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Cited by 23Enzyme Catalysis and ImmobilizationMicrobial bioremediation and biosurfactantsMicrobial Metabolic Engineering and Bioproduction

The DOI matches its Crossref record; 69 of the 69 references it cites were found in a registry or an index; none of the 21 sources its claims cite is checked for support yet.

Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion

Open access
Dodge, Anthony G., Thoma, Calvin J., O’Connor, Madeline R., Wackett, Lawrence P.·2024·mBio
DOI 10.1128/mbio.02785-23PMC PMC10790756
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Abstract

Microbial response to toxic fluoride anion has traditionally been studied by adding inorganic fluoride salts to growth media. Fluoride is known to spontaneously transit the membrane as hydrogen fluoride (HF) and manifests significant toxicity in the cytoplasm. The present study investigated how microbes respond to high levels of HF generated directly in the cytoplasm to better understand potential limits of microbial defluorination of organofluorine compounds. For that purpose, Pseudomonas putida ATCC 12633 was engineered to express a defluorinase enzyme from Delftia acidovorans strain B that had high activity in producing growth-supporting alcohols from organofluorinated compounds of xenobiotic origin. Genome annotation predicted the growth of P. putida ATCC 12633 on all possible chiral products from 2-fluoropropionic acid and α-fluorophenylacetic acid when defluroinases are expressed. The defluorinase was shown to have complete enantioselectivity for ( S )-fluoro substrates via experimental and computational modeling methods. The bacterium grew to a high turbidity with stoichiometric release of fluoride from the ( S )-enantiomers into the medium. The highest yield of fluoride obtained was 50 mM with 2-fluoropropionic acid as the growth substrate. The growth yield was significantly lowered by 41% with α-fluorophenylacetic acid or 2-fluoropropionic acid compared to ( S )-mandelic acid or D-lactic acid, respectively. Fluoride stress was also indicated by longer lag phases, slower growth, and cell morphology changes on fluorinated substrates or the cognate alcohols with NaF in the medium. In total, these studies show the potential for engineering bacterial defluorination of non-natural substrates within limits posed by fluoride stress. IMPORTANCE: Society uses thousands of organofluorine compounds, sometimes denoted per- and polyfluoroalkyl substances (PFAS), in hundreds of products, but recent studies have shown some to manifest human and environmental health effects. As a class, they are recalcitrant to biodegradation, partly due to the paucity of fluorinated natural products to which microbes have been exposed. Another limit to PFAS biodegradation is the intracellular toxicity of fluoride anion generated from C-F bond cleavage. The present study identified a broader substrate specificity in an enzyme originally studied for its activity on the natural product fluoroacetate. A recombinant Pseudomonas expressing this enzyme was used here as a model system to better understand the limits and effects of a high level of intracellular fluoride generation. A fluoride stress response has evolved in bacteria and has been described in Pseudomonas spp. The present study is highly relevant to organofluorine compound degradation or engineered biosynthesis in which fluoride anion is a substrate.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Recombinant *Pseudomonas putida* ATCC 12633 expressing a *Delftia acidovorans* strain B defluorinase grew on non-natural fluorinated substrates and released up to 50 mM fluoride into the medium, showing stress but overall tolerance to cytoplasmically generated fluoride. The study links defluorination activity, enantioselectivity, growth inhibition, and fluoride stress indicators to assess limits for microbial organofluorine degradation.

Key findings

  • The *Delftia* defluorinase had higher activity than *R. palustris* FAcD on α-fluorophenylacetic acid (14.5 ± 0.9 vs 2.5 ± 0.1 µmol min−1 mg−1) and 2-fluoropropionic acid (1.7 ± 0.2 vs 0.063 ± 0.002 µmol min−1 mg−1), and was enantioselective for (S)-fluoro substrates, yielding (R)-alcohol products.
  • Recombinant *P. putida* 12633 grew on racemic α-fluorophenylacetic acid and 2-fluoropropionic acid with stoichiometric fluoride release; the highest fluoride yield was 50 mM with 2-fluoropropionic acid, while 120 mM 2-fluoropropionic acid caused growth arrest and α-fluorophenylacetic acid could not be processed above 40 mM without growth arrest.
  • Growth yield was lowered by 41% on α-fluorophenylacetic acid or 2-fluoropropionic acid compared with the cognate alcohols (R)-mandelic acid or D-lactic acid, respectively; with 10 mM NaF added externally, the drop was 22% for mandelate and 14% for lactate.
  • Fluoride stress was indicated by longer lag phases (14.3 h on α-fluorophenylacetic acid vs 1.2 h on mandelate), slower maximum growth rate (0.12 h−1 vs 0.23 h−1), a 90% CFU reduction during the first 20 h on 2-fluoropropionic acid, and cell shortening from 4 microns to 2 microns.

Methods

  • *P. putida* ATCC 12633 was engineered to constitutively express a codon-optimized *D. acidovorans* strain B defluorinase from plasmid pBBR1MCS-2; the *R. palustris* FAcD was also purified for comparison.
  • Purified enzymes were assayed for fluoride release with a fluoride ion selective electrode; growth was measured as OD600 nm and CFU on fluorinated substrates, cognate alcohols, and cognate alcohols plus NaF, with fluoride release monitored in the medium.
  • Stereochemistry was determined by polarimetry of remaining α-fluorophenylacetic acid, by D-/L-lactate dehydrogenase-coupled NADH formation for 2-fluoropropionic acid product, and by AlphaFold-based active-site modeling and substrate docking; microscopy and CrcB sequence comparisons were also used.

Limitations

  • The paper states that much remains to be learned about fluoride stress responses and that precise intracellular fluoride concentrations are difficult to determine when fluoride is supplied externally; it also notes that the Delftia enzyme was only reactive with monofluorinated compounds tested and lacks an X-ray structure, so an AlphaFold model was used.
  • Evident from the text: fluoride release was measured in the medium as a proxy for intracellular fluoride, and the study used one engineered host–enzyme system with racemic substrates, so direct intracellular fluoride dynamics and generality across other bacteria or defluorinases are not established.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1128/mbio.02785-23

Cites

69 of 69 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 21 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    With 2-fluoropropionic acid, cells grew slowly for up to 30 h and then grew more rapidly before plateauing after 72 h.

    “the cells grew slowly for up to 30 h and subsequently grew more rapidly before plateauing after 72 h”
    Quote in the paperSubstantial claim
  • Findingown data

    No significant difference was observed in poly-hydroxyalkanoate accumulation between cells grown on α-fluorophenylacetic acid or mandelic acid.

    “No significant difference was observed between cells grown on α-fluorophenylacetic acid or mandelic acid.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Cells exposed to sodium fluoride or α-fluorophenylacetic acid in liquid media showed significant cell morphological changes, most pronounced during mid-to-late exponential phase.

    “Cells exposed to sodium fluoride and α-fluorophenylacetic acid in liquid media showed significant cell morphological changes.”
    • Also stated in: § Other indicators of fluoride stress · plain
      “The cell differences were most pronounced during the mid-to-late exponential phase of growth (Fig. S3).”
    Quote in the paperNot a substantial claim
  • Findingown data

    With α-fluorophenylacetic acid releasing 10 mM fluoride internally, the decrease in growth yield was 41%.

    “With a level of α-fluorophenylacetic acid that released 10 mM fluoride internally, the decrease in growth yield was 41%.”
    Quote in the paperSubstantial claim
  • Findingown data

    More than one-third of α-fluorophenylacetic acid (>3.3 mM) was defluorinated early in the growth phase.

    “A separate growth experiment on α-fluorophenylacetic acid determined fluoride release and revealed more than one-third of the substrate had been defluorinated (>3.3 mM) early in the growth phase”
    Quote in the paperSubstantial claim
  • It may state two separate claims as one. 32
  • The quote only partly states this claim. 3
  • Probably not a substantial claim. 22

  • It may state two separate claims as one. The quote only partly states this claim.

    Findingown data

    Growth on α-fluorophenylacetic acid showed a longer lag time of 14.3 h and a lower maximum growth rate of 0.12 h−1 than growth on mandelate.

    “growth on α-fluorophenylacetic acid showed a much longer lag time of 14.3 h and a lower maximum growth rate of 0.12 h.−1”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The time to reach stationary phase was 8 h with sodium fluoride in the medium and 78 h with 2-fluoropropionic acid.

    “Dramatically, the time to reach the stationary phase was 8 h with sodium fluoride in the medium and 78 h with 2-fluoropropionic acid.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    After 168 h, the 50 mM culture ceased increasing in turbidity and had released the near theoretical limit of fluoride, showing complete consumption of one enantiomer of the substrate.

    “After 168 h, the 50 mM culture ceased increasing in turbidity and had released the near theoretical limit of fluoride, showing complete consumption of one enantiomer of the substrate.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    On minimal medium agar plates, P. putida 12633 expressing the recombinant defluorinase showed growth on mandelic acid in 24 h and large uniform colonies in 3 days.

    “The mandelic acid plates showed growth in 24 h, and relatively large, uniform-sized colonies were observed in 3 days.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Cell numbers of recombinant P. putida 12633 increased over the 24 h growth phase on α-fluorophenylacetic acid, indicating the strain remained viable.

    “By this method, the cell numbers increased over the 24 h growth phase, indicating that the strain remained viable.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Fluoride measurements showed complete release of fluoride from one substrate enantiomer, indicating metabolizable substrate was depleted at 72 h with 2-fluoropropionic acid.

    “Fluoride measurements showed a complete release of fluoride from one substrate enantiomer, indicating metabolizable substrate was depleted at 72 h.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    After 48 h, the culture with 50 mM 2-fluoropropionic acid showed the highest OD600 nm and the highest release of fluoride.

    “After 48 h, the culture with 50 mM 2-fluoropropionic acid showed the highest OD600 nm (Fig. 4A) and the highest release of fluoride.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Neither the R. palustris FAcD nor the D. acidovorans defluorinase was reactive with difluorinated carbon centers or with a fluoroolefin substrate.

    “Neither enzyme was reactive with difluorinated carbon centers or with a fluoroolefin substrate.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    On α-fluorophenylacetic acid agar plates, no colonies were observable in the first few days, but growth was seen after 7 days.

    “On α-fluorophenylacetic acid, no colonies were observable in the first few days, but growth was seen after 7 days.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    When cultured on 50-100 mM racemic 2-fluoropropionic acid, the culture took 1-2 weeks to grow to completion and showed irregular growth.

    “When strain 12633 was cultured on 50–100 mM racemic 2-fluoropropionic acid as the sole source of carbon and energy, the culture took 1–2 weeks to grow to completion and showed irregular growth (Fig. 4A).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The recombinant P. putida 12633 grew rapidly on mandelate with a lag time of 1.2 h and a maximum growth rate of 0.23 h−1.

    “The recombinant strain grew rapidly with mandelate, showing only a brief lag time of 1.2 h and a maximum growth rate of 0.23 h−1 as determined via a logarithmic fit using a QurvE computational tool (32) model”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Wirth NT, Funk J, Donati S, Nikel PI. 2023. QurvE: user-friendly software for the analysis of biological growth and fluorescence data. Nat Protoc 18: 2401– 2403. doi: 10.1038/s41596-023-00850-7 37380826 source

  • It may state two separate claims as one.

    Findingown data

    With 50 mM 2-fluoropropionic acid, there was only a minor increase in turbidity over the first 48 h; with 80 and 100 mM, a slight decrease in turbidity was observed over the initial 48 h.

    “With 50 mM, there was only a minor increase in turbidity over the first 48 h. With 80 and 100 mM 2-fluoropropionic acid, a slight decrease in turbidity was observed over the initial 48 h.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The time to reach stationary phase was 9 h with sodium fluoride added and 29 h with α-fluorophenylacetic acid.

    “Moreover, the time to reach the stationary phase was only 9 h with sodium fluoride added and 29 h with α-fluorophenylacetic acid, due to a long lag phase (Table 2) in the case of internally generated fluoride.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Fluoride was released during the initial non-growth period and attained ~8 mM in all cultures by 48 h.

    “Fluoride was released during this initial non-growth period and attained ~8 mM in all cultures by 48 h.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    With exposure to NaF, cells were shorter and narrower, with cell lengths decreasing from 4 microns to 2 microns.

    “With exposure to NaF, cells were discernibly shorter and narrower, with cell lengths decreasing from 4 microns to 2 microns (Fig. S3).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    An attempt to grow the strain on 120 mM 2-fluoropropionic acid resulted in growth arrest, indicating heightened toxicity to fluoride, the substrate, or a combination of both.

    “An attempt to grow the strain on 120 mM of 2-fluoropropionic acid resulted in growth arrest, indicating heightened toxicity to fluoride, the substrate, or a combination of both.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The Delftia defluorinase was only reactive with monofluorinated compounds tested, but had sufficiently broad specificity and high activity to generate a relatively high fluoride flux intracellularly with substrates able to support growth.

    “The Delftia defluorinase was only reactive with monofluorinated compounds that we tested, but it had sufficiently broad specificity and high activity to generate a relatively high fluoride flux intracellularly with substrates able to support growth.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Delftia defluorinase is localized in the cytoplasm of P. putida 12633, so the fluoride it releases is generated intracellularly.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingcitation

    The cell shortening observed here has not previously been reported in response to fluoride.

    “The cell shortening observed here has not previously been reported in response to fluoride but has been observed in Pseudomonas syringae under starvation stress conditions (47).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    After 168 h, the 80 and 100 mM cultures continued to slowly increase in turbidity and release fluoride, attaining near complete release of fluoride of 40 and 50 mM, respectively.

    “After 168 h, the 80 and 100 mM cultures continued to slowly increase in turbidity and release fluoride. Both attained near complete release of fluoride of 40 and 50 mM, respectively.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The rate of the Delftia defluorinase with 2-fluoropropionic acid was nearly an order of magnitude less than with α-fluorophenylacetic acid, allowing a comparison of high flux and lower flux fluoride release on the bacterium.

    “The rate of the enzyme with 2-fluoropropionic acid was nearly an order of magnitude less, allowing a comparison of high flux and lower flux fluoride release on the bacterium.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The AlphaFold model of the Delftia defluorinase active site is consistent with the direct stereochemical determinations and predicts attack by D105 with inversion, converting (S)-fluoro substrates to (R)-alcohols.

    “The enzyme modeling is thus consistent with the direct stereochemical determinations.”
    • Also stated in: § Substrate conversion and stereochemical outcomes · hedged
      “Computational model of the defluorinase active site showing the proposed nucleophilic attack by D105 with inversion of configuration, converting (S)-substrates to (R)-products.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: The AlphaFold model of the Delftia defluorinase accurately represents the active site and the stereochemical course of defluorination.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    Inhibition from fluoride might underlie the long lag phase on α-fluorophenylacetic acid.

    “This suggested that inhibition from fluoride might underlie the long lag phase.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The results show energy-demanding stress due to the cytoplasmic defluorination reaction, while also indicating significant robustness of the recombinant P. putida strain to continue to process the fluorinated substrates to completion.

    “In total, the results show energy-demanding stress due to the cytoplasmic defluorination reaction, while also indicating a significant robustness of the recombinant P. putida strain to continue to process the fluorinated substrates to completion.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Delftia defluorinase is localized in the cytoplasm of P. putida 12633, so the fluoride it releases is generated intracellularly.

  • It may state two separate claims as one.

    Interpretationown data

    Both α-fluorophenylacetic acid and 2-fluoropropionic acid are processed by the Delftia defluorinase with inversion of configuration during replacement of fluorine with hydroxyl.

    “The results indicate that both substrates are processed with the inversion of configuration during the replacement of the fluorine atom with a hydroxyl group.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    A control with sodium chloride did not show distinct cell morphological changes up to 50 mM, suggesting the observations are not due to salt stress and reflect a response to fluoride anion specifically.

    “A control set of experiments with sodium chloride did not show any distinct cell morphological changes with up to 50 mM of the salt, suggesting the observations are not due to salt stress and reflect a response to fluoride anion specifically.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    α-Chlorophenylacetic acid did not show the substantial lag phase observed with α-fluorophenylacetic acid, consistent with the idea that the acidic substrate itself is not inhibitory, but rather the fluoride being released is mediating the growth delay.

    “That substrate did not show the substantial lag phase observed with α-fluorophenylacetic acid, consistent with the idea that the acidic substrate itself is not inhibitory, but rather the fluoride being released is mediating the growth delay.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    This is the first report of microbes generating up to 50 mM fluoride via enzymatic defluorination and responding to high levels of fluoride generated directly in the cytoplasm.

    “To our knowledge, this is the first report of microbes generating up to 50 mM fluoride via enzymatic defluorination and responding to high levels of fluoride generated directly in the cytoplasm.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Delftia defluorinase is localized in the cytoplasm of P. putida 12633, so the fluoride it releases is generated intracellularly.

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisenone given

    Experiments to date have exposed cells to external fluoride by supplementation of the growth medium, and the precise intracellular concentration of fluoride under those conditions is difficult to determine.

    “Experiments to date have exposed cells to external fluoride by supplementation of the growth medium, and the precise intracellular concentration of fluoride at any one time under those conditions is difficult to determine.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    Taken together, the data showed that P. putida 12633 is capable of degrading very significant (50 mM) levels of a fluorinated substrate but, under those conditions, shows extreme stress leading to atypical and slow growth.

    “Taken together, these data showed that P. putida 12633 is capable of degrading very significant (50 mM) levels of a fluorinated substrate but, under those conditions, shows extreme stress leading to atypical and slow growth.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Study design and conductown data

    Enzyme activity was measured as net fluoride release from fluorinated substrates using a fluoride ion selective electrode calibrated with sodium fluoride standards.

    “The activity of the purified enzymes was measured as net fluoride release from fluorinated substrates using a fluoride ion selective electrode interfaced with an Orion Star A214 pH/ISE meter (Thermo Scientific) calibrated with sodium fluoride standards.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    The transformation efficiency was at least 30 CFU/μg plasmid DNA.

    “The transformation efficiency was ≥30 CFU/µg plasmid DNA.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one. The quote only partly states this claim.

    Interpretationown data

    The Delftia defluorinase modeling shows comparable structural features to homologous defluorinases, including a tryptophan residue that can assist fluoride transfer out of the active site.

    “In our modeling, we see comparable structural features in the Delftia defluorinase.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The AlphaFold model of the Delftia defluorinase accurately represents the active site and the stereochemical course of defluorination.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premiseown data

    CrcB proteins have demonstrated fluoride export rates as high as 10,000-100,000 s−1 in liposomes.

    “The latter has demonstrated fluoride export rates as high as 10,000–100,000 s−1.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Once in bulk water, fluoride anion is extensively hydrated, and significant energy would be required to strip off the water and reenter the enzyme fluorine binding site.

    “Once in a bulk water solvent, fluoride anion is known to be extensively hydrated (39, 45), and significant energy would be required to strip off the water and reenter the enzyme fluorine binding site.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [39] Orabi EA, Faraldo-Gómez JD. 2020. New molecular-mechanics model for simulations of hydrogen fluoride in chemistry and biology. J Chem Theory Comput 16: 5105– 5126. doi: 10.1021/acs.jctc.0c00247 32615034 PMC8431972 source
    • cites [45] Zhan CG, Dixon DA. 2004. Hydration of the fluoride anion: structures and absolute hydration free energy from first-principles electronic structure calculations. J Phys Chem A 108: 2020– 2029. doi: 10.1021/jp0311512 source

  • It may state two separate claims as one.

    Interpretationown data

    The rate of the Delftia defluorinase with α-fluorophenylacetic acid (14.5 μmol min−1 mg−1) was as high as many enzymes working on their natural substrates, allowing for a significant flux of fluoride release and significant growth in 24 h.

    “The rate with α-fluorophenylacetic acid (14.5 µmol min−1 mg−1) was as high as many enzymes working on their natural substrates, allowing for a significant flux of fluoride release and significant growth in 24 h.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    The activity and specificity of both lactate dehydrogenases were verified by testing each separately with 50 mM D- or L-lactate as substrate.

    “The activity and specificity of both lactate dehydrogenases were verified by testing each separately in similar reactions using 50 mM D- or L-lactate as the substrate with 1 unit of enzyme.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Premisecitation

    A knockout of the crcB gene, encoding a membrane protein that exports fluoride, makes P. putida KT2440 and other bacteria much more sensitive to fluoride.

    “Previous studies had also shown that a knockout of the crcB gene, encoding a membrane protein that exports fluoride, makes P. putida KT2440 (46) and other bacteria (13) much more sensitive to fluoride.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [46] Calero P, Volke DC, Lowe PT, Gotfredsen CH, O’Hagan D, Nikel PI. 2020. A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida. Nat Commun 11: 5045. doi: 10.1038/s41467-020-18813-x 33028813 PMC7541441 source
    • cites [13] McIlwain BC, Gundepudi R, Koff BB, Stockbridge RB. 2021. The fluoride permeation pathway and anion recognition in Fluc family fluoride channels. Elife 10: e69482. doi: 10.7554/eLife.69482 34250906 PMC8315801 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Protein concentration was measured via the Bradford method with a BSA standard curve.

    “The protein concentration was measured via the Bradford method with the Bio-Rad (Hercules, CA, USA) Protein Assay Dye Reagent and a standard curve prepared from a commercial bovine serum albumin (BSA) standard (Thermo Scientific).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductnone given

    The purity of protein fractions was assessed by SDS-PAGE, and fractions of equivalent purity were pooled.

    “The purity of fractions was assessed by SDS-PAGE, and linear gradient fractions of equivalent purity were pooled.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductown data

    The presence of the expression cassettes was verified by Sanger sequencing.

    “Plasmids were isolated from kanamycin-resistant clones using a QIAGEN plasmid miniprep kit, and the presence of the expression cassettes was verified by Sanger sequencing (ACGT, Inc., Wheeling, IL, USA).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    P. putida strains have become a focus of biocatalysis and biodegradation, including on the industrial scale, because of their resistance to various stresses other than fluoride stress.

    “P. putida strains have become a focus of biocatalysis and biodegradation, including on the industrial scale (48–50), because of their resistance to various stresses other than fluoride stress.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [48] Retallack DM, Jin H, Chew L. 2012. Reliable protein production in a Pseudomonas fluorescens expression system. Protein Expr Purif 81: 157– 165. doi: 10.1016/j.pep.2011.09.010 21968453 source
    • cites [49] Nikel PI, de Lorenzo V. 2018. Pseudomonas putida as a functional chassis for industrial biocatalysis: from native biochemistry to trans-metabolism. Metab Eng 50: 142– 155. doi: 10.1016/j.ymben.2018.05.005 29758287 source
    • cites [50] Craig K, Johnson BR, Grunden A. 2021. Leveraging Pseudomonas stress response mechanisms for industrial applications. Front Microbiol 12: 660134. doi: 10.3389/fmicb.2021.660134 34040596 PMC8141521 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    A recent effort sought to define the system-wide transcriptional regulatory network of P. putida KT2440, but that study did not include a response to stress from fluoride or organofluorine compound degradation.

    “A recent effort sought to define the system-wide transcriptional regulatory network of P. putida KT2440 (58), but that study did not include a response to stress from fluoride or organofluorine compound degradation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [58] Lim HG, Rychel K, Sastry AV, Bentley GJ, Mueller J, Schindel HS, Larsen PE, Laible PD, Guss AM, Niu W, Johnson CW, Beckham GT, Feist AM, Palsson BO. 2022. Machine-learning from Pseudomonas putida KT2440 transcriptomes reveals its transcriptional regulatory network. Metab Eng 72: 297– 310. doi: 10.1016/j.ymben.2022.04.004 35489688 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Cell shortening has been observed in Pseudomonas syringae under starvation stress conditions.

    “The cell shortening observed here has not previously been reported in response to fluoride but has been observed in Pseudomonas syringae under starvation stress conditions (47).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Monier J-M, Lindow SE. 2003. Pseudomonas syringae responds to the environment on leaves by cell size reduction. Phytopathology 93: 1209– 1216. doi: 10.1094/PHYTO.2003.93.10.1209 18944318 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    A previous study with P. putida KT2440 used a Tn-Seq protocol to identify genes involved in mitigating fluoride toxicity.

    “A previous study with P. putida KT2440 used a Tn-Seq protocol to identify genes involved in mitigating fluoride toxicity (9).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Calero P, Gurdo N, Nikel PI. 2022. Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride. Environ Microbiol 24: 5082– 5104. doi: 10.1111/1462-2920.16110 35726888 PMC9796867 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Mitigation of fluoride stress is multi-faceted, with some bacteria sensing toxic fluoride with a riboswitch, producing enzymes less sensitive to fluoride, inducing fluoride-specific membrane export proteins, and carrying out other as-yet-to-be defined functions.

    “Mitigation of fluoride stress has been shown to be multi-faceted, with some bacteria sensing toxic fluoride with a riboswitch, producing enzymes less sensitive to fluoride, inducing fluoride-specific membrane export proteins, and carrying out other as-yet-to-be defined functions (5, 8, 9).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [5] Marquis RE, Clock SA, Mota-Meira M. 2003. Fluoride and organic weak acids as modulators of microbial physiology. FEMS Microbiol Rev 26: 493– 510. doi: 10.1111/j.1574-6976.2003.tb00627.x 12586392 source
    • cites [8] Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335: 233– 235. doi: 10.1126/science.1215063 22194412 PMC4140402 source
    • cites [9] Calero P, Gurdo N, Nikel PI. 2022. Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride. Environ Microbiol 24: 5082– 5104. doi: 10.1111/1462-2920.16110 35726888 PMC9796867 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Digestion was verified by agarose gel electrophoresis, and the 4.3 kb linearized vector backbone band was excised and purified.

    “Digestion was verified by agarose gel electrophoresis, and the 4.3 kb linearized vector backbone band was excised and purified using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Solvent stress in P. putida S1 was found to decrease growth yield by 26%.

    “By comparison, solvent stress in P. putida S1 was found to decrease growth yield by 26% (57).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [57] Isken S, Derks A, Wolffs PF, de Bont JA. 1999. Effect of organic solvents on the yield of solvent-tolerant Pseudomonas putida S12. Appl Environ Microbiol 65: 2631– 2635. doi: 10.1128/AEM.65.6.2631-2635.1999 10347053 PMC91388 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Specific activity was calculated from fluoride release in triplicate reactions at three different time points.

    “Specific activity was calculated from fluoride release in triplicate reactions at three different time points.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductown data

    Growth yields of the P. putida ATCC 12633 Delftia FAcD expression strain on fluorinated or non-fluorinated analogs as sole carbon sources were determined in triplicate.

    “Growth yields of the P. putida ATCC 12633 Delftia FAcD expression strain growing on fluorinated or non-fluorinated analogs as sole carbon sources were determined in triplicate”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    There is a parallel interest in using Pseudomonas as a vector for the biosynthesis of new fluorinated compounds, some of which may serve as substitutes for synthetic fluorinated chemicals.

    “There is a parallel interest in using Pseudomonas as a vector for the biosynthesis of new fluorinated compounds (9, 46, 59), some of which may serve as substitutes for synthetic fluorinated chemicals.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [9] Calero P, Gurdo N, Nikel PI. 2022. Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride. Environ Microbiol 24: 5082– 5104. doi: 10.1111/1462-2920.16110 35726888 PMC9796867 source
    • cites [46] Calero P, Volke DC, Lowe PT, Gotfredsen CH, O’Hagan D, Nikel PI. 2020. A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida. Nat Commun 11: 5045. doi: 10.1038/s41467-020-18813-x 33028813 PMC7541441 source
    • cites [59] Carvalho MF, Oliveira RS. 2017. Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme. Crit Rev Biotechnol 37: 880– 897. doi: 10.1080/07388551.2016.1267109 28049355 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Lag phase determinations were done with 10 replicates for each treatment.

    “Aliquots (0.1 mL) of media inoculated with exponential-phase cells were dispensed into wells (10 replicates for each treatment), and then, the plate was shaken at 30°C with OD600 nm readings taken every 20 min.”
    Quote in the paperNot a substantial claim
51 of 51

  • The paper relies on this without support.

    The Delftia defluorinase is localized in the cytoplasm of P. putida 12633, so the fluoride it releases is generated intracellularly.

    “since the enzyme is intracellular, all of the fluoride anion is generated intracellularly”

  • The authors state this as an assumption.

    The AlphaFold model of the Delftia defluorinase accurately represents the active site and the stereochemical course of defluorination.

    “We used an AlphaFold (36, 37) model of the Delftia enzyme, for which an X-ray structure is not currently available, to infer if the defluorination reaction would also yield a product with inverted stereochemistry.”

  • The paper relies on this without support.

    The pre-existing AlphaFold structure of the Delftia defluorinase is accurate enough for modeling enzyme–ligand interactions.

    “The pre-existing AlphaFold (37, 38) structure of the Delftia defluorinase (UniProt accession Q01398) was used for modeling interactions between the enzyme and different fluorinated ligands.”

  • The paper relies on this without support.

    The 251 Pseudomonas spp. genomes used are representative of the genus for analyzing CrcB conservation.

    “Protein sequences encoded in a previously curated set of 251 Pseudomonas spp. genomes (68) were downloaded, and potential CrcB proteins were identified using the CrcB-like protein, Camphor Resistance (CrcB) hidden Markov model (InterPro PF02537) with a gathering threshold of 32.9.”

  • The paper relies on this without support.

    The presence of genes identified in P. putida KT2440 Tn-Seq in ATCC 12633 implies they perform similar fluoride-mitigation functions in ATCC 12633.

    “The genes identified in that study were found here to also be present in the genome of P. putida ATCC 12633 (Table S1).”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Abstract names a different mandelic acid enantiomer as the comparator

    Minor

    The abstract attributes the 41% growth-yield reduction to a comparison with (S)-mandelic acid, but the Table 2 value supporting that reduction uses (R)-mandelic acid.

  • Engineered strain is identified by two ATCC numbers

    Minor

    The introduction calls the engineered strain 12663, whereas the paper otherwise identifies the selected and engineered P. putida strain as ATCC 12633.

2 of 2

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Smart OS Sharff A Holstein J Womack TO Flensburg C Keller P Paciorek W Vonrhein C Bricogne G. 2021. Grade2 version 1.4.0. Cambridge United Kingdom: Global Phasing Ltd. Available from: https://grade.globalphasing.org/cgi-bin/grade2_server.cgi
  2. Eddy SR. 2020. Biological sequence analysis using profile hidden Markov models, version 3.3.2. Howard Hughes Medical Institute, Chevy Chase Maryland. http://eddylab.org/software/hmmer/Userguide.pdf.

Cited by

Sorted by citations

Related

Metadata

Authors
Dodge, A. G., Thoma, C. J., O’Connor, M. R., & Wackett, L. P.
Affiliations
University of Minnesota (US)
Corresponding author
Wackett, Lawrence P.
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2024-01-16
Volume
15
Issue
1
Pages
e0278523-e0278523
ISSN
2150-7511 (ISSN-L)
DOI
10.1128/mbio.02785-23
PMC
PMC10790756
OpenAlex
W4389488837
Cited by
23
References
69
Citations by year
2026: 8 · 2025: 7 · 2024: 8
FWCI
1.78
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
MnDRIVE Industry and the Environment Program
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Physical Sciences › Environmental Science › Pollution › Microbial bioremediation and biosurfactants
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
Pseudomonas putida, Delftia acidovorans, fluoride toxicity, organofluorine compounds, per- and polyfluoroalkyl substances, biodegradation, enantioselectivity, C–F bond cleavage, fluoride stress
MeSH terms
Biodegradation, Environmental, Fluorides, Fluoroacetates, Fluorocarbons, Humans, Pseudomonas

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