaffected, and that deletion may be caused by genetic rearrangements of the IS91-like elements. Two other dichlorprop-degrading bacterial strains, Rhodoferax sp. strain P230 and Sphingobium herbicidovorans MH, were shown to carry rdpA genes of high similarity to rdpA from strain MC1, but sdpA was not detected. This suggested that rdpA gene products are involved in the degradation of R-dichlorprop in these strains.
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Regulation of catabolic enzymes during long-term exposure of Delftia acidovorans MC1 to chlorophenoxy herbicides.
Microbiology, 2004Co-Authors: Dirk Benndorf, Ian Davidson, Wolfgang BabelAbstract:Delftia acidovorans MC1 is able to grow on chlorophenoxy herbicides such as 2,4-dichlorophenoxypropionic acid (2,4-DCPP) and 2,4-dichlorophenoxyacetic acid as sole sources of carbon and energy. High concentrations of the potentially toxic organics inhibit the productive degradation and poison the organism. To discover the target of chlorophenoxy herbicides in D. acidovorans MC1 and to recognize adaptation mechanisms, the response to chlorophenoxy acids at the level of proteins was analysed. The comparison of protein patterns after chemostatic growth on pyruvate and 2,4-DCPP facilitated the discovery of several proteins induced and repressed due to the substrate shifts. Many of the induced enzymes, for example two chlorocatechol 1,2-dioxygenases, are involved in the metabolism of 2,4-DCPP. A stronger induction of some catabolic enzymes (chlorocatechol 1,2-dioxygenase TfdCII, chloromuconate cycloisomerase TfdD) caused by an instant increase in the concentration of 2,4-DCPP resulted in increased rates of productive detoxification and finally in resistance of the cells. Nevertheless, the decrease of the (S)-2,4-DCPP-specific 2-oxoglutarate-dependent dioxygenase in 2D gels reveals a potential bottleneck in 2,4-DCPP degradation. Well-known heat-shock proteins and oxidative-stress proteins play a minor role in adaptation, because apart from DnaK only a weak or no induction of the proteins GroEL, AhpC and SodA was observed. Moreover, the modification of elongation factor Tu (TufA), a strong decrease of asparaginase and the induction of the hypothetical periplasmic protein YceI point to additional resistance mechanisms against chlorophenoxy herbicides.
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Delftia acidovorans mc1 resists high herbicide concentrations a study of nutristat growth on rs 2 2 4 dichlorophenoxy propionate and 2 4 dichlorophenoxyacetate
Bioscience Biotechnology and Biochemistry, 2004Co-Authors: Roland H Muller, Wolfgang BabelAbstract:Delftia acidovorans MC1 was continuously cultivated under nutristat conditions with elevated concentrations of the herbicides (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP] and 2,4-dichlorophenoxyacetate (2,4-D). The presence of 1-5 mM of either of these compounds did not essentially inhibit growth. Moreover, substrate consumption was not essentially affected at pH values of 7.0-9.0 selected by reason of alkaline in situ conditions found e.g. on contaminated building rubble but was decreased at pH 9.3. The adenylate energy charge declined to some degree as the herbicide concentration rose, the extent of this increasing as the pH rose. This was caused by an increase in the concentration of ADP and in particular AMP, in contrast to the fairly constant ATP level of around 4 nmol/mg dry mass with (RS)-2,4-DP and 2 nmol/mg with 2,4-D. Comparison of the individual growth parameters with theoretical data taking into account maintenance coefficients of 0.48 mmol (RS)-2,4-DP/g*h and 0.6 mmol 2,4-D/g*h revealed that the culture followed purely kinetic rules. This excludes the necessity of using substrate to a significant extent to satisfy extra efforts in energy for homeostasic work under these accentuated conditions.
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a transposon encoding the complete 2 4 dichlorophenoxyacetic acid degradation pathway in the alkalitolerant strain Delftia acidovorans p4a
Microbiology, 2003Co-Authors: Doreen Hoffmann, Roland H Muller, Sabine Kleinsteuber, Wolfgang BabelAbstract:The bacterial strain Delftia acidovorans P4a, isolated from an extreme environment (heavily contaminated with organochlorines, highly alkaline conditions in an aqueous environment), was found to mineralize 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid under alkaline conditions. Screening a genomic DNA library of the alkalitolerant strain for 2,4-D genes revealed the presence of the two 2,4-D gene clusters tfdCDEF and tfdC(II)E(II)BKA, tfdR genes being located in the vicinity of each tfd gene cluster. The results showed that the putative genes of the complete 2,4-D degradation pathway are organized in a single genomic unit. Sequence similarities to homologous gene clusters indicate that the individual tfd elements of strain P4a do not share a common origin, but were brought together by recombination events. The entire region is flanked by insertion elements of the IS1071 and IS1380 families, forming a transposon-like structure of about 30 kb, of which 28.4 kb were analysed. This element was shown to be located on the bacterial chromosome. The present study provides the first reported case of a chromosomally located catabolic transposon which carries the genes for the complete 2,4-D degradation pathway.
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purification and characterisation of the enantiospecific dioxygenases from Delftia acidovorans mc1 initiating the degradation of phenoxypropionate and phenoxyacetate herbicides
Acta Biotechnologica, 2003Co-Authors: A Westendorf, Roland H Muller, Wolfgang BabelAbstract:After cultivation on (R,S)-2-(2,4-dichlorophenoxy)propionate, two α-ketoglutarate-dependent dioxygenases were isolated and purified from Delftia acidovorans MCl, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R-enantiomer of substituted phenoxypropionate derivatives: the K m values were 55 μM and 30 μM, the k cat values 55 min -1 and 34 min -1 with (R)-2-(2,4-dichlorophenoxy)propionate [(R)-2,4-DP] and (R)-2-(4-chloro-2-methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S-enantiomers with K m values of 49 μM and 22 μM, and k cat values of 50 min -1 and 46 min -1 with (S)-2-(2,4-dichlorophenoxy)propionate [(S)-2,4-DP] and (S)-2-(4-chloro-2-methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4-dichlorophenoxyacetate: K m = 123 μM, k cat = 36 min -1 ) with significant activity. As the second substrate, only α-ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K i values of 3 mM with (R)-2,4-DP and 1.5 mM with (S)-2,4-DP. The reaction was strictly dependent on the presence of Fe 2+ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).
Roland H Muller - One of the best experts on this subject based on the ideXlab platform.
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uptake of the herbicide 2 4 dichlorophenoxyacetate 2 4 d by Delftia acidovorans mc1 complex kinetic characteristics in dependence of ph and growth substrate
2016Co-Authors: Roland H Muller, Doreen HoffmannAbstract:The uptake of the herbicide 2,4-dichlorophenoxyacetate (2,4-D) by the bacterial strain Delftia acidovorans MC1 was studied using 14 C-labeled compound. Implication of active transport was suggested due to the effect of carbonylcyanide m-chlorophenylhydrazone (CCCP) as an uncoupler of the proton motive force, the presence of which reduced the uptake rate by up to 90%. Kinetic characteristics revealed a complex pattern that was strongly affected by the external pH. With 2,4-D-grown cells, the uptake characteristics followed a hyperbolic shape at pH 6.8 showing an intermediary plateau at ca. 20-100 µM 2,4-D. In contrast, the kinetics at pH 7.5 and 8.5 revealed a sigmoidal pattern. The maximum rate was obtained at around 400 µM 2,4-D, and amounted to 15-20 nmol/min*mg protein. Higher substrate concentrations led to inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP], the uptake rate increased to about 65 nmol/min*mg protein which hints at substrate-dependent induction of specific carrier(s). With 2,4-D-grown cells, such a high rate was obtained only after cloning and expression of the tfdK gene, which encodes a specific transporter for 2,4-D. The uptake pattern of 2,4-D changed with mutant strains of MC1 that were phenotypically deficient of cleavage activity to ether bond in phenoxyalkanoate herbicides. Apparently, genes coding for proteins in uptake function were in addition deleted. With strains that lacked (R)-2,4-DP cleavage, the plateau disappeared and the kinetics followed a more continuous pattern. Strains that lacked (S)-2,4-DP cleavage showed faint 2,4-D uptake at all. The present picture hints at three proteins that are involved in 2,4-D uptake by active transport. With degradation-negative mutants of strain MC1, the influx of 2,4-D proceeded at a low rate. It was linearly dependent on the 2,4-D concentration correspondding to a micro-molar rate constant of 1.08*10 -5 min -1 mg protein -1 . The latter hints at 2,4
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adaptation of Delftia acidovorans for degradation of 2 4 dichlorophenoxyacetate in a microfluidic porous medium
Biodegradation, 2014Co-Authors: Sabine Leibeling, Roland H Muller, Hongkyu Yoon, Changyong Zhang, Charles J Werth, Julie L ZillesAbstract:Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.
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declining capacity of starving Delftia acidovorans mc1 to degrade phenoxypropionate herbicides correlates with oxidative modification of the initial enzyme
Environmental Science & Technology, 2010Co-Authors: Sabine Leibeling, Frank Schmidt, Nico Jehmlich, Martin Von Bergen, Roland H Muller, Hauke HarmsAbstract:Bioremediation relies on the stability of enzymatic activities, particularly when bioavailable contaminant concentrations do not permit much renewal of microbial biomass. Starving Delftia acidovorans MC1 were found to lose specific degradation activity, while accumulating variants of the α-ketoglutarate-dependent dioxygenase RdpA, the enzyme initiating the degradation of (RS)-2-(2,4-dichlorophenoxy)propionate. These variants differed in their pI and originated from post-translational modification, since there is only one rdpA gene in the genome. It was tested if RdpA modification resulted from carbonylation by reactive oxygen species, known side products of dioxygenase reactions. Carbonylated amino acids in proteins of starved cells were specifically derivatized with 2,4-dinitrophenylhydrazine. Subsequent immunolabeling of the resulting hydrazones and mass spectrometry of tryptic digests confirmed different levels of carbonylation of RdpA.
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uptake kinetics of 2 4 dichlorophenoxyacetate by Delftia acidovorans mc1 and derivative strains complex characteristics in response to ph and growth substrate
Bioscience Biotechnology and Biochemistry, 2006Co-Authors: Roland H Muller, Doreen HoffmannAbstract:The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20-100 microM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 microM 2,4-D at either pH, and amounted to 15-20 nmol/min x mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min x mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.
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2 4 dichlorophenoxyacetic acid 2 4 d utilization by Delftia acidovorans mc1 at alkaline ph and in the presence of dichlorprop is improved by introduction of the tfdk gene
Biodegradation, 2006Co-Authors: Doreen Hoffmann, Roland H MullerAbstract:Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μmax) of 0.158 h−1. The half-maximum rate-associated substrate concentration (Ks) was 45 μM. At pH 8.5 μmax was only 0.05 h−1 and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μmax with dichlorprop was around 0.2 h−1 at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μmax of 0.147 h−1 and Ks of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h−1 at pH 6.8 and up to D = 0.2 h−1 at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.
Doreen Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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uptake of the herbicide 2 4 dichlorophenoxyacetate 2 4 d by Delftia acidovorans mc1 complex kinetic characteristics in dependence of ph and growth substrate
2016Co-Authors: Roland H Muller, Doreen HoffmannAbstract:The uptake of the herbicide 2,4-dichlorophenoxyacetate (2,4-D) by the bacterial strain Delftia acidovorans MC1 was studied using 14 C-labeled compound. Implication of active transport was suggested due to the effect of carbonylcyanide m-chlorophenylhydrazone (CCCP) as an uncoupler of the proton motive force, the presence of which reduced the uptake rate by up to 90%. Kinetic characteristics revealed a complex pattern that was strongly affected by the external pH. With 2,4-D-grown cells, the uptake characteristics followed a hyperbolic shape at pH 6.8 showing an intermediary plateau at ca. 20-100 µM 2,4-D. In contrast, the kinetics at pH 7.5 and 8.5 revealed a sigmoidal pattern. The maximum rate was obtained at around 400 µM 2,4-D, and amounted to 15-20 nmol/min*mg protein. Higher substrate concentrations led to inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP], the uptake rate increased to about 65 nmol/min*mg protein which hints at substrate-dependent induction of specific carrier(s). With 2,4-D-grown cells, such a high rate was obtained only after cloning and expression of the tfdK gene, which encodes a specific transporter for 2,4-D. The uptake pattern of 2,4-D changed with mutant strains of MC1 that were phenotypically deficient of cleavage activity to ether bond in phenoxyalkanoate herbicides. Apparently, genes coding for proteins in uptake function were in addition deleted. With strains that lacked (R)-2,4-DP cleavage, the plateau disappeared and the kinetics followed a more continuous pattern. Strains that lacked (S)-2,4-DP cleavage showed faint 2,4-D uptake at all. The present picture hints at three proteins that are involved in 2,4-D uptake by active transport. With degradation-negative mutants of strain MC1, the influx of 2,4-D proceeded at a low rate. It was linearly dependent on the 2,4-D concentration correspondding to a micro-molar rate constant of 1.08*10 -5 min -1 mg protein -1 . The latter hints at 2,4
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uptake kinetics of 2 4 dichlorophenoxyacetate by Delftia acidovorans mc1 and derivative strains complex characteristics in response to ph and growth substrate
Bioscience Biotechnology and Biochemistry, 2006Co-Authors: Roland H Muller, Doreen HoffmannAbstract:The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20-100 microM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 microM 2,4-D at either pH, and amounted to 15-20 nmol/min x mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min x mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.
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2 4 dichlorophenoxyacetic acid 2 4 d utilization by Delftia acidovorans mc1 at alkaline ph and in the presence of dichlorprop is improved by introduction of the tfdk gene
Biodegradation, 2006Co-Authors: Doreen Hoffmann, Roland H MullerAbstract:Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μmax) of 0.158 h−1. The half-maximum rate-associated substrate concentration (Ks) was 45 μM. At pH 8.5 μmax was only 0.05 h−1 and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μmax with dichlorprop was around 0.2 h−1 at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μmax of 0.147 h−1 and Ks of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h−1 at pH 6.8 and up to D = 0.2 h−1 at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.
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a transposon encoding the complete 2 4 dichlorophenoxyacetic acid degradation pathway in the alkalitolerant strain Delftia acidovorans p4a
Microbiology, 2003Co-Authors: Doreen Hoffmann, Roland H Muller, Sabine Kleinsteuber, Wolfgang BabelAbstract:The bacterial strain Delftia acidovorans P4a, isolated from an extreme environment (heavily contaminated with organochlorines, highly alkaline conditions in an aqueous environment), was found to mineralize 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid under alkaline conditions. Screening a genomic DNA library of the alkalitolerant strain for 2,4-D genes revealed the presence of the two 2,4-D gene clusters tfdCDEF and tfdC(II)E(II)BKA, tfdR genes being located in the vicinity of each tfd gene cluster. The results showed that the putative genes of the complete 2,4-D degradation pathway are organized in a single genomic unit. Sequence similarities to homologous gene clusters indicate that the individual tfd elements of strain P4a do not share a common origin, but were brought together by recombination events. The entire region is flanked by insertion elements of the IS1071 and IS1380 families, forming a transposon-like structure of about 30 kb, of which 28.4 kb were analysed. This element was shown to be located on the bacterial chromosome. The present study provides the first reported case of a chromosomally located catabolic transposon which carries the genes for the complete 2,4-D degradation pathway.
Ameesha Shetty - One of the best experts on this subject based on the ideXlab platform.
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complete genome sequence of the phenanthrene degrading soil bacterium Delftia acidovorans cs1 4
Standards in Genomic Sciences, 2015Co-Authors: Ameesha Shetty, Vidya De Gannes, Chioma C Obi, Susan Lucas, Alla Lapidus, Jan Fang Cheng, Lynne Goodwin, Samuel Pitluck, Linda Peters, Natalia MikhailovaAbstract:Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs in two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3’ end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.
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effects of outer membrane vesicle formation surface layer production and nanopod development on the metabolism of phenanthrene by Delftia acidovorans cs1 4
PLOS ONE, 2014Co-Authors: Ameesha Shetty, William J HickeyAbstract:Nanopods are extracellular structures arising from the convergence of two widely distributed bacterial characteristics: production of outer membrane vesicles (OMV) and formation of surface layers (S-layers). Nanopod production is driven by OMV formation, and in Delftia acidovorans Cs1-4 growth on phenanthrene induces OMV/nanopod formation. While OMV production has been associated with many functions, particularly with pathogens, linkage to biodegradation has been limited to a membrane stress response to lipophilic compounds. The objectives of this study were to determine: 1.) Whether induction of nanopod formation was linked to phenanthrene metabolism or a non-specific membrane stress response, and 2.) The relative importance of OMV/nanopod formation vs. formation of the S-layer alone to phenanthrene utilization. Membrane stress response was investigated by quantifying nanopod formation following exposure to compounds that exceeded phenanthrene in membrane stress-inducing potential. Naphthalene did not induce nanopod formation, and toluene was a weak inducer compared to phenanthrene (two- vs. six-fold increase, respectively). Induction of nanopod formation by growth on phenanthrene was therefore linked to phenanthrene metabolism and not a membrane stress response. Impacts on phenanthrene biodegradation of OMV/nanopod production vs. S-layer formation were assessed with D. acidovorans Cs1-4 mutants deficient in S-layer formation or OMV/nanopod production. Both mutants had impaired growth on phenanthrene, but the loss of OMV/nanopod production was more significant than loss of the S-layer. The S-layer of D. acidovorans Cs1-4 did not affect phenanthrene uptake, and its primary role in phenanthrene biodegradation process appeared to be enabling nanopod development. Nanopods appeared to benefit phenanthrene biodegradation by enhancing cellular retention of metabolites. Collectively, these studies established that nanopod/OMV formation was an essential characteristic of the D. acidovorans Cs1-4 phenanthrene degradation process. This report thus established a new dimension in the area of biodegradation, namely, the involvement of extracellular structures as elements supporting metabolic processes underlying biodegradation.
Hongkyu Yoon - One of the best experts on this subject based on the ideXlab platform.
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adaptation of Delftia acidovorans for degradation of 2 4 dichlorophenoxyacetate in a microfluidic porous medium
Biodegradation, 2014Co-Authors: Sabine Leibeling, Roland H Muller, Hongkyu Yoon, Changyong Zhang, Charles J Werth, Julie L ZillesAbstract:Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.