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Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride
Open accessAbstract
The presence of mineral fluoride (F − ) in the environment has both a geogenic and anthropogenic origin, and the halide has been described to be toxic in virtually all living organisms. While the evidence gathered in different microbial species supports this notion, a systematic exploration of the effects of F − salts on the metabolism and physiology of environmental bacteria remained underexplored thus far. In this work, we studied and characterized tolerance mechanisms deployed by the model soil bacterium Pseudomonas putida KT2440 against NaF. By adopting systems‐level omic approaches, including functional genomics and metabolomics, we gauged the impact of this anion at different regulatory levels under conditions that impair bacterial growth. Several genes involved in halide tolerance were isolated in a genome‐wide Tn ‐Seq screening—among which crcB , encoding an F − ‐specific exporter, was shown to play the predominant role in detoxification. High‐resolution metabolomics, combined with the assessment of intracellular and extracellular pH values and quantitative physiology experiments, underscored the key nodes in central carbon metabolism affected by the presence of F − . Taken together, our results indicate that P . putida undergoes a general, multi‐level stress response when challenged with NaF that significantly differs from that caused by other saline stressors. While microbial stress responses to saline and oxidative challenges have been extensively studied and described in the literature, very little is known about the impact of fluoride (F − ) on bacterial physiology and metabolism. This state of affairs contrasts with the fact that F − is more abundant than other halides in the Earth crust (e.g. in some soils, the F − concentration can reach up to 1 mg g soil −1 ). Understanding the global effects of NaF treatment on bacterial physiology is not only relevant to unveil distinct mechanisms of detoxification but it could also guide microbial engineering approaches for the target incorporation of fluorine into value‐added organofluorine molecules. In this regard, the soil bacterium P . putida constitutes an ideal model to explore such scenarios, since this species is particularly known for its high level of stress resistance against a variety of physicochemical perturbations.
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Written from the paper’s full text.
TL;DR
The paper characterizes how *Pseudomonas putida* KT2440 responds to mineral fluoride (NaF), showing that the CrcB exporter is the dominant detoxification mechanism and that NaF causes a multi-level stress response distinct from other saline stressors. Tn-Seq, physiology, pH biosensors, and metabolomics together indicate membrane/redox/central-carbon-metabolism disruption and intracellular acidification.
Key findings
- Tn-Seq identified *crcB* (PP_4001) as the top fluoride-tolerance gene, with log2(FC) = −2.79 and p = 0.0151; the authors conclude CrcB is the predominant F− detoxification mechanism.
- Wild-type *P. putida* KT2440 tolerated NaF with MICs in the range of 75 mM in M9 minimal medium and 125 mM in LB; the Δ*crcB* mutant was impaired by as little as 0.5 mM NaF in M9, about 100-fold lower than wild type, and its LB MIC shifted 10-fold higher.
- Additional Tn-Seq hits included *rarA*, *prtR*, *lapA*, *lapD*, *flhA*, *yijP*, and LPS/flagella/biofilm-related functions; insertions in *orn*, *dusB*, and *vacB* were enriched under NaF.
- Metabolomics showed NaF-dependent re-organization of central carbon metabolism: sugar phosphates accumulated, PEP was depleted, citrate decreased about threefold in wild type and fourfold in Δ*crcB*, NADPH/NADH were depleted, and L-tyrosine and L-methionine accumulated, especially in Δ*crcB*.
- Intracellular pH decreased with higher NaF concentrations; the authors argue HF is the toxic, membrane-permeant form, and Δ*crcB* showed cytoplasmic acidification even at low NaF.
Methods
- *P. putida* KT2440 was grown in LB or M9 minimal medium with 5 g L−1 glucose; NaF was used as the stressor, with MIC/growth assays, quantitative physiology, and in-frame deletion mutants of Tn-Seq candidates.
- Genome-wide Tn-Seq used a mini-Tn5 insertion library under control and 50 mM NaF conditions, followed by Illumina MiSeq sequencing and ESSENTIALS analysis; genes with log2(FC) < −2 or > 2 and p < 0.05 were selected.
- Intracellular F− was measured with the fluoride-responsive riboswitch biosensor pS441·FRSv1; intracellular pH with the PHP biosensor; and targeted LC–MS/MS metabolomics was performed after NaF addition (50 mM for wild type, 1 mM for Δ*crcB) over 0–5400 s, with two biological replicates and technical duplicates.
Limitations
- Not stated in the paper: the article has no dedicated limitations section.
- Evident from the text: Tn-Seq selection used 50 mM NaF, mutant phenotyping used 25 mM NaF, and metabolomics used different NaF concentrations for wild type (50 mM) and Δ*crcB* (1 mM) to match growth inhibition, which may complicate direct strain-to-strain comparisons.
- Evident from the text: the authors note that *orn*, *dusB*, and *vacB* may have pleiotropic roles in general homeostasis rather than a specific NaF response, and the metabolomics experiments were performed with two biological replicates.
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Claims the paper makes
0 of 63 cited sources checked · 0 supported · 0 flagged
- Findingown data
Both kinetic parameters, λ and μmax, of P. putida KT2440 are affected by 50 mM NaF in shaken-flask cultures.
“Both kinetic parameters were affected by the presence of the halide in the medium”
Quote in the paperSubstantial claim - Findingown data
Both μ max and the final cell density were severely affected in cultures of P. putida ΔcrcB when the medium was supplemented with 25 mM NaF, essentially inhibiting bacterial growth.
“As expected, both μ max and the final cell density (OD600, optical density measured at 600 nm) were severely affected in cultures of P. putida ΔcrcB when the medium was supplemented with 25 mM NaF—which essentially inhibited bacterial growth under these conditions.”
Quote in the paperSubstantial claim - Findingown data
LapD was also isolated in our screening.
“LapD was also isolated in our screening, probably due to its functional relationship with LapA.”
Quote in the paperNot a substantial claim - Findingown data
Under acid stress (HCl) and oxidative stress (H2O2 or paraquat), the growth pattern of wild-type P. putida and ΔcrcB was similar.
“The growth pattern of both strains was similar under all three stressful conditions (i.e. both the wild‐type strain and the ΔcrcB strain displayed a similar sensitivity to the addition of HCl, H2O2 or paraquat)”
Quote in the paperSubstantial claim - Findingown data
The ORFs identified in the Tn-Seq screening are scattered throughout the genome of P. putida KT2440.
“The ORFs identified in this screening experiment are scattered throughout the genome of P. putida KT2440 [Figure 2(D)]”
Quote in the paperNot a substantial claim
- Findingown data
P. putida KT2440 recovered faster from NaF stress in rich medium than in M9 minimal medium.
“yet P. putida recovered faster in rich medium than in M9 minimal medium.”
Quote in the paperNot a substantial claim - Findingown data
P. putida ΔrarA showed a twofold increase in fluorescence output compared to the wild-type strain after 10 h of incubation with 1 mM NaF.
“The only exception was P. putida ΔrarA, which already showed a twofold increase in the fluorescence output compared to the wild‐type strain after 10 h of incubation with 1 mM NaF.”
Quote in the paperSubstantial claim - Findingown data
The loss of function of five genes increases the growth of P. putida KT2440 in the presence of F−.
“Additionally, the loss of function of five genes was identified to increase the growth of P. putida KT2440 in the presence of F−, as Tn5 insertions in these sequences were more represented in cultures added with NaF than in control experiments [Figure 2(F) and Table S2 in the Supporting Information].”
Quote in the paperSubstantial claim - Findingown data
In LB medium, P. putida KT2440 cultures were strongly affected by NaF during early exponential growth, with normalized growth values immediately dropping by >90%.
“LB medium cultures, in contrast, were strongly affected by the presence of NaF during early exponential growth (with normalized growth values immediately dropping by >90%)”
Quote in the paperNot a substantial claim - Findingown data
In M9 minimal medium with glucose, normalized growth of P. putida KT2440 in the presence of NaF had a relatively stable behaviour, with normalized growth values gradually decreasing over the first 10 h.
“Cultures grown in M9 minimal medium with glucose had a relatively stable behaviour, as judged by normalized growth values that gradually decreased over the first 10 h.”
Quote in the paperNot a substantial claim - Findingown data
Intermediate growth phenotypes between those of the wild-type strain and the ΔcrcB mutant could be detected in cultures of the remaining mutants.
“Intermediate growth phenotypes between those of the wild‐type strain and the ΔcrcB mutant could be detected in cultures of the remaining mutants.”
Quote in the paperNot a substantial claim - Findingown data
Final OD600 values after 24 h in shaken-flask cultures of P. putida KT2440 with 50 mM NaF are similar to those in control cultures without added F−.
“although the final OD600 values (upon 24 h of incubation) in these experiments were remarkably similar to those reached in control cultures, without any added F− [Figure 1(C,D)].”
Quote in the paperSubstantial claim - Findingown data
P. putida KT2440 tolerates NaF within different concentration ranges depending on the culture medium (LB vs M9 minimal medium with glucose).
“After 20 h of incubation, we found that P. putida tolerated NaF within different concentration ranges depending on the culture medium used for the experiments [Figure 1(A)].”
Quote in the paperSubstantial claim - Findingown data
A clear msfGFP signal corresponding to a significant intracellular F− level could be observed in cultures of P. putida ΔcrcB/pS441·FRSv1 exposed to the salt even when NaF was added at concentrations as low as 0.25 mM.
“Expectedly, a clear msfGFP signal corresponding to a significant intracellular F− level could be observed in cultures of P. putida ΔcrcB/pS441·FRSv1 exposed to the salt—even when NaF was added at concentrations as low as 0.25 mM [Figure 3(C)].”
Quote in the paperSubstantial claim- Rests on an assumption: CrcB functions as a fluoride efflux transporter in P. putida KT2440, so that loss of CrcB leads to intracellular F− accumulation.
- Findingown data
prtR (PP_2889) is involved in the F− resistance phenotype observed in the Tn-Seq experiments.
“We also discovered that prtR (PP_2889) was involved in the resistance phenotype in these experiments.”
Quote in the paperNot a substantial claim - Findingown data
Several independent insertions in orn, dusB and vacB were enriched in the presence of F−, up to 15 insertions in the case of vacB.
“These genes included orn, dusB and vacB, in which several independent insertions (up to 15 in the case of vacB) were enriched in the presence of F−.”
Quote in the paperNot a substantial claim - Findingown data
The gene encoding the F− exporter CrcB had the top score (log2(FC) = −2.79, p-value = 0.0151) among the functions expected to confer NaF resistance in the Tn-Seq screen.
“The gene encoding the F− exporter CrcB had a top score [log2(FC) = −2.79 and p‐value = 0.0151; Table 2] among the functions expected to confer NaF resistance.”
Quote in the paperSubstantial claim - Findingown data
NaF treatment essentially did not affect the lag phase duration (λ) of P. putida KT2440 in M9 minimal medium containing glucose as the carbon source.
“while this growth parameter was essentially unaffected in M9 minimal medium containing glucose as the carbon substrate [Figure 1(F)].”
Quote in the paperNot a substantial claim - Findingown data
In P. putida ΔcrcB, growth was impaired when M9 minimal medium was supplemented with as little as 0.5 mM NaF after 20 h.
“finding that bacterial growth was impaired when M9 minimal medium is supplemented with as little as 0.5 mM NaF [Figure 4(A)].”
Quote in the paperSubstantial claim - Findingown data
At 125 mM NaF, growth of P. putida KT2440 is similarly and dramatically affected in both LB and M9 minimal medium, with >90% reduction in OD600 values.
“When the halide was added to the media at 125 mM, bacterial growth was similarly (and dramatically) affected in both culture conditions [Figure 1(A)], with a >90% reduction in OD600 values.”
Quote in the paperSubstantial claim - Findingown data
NaF treatment reduced the maximum specific growth rate (μmax) of P. putida KT2440 by 43% in M9 minimal medium.
“μ max was reduced by 62% and 43% upon NaF treatment in LB and M9 minimal medium, respectively.”
Quote in the paperSubstantial claim - Findingown data
The rest of the mutants in genes identified in the screening could stably maintain low intracellular F− levels when NaF was fed at concentrations up to 1 mM.
“The rest of the mutants in genes identified in our screening, in contrast, could stably maintain low intracellular F− levels when NaF was fed at concentrations up to 1 mM.”
Quote in the paperSubstantial claim - Findingown data
High-resolution metabolomics combined with the assessment of intracellular and extracellular pH and quantitative physiology identified the key nodes in central carbon metabolism affected by the presence of F−.
“High‐resolution metabolomics, combined with the assessment of intracellular and extracellular pH values and quantitative physiology experiments, underscored the key nodes in central carbon metabolism affected by the presence of F−.”
Quote in the paperSubstantial claim - Findingown data
At the highest NaF concentration tested, a P. putida mutant showed a fluorescence signal sevenfold higher than the control strain after 15 h of F− exposure.
“At the highest NaF concentration tested, this mutant showed a fluorescence signal sevenfold higher than the control strain after 15 h of F− exposure.”
Quote in the paperSubstantial claim - Findingown data
NaF treatment increased the lag phase duration (λ) of P. putida KT2440 by 3.5-fold in LB medium.
“λ increased by 3.5‐fold in LB medium added with F−”
Quote in the paperSubstantial claim - Findingown data
NaF treatment reduced the maximum specific growth rate (μmax) of P. putida KT2440 by 62% in LB medium.
“μ max was reduced by 62% and 43% upon NaF treatment in LB and M9 minimal medium, respectively.”
Quote in the paperSubstantial claim - Findingown data
Under the Tn-Seq culture conditions (M9 minimal medium with glucose, with or without 50 mM NaF), the maximum specific growth rate of the mini-Tn5 libraries dropped from 0.46 h−1 in the control to 0.15 h−1 in the presence of NaF.
“Under these culture conditions, μ max dropped from 0.46 h−1 in the control experiment to 0.15 h−1 in the presence of NaF [Figure 2(C)], consistent with our previous observations in wild‐type P. putida cultures (Figure 1).”
Quote in the paperSubstantial claim - Findingown data
The F− concentration that impairs growth of P. putida ΔcrcB is ca. 100-fold lower than the amount needed to inhibit growth of wild-type P. putida KT2440.
“This is an F− concentration ca. 100‐fold lower than the amount needed to inhibit the growth of wild‐type P. putida KT2440.”
Quote in the paperSubstantial claim - Findingown data
Several genes are important for the growth of P. putida KT2440 when exposed to high F− concentrations, as identified by the Tn-Seq screen.
“Several genes were found to be important for the growth of P. putida KT2440 when exposed to high F− concentrations [Figure 2(D); Table 1 and Table S2 in the Supporting Information].”
Quote in the paperSubstantial claim - Findingown data
In M9 minimal medium with glucose, P. putida KT2440 growth is impaired even at the lowest NaF concentration tested.
“yet bacterial growth in M9 minimal medium with glucose was impaired even at the lowest NaF concentration tested.”
Quote in the paperSubstantial claim - Findingown data
P. putida strains carrying mutations in orn, dusB and vacB showed reduced-fitness phenotypes in the tested conditions of F− when compared to the wild-type strain.
“The P. putida strains carrying mutations in the genes orn, dusB and vacB, which were identified in the Tn‐seq screening to have a better growth when disrupted, also showed reduced‐fitness phenotypes in the tested conditions of F− when compared to cultures of the wild‐type strain [Figure 2(F)].”
Quote in the paperSubstantial claim - Findingown data
A genome-wide Tn-Seq screening isolated several genes involved in halide tolerance.
“Several genes involved in halide tolerance were isolated in a genome‐wide Tn‐Seq screening”
Quote in the paperSubstantial claim - Findingown data
At the highest NaF concentration tested, P. putida ΔrarA showed a fluorescence signal sevenfold higher than the wild-type strain.
“At the highest NaF concentration tested, this mutant showed a fluorescence signal sevenfold higher”
Quote in the paperSubstantial claim - Findingown data
crcB, encoding an F−-specific exporter, plays the predominant role in F− detoxification.
“among which crcB, encoding an F−‐specific exporter, was shown to play the predominant role in detoxification”
Quote in the paperSubstantial claim - Findingown data
Two genes selected in the Tn-Seq experiments, fleQ (PP_4373) and flhA (PP_4344), were involved in the regulation and synthesis of flagella.
“Two other genes selected in the Tn‐Seq experiments were involved in the regulation and synthesis of flagella, i.e. fleQ (PP_4373) and flhA (PP_4344).”
Quote in the paperNot a substantial claim - Findingown data
Two genes related to biofilm formation, lapA (PP_0168) and lapD (PP_0165), were recognized in the experiment.
“Furthermore, two genes related to biofilm formation were recognized in the experiment, lapA (PP_0168) and lapD (PP_0165).”
Quote in the paperNot a substantial claim - Findingown data
In LB medium, P. putida KT2440 can grow in the presence of 75 mM NaF to final OD600 values >70% of those in control experiments without F−.
“Cells could grow in the presence of 75 mM NaF in LB medium up to final OD600 values >70% of that registered in control experiments (i.e. no F− in the medium)”
Quote in the paperSubstantial claim - Findingown data
P. putida ΔrarA displayed a reduced (ca. 50%) μ max value in the presence of NaF compared to the control experiment.
“Pseudomonas putida ΔrarA, for instance, displayed a reduced (ca. 50%) μ max value in the presence of NaF compared to the control experiment.”
Quote in the paperNot a substantial claim - Findingreasoning
In media containing 1 mM NaF, P. putida ΔcrcB was able to grow only when the plates were basified by the growth of the wild-type strain.
“In the media containing 1 mM NaF, P. putida ΔcrcB was able to grow only when the plates were basified by the growth of the wild‐type strain.”
Quote in the paperSubstantial claim - Findingown data
Screening a genome-wide Tn-Seq library in P. putida singled out CrcB, the widespread F− exporter, as the predominant mechanism for alleviating the effects of the halide.
“We have extended the analysis of toxicity tolerance by screening a genome‐wide Tn‐Seq library in P. putida, which singled out CrcB (the widespread F− exporter) as the predominant mechanism for alleviating the effects of the halide.”
Quote in the paperSubstantial claim - Findingown data
NADPH and NADH seemed to be depleted in the presence of F−.
“Redox power was likewise affected by the halide, especially at the level of NADPH and NADH, as both seemed to be depleted in the presence of F− (Figure S5).”
Quote in the paperSubstantial claim - Findingown data
The general trend observed was a lower intracellular pH in cells exposed to higher NaF concentrations (inverse relationship between NaF concentration and intracellular pH).
“The general trend observed was a lower intracellular pH in cells exposed to higher NaF concentrations (i.e. we detected an inverse relationship between the two parameters studied”
Quote in the paperSubstantial claim - Findingown data
The gene encoding an NADP+-dependent alcohol dehydrogenase (CurA, PP_2476) was identified in the Tn-Seq screening as relevant for survival in the presence of F−.
“Interestingly, the gene encoding an NADP+‐dependent alcohol dehydrogenase (CurA, PP_2476) was also identified in the Tn‐Seq screening as to be relevant for survival in the presence of F−”
Quote in the paperNot a substantial claim - Findingown data
L-tyrosine accumulated at elevated levels that inversely correlate with the content of its metabolic precursor, PEP.
“This aromatic amino acid was accumulated at elevated levels that inversely correlate with the content of its metabolic precursor, PEP.”
Quote in the paperNot a substantial claim - Findingown data
The growth of the crcB mutant was impaired in M9 minimal medium with glucose, either co-cultured with the wild-type or by itself.
“Conversely, the growth of the crcB mutant was impaired in M9 minimal medium with glucose, either co‐cultured with the wild‐type or by itself [Figure 4(H)]”
Quote in the paperSubstantial claim - Findingown data
An imbalance of TCA cycle intermediates was seen in both P. putida strains tested when F− was present in the culture medium.
“In any case, an imbalance of the TCA cycle intermediates could be seen in the two P. putida strains tested when F− was present in the culture medium.”
Quote in the paperNot a substantial claim - Findingown data
The gene encoding the B12-dependent methionine synthase MetH seemed to be relevant for F− tolerance in the Tn-Seq experiment.
“We found that the gene encoding the last enzyme involved in l‐methionine synthesis, the B12‐dependent methionine synthase MetH, seemed to be relevant for F− tolerance in the Tn‐Seq experiment (Table 2)”
Quote in the paperSubstantial claim - Findingreasoning
In LB broth, the growth of P. putida mediated a change in the pH towards basicity, detected as phenol red turned fuchsia.
“In a rich medium, such as LB broth, the growth of P. putida mediated a change in the pH towards basicity, which could be easily detected as phenol red turned fuchsia under these conditions [Figure 4(G)]”
Quote in the paperNot a substantial claim - Findingcitation
In M9 minimal medium with glucose, the growth of P. putida cells acidified the medium.
“On the contrary, in M9 minimal medium with glucose as a carbon source, the growth of cells acidified the medium”
Quote in the paperSubstantial claim - Findingown data
Metabolites within upper glycolysis accumulated over time upon NaF addition in both P. putida KT2440 and the ΔcrcB mutant.
“In general, we detected a trend where metabolites within upper glycolysis (e.g. G6P, sedoheptulose 7‐phosphate, fructose 6‐phosphate and ribose 5‐phosphate) accumulated over time upon NaF addition in both P. putida strains.”
Quote in the paperNot a substantial claim - Findingreasoning
PEP was depleted over time upon NaF addition.
“Moreover, we observed depletion of PEP over time”
Quote in the paperNot a substantial claim - Findingown data
Some key glycolytic metabolites, including glucose 6-phosphate and phosphoenolpyruvate, were affected by F− treatment.
“Some key glycolytic metabolites were affected by the F− treatment [Figure 5(B)], e.g. glucose 6‐phosphate (G6P, Figure S2) and phosphoenolpyruvate (PEP, Figure S3).”
Quote in the paperNot a substantial claim - Premisecitation
HF crosses biological membranes through water channels, including aquaporins.
“HF is such a small molecule that it would cross biological membranes through water channels, including aquaporins (Marquis et al., 2003).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Marquis et al., 2003 source
- Interpretationreasoning
The results so far indicate a general impact of F− on the cell physiology.
“The results so far indicate a general impact of F− on the cell physiology, and prompted the question of how are the genes identified in the Tn‐Seq experiment involved at the molecular level.”
Quote in the paperSubstantial claim - Premisecitation
The FRS can be indirectly used as a means to assess F− concentrations owing to the conformation change in the secondary mRNA structure in the presence of the anion.
“The FRS can be indirectly used as a means to assess F− concentrations owing to the conformation change in the secondary mRNA structure in the presence of the anion.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Calero et al., 2020 source
- Interpretationown data
The genes isolated in the Tn-Seq experiments are involved in the response of P. putida KT2440 to F− treatment.
“Taken together, these experiments show that the genes isolated in the Tn‐Seq experiments are indeed involved in the response of P. putida KT2440 to F− treatment.”
Quote in the paperSubstantial claim - Interpretationown data
CrcB is not directly involved in the tolerance towards HCl, H2O2, or paraquat stressors.
“which indicates that CrcB is not directly involved in the tolerance towards these stressors.”
Quote in the paperSubstantial claim - Interpretationown data
F− treatment is connected to diverse physiological and regulatory functions in P. putida KT2440.
“Taken together, these results indicate that F− treatment is connected to diverse physiological and regulatory functions in P. putida KT2440—even when the actual number of genes involved in the tolerance phenotype is relatively small, as is the type and number of functional categories to which these genes belong.”
Quote in the paperSubstantial claim - Interpretationown data
The low msfGFP output of the wild-type strain indicates efficient F− secretion via CrcB.
“The wild‐type strain, in contrast, tolerated the NaF challenge even at 15 mM, displaying a low msfGFP output that indicates efficient F− secretion via CrcB.”
Quote in the paperSubstantial claim- Rests on an assumption: CrcB functions as a fluoride efflux transporter in P. putida KT2440, so that loss of CrcB leads to intracellular F− accumulation.
- Interpretationown data
CrcB plays a central role in F− detoxification.
“At this point, it was clear that CrcB plays a central role in F− detoxification, as knocking out the gene encoding this exporter led to a significant intracellular accumulation of F− that impaired growth even at very low NaF concentrations.”
Quote in the paperSubstantial claim - Premisecitation
yijP (PP_2579) and PP_1779 are involved in the synthesis and export of lipopolysaccharides.
“Finally, two genes recognized as being involved in the synthesis and export of lipopolysaccharides, i.e. yijP (PP_2579) and PP_1779 (Cuthbertson et al., 2010; Mumm et al., 2016), were likewise isolated in this experiment.”
Quote in the paperNot a substantial claim0 of 2 sources checked - Study design and conductown data
The 25 mM NaF concentration (half that used in the Tn-Seq experiments) was selected to still observe the negative impact of F− on cell physiology without completely suppressing bacterial growth.
“Note that this NaF concentration (half the amount used in the Tn‐Seq experiments) was selected as to still observe the negative impact of F− on the cell physiology without completely suppressing bacterial growth according to the preliminary experiments shown in Figure 1—these conditions are especially relevant for characterizing mutants affected in tolerance against NaF.”
Quote in the paperSubstantial claim - Premisecitation
The intracellular F− concentration in P. putida strains carrying pS441·FRSv1 is correlated with the msfGFP fluorescence output.
“The intracellular F− concentration in these strains was correlated to the msfGFP fluorescence output.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Calero et al., 2020 source
- Premisecitation
The conformational transition of the FRS exposes a buried ribosome binding site, leading to the translation of the reporter msfGFP.
“This conformational transition exposes a (buried) ribosome binding site, leading to the translation of the reporter (i.e. msfGFP, a monomeric superfolder GFP).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Calero et al., 2020 source
- Premisereasoning
The effects of F− salts on the physiology of Pseudomonas putida remained unstudied prior to this work.
“yet the effects of these salts on the bacterial physiology remain unstudied”
Quote in the paperSubstantial claim0 of 2 sources checked - Interpretationreasoning
F− exerts an impact on P. putida KT2440 growth unique to this halide.
“These observations indicate that F− exerts an impact on bacterial growth unique to this halide.”
Quote in the paperSubstantial claim - Interpretationown data
CrcB-dependent efflux of F− ions is by far the predominant molecular mechanism against F− toxicity.
“Taken together, these results indicate that CrcB‐dependent efflux of F− ions is by far the predominant molecular mechanism against toxicity.”
Quote in the paperSubstantial claim - Study design and conductown data
There was no significant positional bias in the Tn-Seq analysis.
“which indicates that there was no significant positional bias in the Tn‐Seq analysis”
Quote in the paperNot a substantial claim - Interpretationreasoning
The depletion of PEP highlights inhibition of the enolase enzyme, a previously identified target of NaF toxicity.
“highlighting the inhibition of the enolase enzyme (which catalyses the penultimate step of the EMP pathway, converting 3‐phosphoglycerate to PEP), one of the targets of NaF toxicity previously identified in in vitro studies (Qin et al., 2006).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Qin et al., 2006 source
- Interpretationown data
L-methionine levels play a role in the response to the halide.
“providing evidence that l‐methionine levels play a role in the response to the halide.”
Quote in the paperSubstantial claim - Interpretationcitation
The time-dependent accumulation of L-tyrosine in the ΔcrcB mutant may be explained as a mechanism of defence against oxidative stress.
“The time‐dependent accumulation of l‐tyrosine in the ΔcrcB mutant may also be explained as a mechanism of defence against oxidative stress as pointed previously (Liu et al., 2020; Zhang et al., 2021).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Liu et al., 2020
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Zhang et al., 2021
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationreasoning
The identification of CurA, together with physiology experiments, could point to F− causing an effect on redox balance.
“which, together with the evidence gathered in physiology experiments, could point to the fact that the F− causes an effect in the redox balance (Table 2).”
Quote in the paperNot a substantial claim - Interpretationreasoning
The observations relate to biogeochemical processes comprising F− salts and other elements in groundwater and soil.
“These observations also relate to biogeochemical processes comprising F− salts and other elements in groundwater and soil, suggesting that F− concentration should be considered when evaluating microbial response to environmental conditions.”
Quote in the paperNot a substantial claim - Interpretationcitation
The accumulation of sugar phosphates upon NaF addition can be explained by a general inhibitory effect of the halide on glycolytic enzymes.
“This accumulation of sugar phosphates can be explained by a general inhibitory effect of glycolytic enzymes by the halide.”
Quote in the paperNot a substantial claim - Interpretationcitation
The metabolic response of P. putida to sub-lethal NaF invokes a network-wide re-arrangement of key metabolites within central carbon metabolism that in some cases resembles alterations provoked by acid stress.
“Indeed, our findings indicate that the metabolic response of P. putida to a challenge with sub‐lethal concentrations of NaF invokes a network‐wide re‐arrangement of key metabolites within central carbon metabolism that, in some cases, resembles the alterations provoked by acid stress (Akkaya et al., 2018; Corona et al., 2019; Reva et al., 2006).”
Quote in the paperSubstantial claim0 of 3 sources checked - Study design and conductreasoning
In the metabolomics experiments, a NaF concentration that would cause a similar reduction in the specific growth rate for P. putida KT2440 and the ΔcrcB mutant (50 and 1 mM, respectively) was used.
“Due to the significant differences in F− sensitivity of either strain, in these experiments we used a NaF concentration that would cause a similar reduction in the specific growth rate for P. putida KT2440 and the ΔcrcB mutant (50 and 1 mM,”
Quote in the paperSubstantial claim - Study design and conductown data
NaF concentrations of 50 mM for P. putida KT2440 and 1 mM for the ΔcrcB mutant were used to cause a similar reduction in specific growth rate.
“we used a NaF concentration that would cause a similar reduction in the specific growth rate for P. putida KT2440 and the ΔcrcB mutant (50 and 1 mM, respectively; see Figures 1 and 4).”
Quote in the paperSubstantial claim - Study design and conductown data
No significant changes in the fluorescence ratio were detected in the absence of NaF.
“Expectedly, no significant changes in the fluorescence ratio were detected in the absence of NaF.”
Quote in the paperNot a substantial claim - Interpretationown data
The inverse relationship may indicate that the HF ↔ H+ + F− equilibrium is displaced towards dissociation in the cytoplasmic milieu.
“which may indicate that the HF ↔ H+ + F− equilibrium is displaced towards dissociation in the cytoplasmic milieu).”
Quote in the paperNot a substantial claim - Interpretationcitation
The pH change towards basicity in LB is probably due to amino acids and small peptides being the main carbon source, thereby releasing ammonia.
“probably since amino acids and small peptides are the main source of carbon, thereby releasing ammonia (Sánchez‐Clemente et al., 2018).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Sánchez‐Clemente et al., 2018 source
- Premisenone given
Changes in the medium pH affect bacterial growth in the presence of F− because HF enters the cells more efficiently than the anionic form.
“Changes in the medium pH, in turn, affect bacterial growth in the presence of F− because HF enters the cells more efficiently than the anionic form.”
Quote in the paperSubstantial claim - Interpretationcitation
The acidification of M9 minimal medium with glucose is due to gluconate and 2-ketogluconate formation.
“as a consequence of gluconate and 2‐ketogluconate formation (Nikel et al., 2015b), thus turning the pH indicator into a yellow hue.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Nikel et al., 2015b
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- It may state two separate claims as one. 35
- The quote only partly states this claim. 1
- Probably not a substantial claim. 42
It may state two separate claims as one.
Findingown dataThe wild-type strain tolerated the NaF challenge even at 15 mM, displaying a low msfGFP output.
“The wild‐type strain, in contrast, tolerated the NaF challenge even at 15 mM, displaying a low msfGFP output that indicates efficient F− secretion via CrcB.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe ΔcrcB cells could not grow at 15 mM NaF, and as a consequence, the biosensor output could not be reliably assessed in these conditions.
“The ΔcrcB cells could not grow at 15 mM NaF, and as a consequence, the biosensor output could not be reliably assessed in these conditions.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown datararA (PP_4002), the upstream gene in the operon where crcB is located, was also identified as important for F− tolerance, with three independent mini-Tn5 insertions more represented in the control than in the +NaF stress condition.
“Interestingly, the upstream gene in the operon where crcB is located, rarA (PP_4002), was also identified as important for F− tolerance. Three independent mini‐Tn5 insertions were found to be more represented in the control experiment than in the stress (+NaF) condition.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataWhen using rich LB medium, the MIC for F− of P. putida ΔcrcB shifted to a 10-fold higher concentration, still significantly lower than the NaF that would impair growth of strain KT2440.
“When using rich LB medium, the MIC shifted to a 10‐fold higher F− concentration—still significantly lower than the NaF that would impair growth of strain KT2440.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataA slight increase in the msfGFP readout was detected for the rest of the P. putida mutants, but the differences were not significant compared to control experiments.
“A slight increase in the msfGFP readout could also be detected for the rest of the P. putida mutants [Figure 3(C)]—yet the differences were not significant compared to control experiments.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe MIC of NaF for P. putida KT2440 lies in the range of 75 mM in M9 minimal medium and 125 mM in LB medium.
“Hence, the MIC of NaF lies in the range of 75 mM in M9 minimal medium and 125 mM in LB medium.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataF− treatment did not affect μ max of P. putida ΔlapA and P. putida ΔlapD, although the final OD600 was reduced by >30% compared to NaF-free control experiments.
“F− treatment did not affect μ max of P. putida ΔlapA and P. putida ΔlapD, although the final OD600 was reduced by >30% in comparison to that in NaF‐free control experiments.”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Findingown dataGrowth patterns of P. putida KT2440 in M9 minimal medium with glucose are similarly affected by KF as by NaF.
“However, a similar impact on the growth patterns of strain KT2440 was observed when the cells were incubated in the presence of different KF concentrations in M9 minimal medium with glucose (Figure S1 in the Supporting Information).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataAddition of 50 mM NaF to M9 minimal medium with glucose as the carbon source has a significant impact on P. putida cell physiology, but still permits bacterial growth to significant levels.
“the addition of 50 mM NaF to M9 minimal medium with glucose as the carbon source had a significant impact on the cell physiology, but it still permits bacterial growth to significant levels.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown datayijP (PP_2579) and PP_1779 were isolated in this experiment.
“Finally, two genes recognized as being involved in the synthesis and export of lipopolysaccharides, i.e. yijP (PP_2579) and PP_1779 (Cuthbertson et al., 2010; Mumm et al., 2016), were likewise isolated in this experiment.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataP. putida KT2440 cells can tolerate NaCl up to 0.5 M, and some growth is detected even in LB medium supplemented with 1 M NaCl.
“In this case, cells could tolerate the salt up to 0.5 M, and some growth could be detected even when LB medium was supplemented with 1 M NaCl [Figure 1(B)].”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe qualitative differences in intracellular pH before and after addition of NaF were similar in both strains, even though the F− concentrations that elicited such response in P. putida ΔcrcB were much lower than those used for the wild-type.
“The qualitative differences in intracellular pH before and after addition of NaF were similar in both strains, even though the F− concentrations that elicited such response in P. putida ΔcrcB were much lower than those used for the wild‐type.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataUpon addition of NaF, an almost immediate increase in the pH was observed in all cultures, with a clear difference between experimental conditions.
“After spiking the cultures with the salt, however, an almost immediate increase in the pH was observed in all cultures, with a clear difference observed between the experimental conditions.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataF− affected TCA cycle metabolites, with citrate depletion of threefold in P. putida KT2440 and fourfold in the ΔcrcB mutant.
“F− also affected the metabolites of the TCA cycle, with a threefold and fourfold change citrate depletion in P. putida KT2440 and the ΔcrcB mutant, respectively (Figure S4).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataL-tyrosine and L-methionine accumulated when F− was present in the medium, and this effect was especially evident when CrcB was removed.
“two amino acids accumulated when F− was present in the medium, i.e. l‐tyrosine and l‐methionine—and this effect was especially evident when the F− transporter CrcB was removed (Figures S6 and S7).”
Quote in the paperSubstantial claimProbably not a substantial claim.
PremisecitationF− negatively affects the performance of a number of key enzymes in central carbon metabolism.
“A conclusion shared by these studies is that F− negatively affects the performance of a number of key enzymes in central carbon metabolism.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
PremisecitationF− salts inhibit enolase and F-ATPase, and also exert an inhibitory impact on pyrophosphatases and catalases.
“A critical example of this sort is the inhibition of both enolase and F‐ATPase, although F− salts also exert an inhibitory impact on pyrophosphatases and catalases (Baykov et al., 2000; Li, 2003; Ma et al., 2014; Marquis et al., 2003; Qin et al., 2006; Samygina et al., 2007; Wackett, 2021b, 2021c).”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites Baykov et al., 2000 source
- cites Li, 2003
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Ma et al., 2014
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
Premisenone givenMineral fluoride is toxic in virtually all living organisms.
“the halide has been described to be toxic in virtually all living organisms”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper may give no reason why it holds.
Premisenone givenA systematic exploration of the effects of F− salts on the metabolism and physiology of environmental bacteria had remained underexplored.
“a systematic exploration of the effects of F− salts on the metabolism and physiology of environmental bacteria remained underexplored thus far”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationTwo unrelated classes of F−-detoxifying transporters have been described: the CLCF-type F−/H+ antiporter (a subclass of the CLC superfamily, including the EriCF transporter of Pseudomonas syringae) and the 'Fluc' family of small membrane proteins.
“Two unrelated classes of F−‐detoxifying transporters have been described thus far: (i) the CLCF‐type F−/H+ antiporter, a subclass of the widespread CLC (voltage‐gated chloride channel) superfamily of anion‐transport proteins (Stockbridge et al., 2012)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Stockbridge et al., 2012 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationCrcB is known to be involved in F− detoxification in several bacterial species.
“given the known involvement of CrcB in F− detoxification in several bacterial species (McIlwain et al., 2021)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites McIlwain et al., 2021 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationPseudomonas putida is tolerant towards different stress conditions, e.g. solvents and toxic aromatic compounds.
“Pseudomonas putida is well known for its tolerance towards different stress conditions, e.g. solvents (Bitzenhofer et al., 2021; Kieboom et al., 1998; Mosqueda et al., 1999) and toxic aromatic compounds (Calero et al., 2018; Fernández‐Cabezón et al., 2022)”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenIn the time-course high-resolution metabolomic analysis, two biological replicates were carried out with technical duplicates for each experiment.
“Two biological replicates were carried out as indicated above, with technical duplicates for each experiment.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataIn the intracellular pH homeostasis assay, measurements were carried out every 10 min on three independent biological replicates.
“Measurements were carried out every 10 min on three independent biological replicates, and the fluorescence ratios at 405 nm/485 nm were plotted and compared across conditions.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationDusB is a tRNA dihydrouridine synthase.
“DusB is identified as a tRNA dihydrouridine synthase (Belda et al., 2016).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Belda et al., 2016 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataThe size distribution of the Tn-Seq gDNA libraries was validated by qPCR and with the Agilent RNA 6000 Pico kit and Agilent 2100 Bioanalyzer.
“Library size distribution was validated by qPCR, using the KAPA qPCR quantification kit, the Agilent RNA 6000 Pico kit and the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA).”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe 'Fluc' family of small membrane proteins comprises the CrcB F− channel of Escherichia coli.
“a group of small membrane proteins generically known as the ‘Fluc’ family, which comprises the CrcB F− channel of Escherichia coli (Last et al., 2016; Stockbridge et al., 2013)”
Quote in the paperNot a substantial claim0 of 2 sources checkedIt may state two separate claims as one.
Interpretationown dataA recovery effect, potentially F− detoxification, occurs during exponential growth and enables P. putida KT2440 to cope with NaF stress and reach final biomass densities within the range of untreated cultures.
“These observations would suggest that a recovery effect (e.g. halide detoxification) occurs during exponential growth, enabling the cells to cope with the stressful conditions to reach a final biomass density within the range of untreated cultures.”
- Also stated in: § Assessing the impact of F− on the growth of wild‐type P. putida KT2440 · hedged
“Prompted by these results, which point to the existence of a potential F− detoxification mechanism, we investigated the genome‐wide genetic basis of the observed phenotypes as indicated in the next section.”
Quote in the paperSubstantial claim- Also stated in: § Assessing the impact of F− on the growth of wild‐type P. putida KT2440 · hedged
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationRarA has been previously annotated as a recombination factor and described as having a role in the tolerance of P. putida towards acid stress brought about by HCl treatment.
“RarA has been previously annotated as a recombination factor, and it has been described as having a role in the tolerance of P. putida towards the acid stress brought about by HCl treatment (Reva et al., 2006).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Reva et al., 2006 source
Probably not a substantial claim. It may state two separate claims as one.
PremisecitationHF hydrolyzes to H+ + F− upon being transported to the cytoplasm, decreasing intracellular pH and mediating acid stress.
“some reports indicate that HF hydrolyzes to H+ + F− upon the acid form is transported to the cytoplasm, thereby decreasing the intracellular pH and mediating acid stress due to the resulting HF ↔ H+ + F− equilibrium (Johnston & Strobel, 2020).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Johnston & Strobel, 2020 source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationLapA modifies the hydrophobicity of cell membranes and is necessary for both initial attachment of P. putida and mature biofilm formation.
“LapA modifies the hydrophobicity of cell membranes and is necessary for both initial attachment of P. putida and mature biofilm formation (El‐Kirat‐Chatel et al., 2014).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites El‐Kirat‐Chatel et al., 2014
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
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 givenAll experiments reported in the article were independently repeated at least twice, and mean values with standard deviations are presented.
“All the experiments reported in this article were independently repeated at least twice (as indicated in the corresponding figure or table legend), and the mean value of the corresponding parameter ± standard deviation is presented.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationLapA is a massive surface adhesion protein (the largest polypeptide in strain KT2440) that participates in assembling cells during biofilm formation.
“LapA is a massive surface adhesion protein (the largest polypeptide in strain KT2440) that participates in assembling cells during biofilm formation (Benedetti et al., 2016; López‐Sánchez et al., 2016).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Benedetti et al., 2016
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites López‐Sánchez et al., 2016 source
Probably not a substantial claim.
PremisecitationMineral F− is released during microbial biodegradation of organofluorines.
“Additionally, mineral F− is released during microbial biodegradation of organofluorines (Bygd et al., 2021; Neilson & Allard, 2002; Wackett, 2021a; Wackett, 2022; Wirth & Nikel, 2021)”
Quote in the paperSubstantial claim0 of 3 sources checkedProbably not a substantial claim.
Study design and conductnone givenThe correctness of gene deletions constructed in P. putida was verified by DNA sequencing.
“The correctness of gene deletions was verified by DNA sequencing.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe transposon-insertion library used consists of a pooled mixture of individual P. putida clones carrying two independent mini-Tn7 and mini-Tn5 transposons.
“This transposon‐insertion library consists of a pooled mixture of individual P. putida clones carrying two independent (mini‐Tn7 and mini‐Tn5) transposons (Calero et al., 2018).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Calero et al., 2018
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationprtR was also identified in a high-throughput screening towards enhanced fitness for aromatic substrate conversion by engineered P. putida strains.
“This gene was also identified in a high‐throughput screening towards enhanced fitness for aromatic substrates conversion by engineered P. putida strains (Eng et al., 2021).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Eng et al., 2021 source
Probably not a substantial claim. It may state two separate claims as one.
PremisecitationThe fluorescence biosensor borne by plasmid pS2513·PHP is a non-invasive, pH-sensitive engineered variant of GFP.
“Changes in the intracellular change of pH in the presence of NaF were studied by using a non‐invasive, pH‐sensitive fluorescence biosensor (an engineered variant of GFP) borne by plasmid pS2513·PHP (Arce‐Rodríguez et al., 2019).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Arce‐Rodríguez et al., 2019 source
Probably not a substantial claim.
PremisecitationThe mini-Tn5 transposon module interrupts genes at different positions of the open reading frame in a random fashion.
“The mini‐Tn5 transposon module interrupts genes at different positions of the open reading frame (ORF) in a random fashion (Martínez‐García et al., 2014)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Martínez‐García et al., 2014 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataIn the fluoride-responsive fluorescent biosensor assay, measurements were carried out every 10 min on three independent biological replicates.
“Measurements were carried out every 10 min on three independent biological replicates, and the normalized fluorescence values at 10 and 15 h after NaF addition were plotted and compared across conditions.”
Quote in the paperNot a substantial claimProbably not a substantial claim.
PremisereasoningPart of the non-lethal insertion mutants will display a phenotype under specific growth conditions for which the cognate function is relevant.
“therefore part of the (non‐lethal) insertion mutants will display a phenotype under specific growth conditions for which the cognate function is relevant.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductreasoningThe identification of the short 374 bp crcB gene served as an internal quality control feature indicating that the mini-Tn5 insertion library had enough quality to identify other genes related to F− toxicity.
“The identification of such a short length (374 bp) gene also served as an internal quality control feature—indicating that the insertion library had enough quality to be used to identify other genes related to F− toxicity.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationLapD is a transmembrane protein that prevents LapA hydrolysis by inhibiting the activity of the LapA-specific periplasmic protease LapG.
“LapD has been previously described as a transmembrane protein that prevents LapA hydrolysis (which would trigger biofilm dispersal) by inhibiting the activity of the LapA‐specific periplasmic protease LapG (Gjermansen et al., 2010; Yousef‐Coronado et al., 2011).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Gjermansen et al., 2010
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Yousef‐Coronado et al., 2011 source
Probably not a substantial claim. It may state two separate claims as one.
PremisecitationF− toxicity is dependent on the pH of the surrounding medium, because HF is probably the form that diffuses across cell membranes.
“F− toxicity has been described to be dependent on the pH of the surrounding medium, due to the fact that hydrogen fluoride (HF) is probably the form that diffuses across cell membranes.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe identification of general response functions in high-throughput screens is a common feature of functional-genomic procedures.
“The identification of general response functions in high‐throughput screens is a common feature of functional‐genomic procedures, and it has been described in several articles where P. putida mutants are selected by enhanced fitness under specific culture conditions (Eng et al., 2021; Thompson et al., 2019, 2020).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Eng et al., 2021 source
- cites Thompson et al., 2019, 2020
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
InterpretationreasoningLapD was isolated probably due to its functional relationship with LapA.
“LapD was also isolated in our screening, probably due to its functional relationship with LapA.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationPrtR is annotated as a transmembrane σ factor and has been previously described in P. fluorescens to control the formation of extracellular proteases and siderophores, as well as temperature-dependent lipases and proteases.
“PrtR is annotated as a transmembrane σ factor, and it has been previously described to display a role in P. fluorescens in controlling the formation of extracellular proteases and siderophores, as well as temperature‐dependent lipases and proteases (Burger et al., 2000; Llamas et al., 2014; Song et al., 2014).”
Quote in the paperNot a substantial claim0 of 3 sources checkedIt may state two separate claims as one.
Interpretationown dataPseudomonas putida undergoes a general, multi-level stress response when challenged with NaF that significantly differs from that caused by other saline stressors or halides, leading to a significant restructuring of central carbon metabolism towards counteracting the toxic effects of fluoride salts.
“our results indicate that P. putida undergoes a general, multi‐level stress response when challenged with NaF that significantly differs from that caused by other saline stressors.”
- Also stated in: § INTRODUCTION · hedged
“Our results indicate that the F− ion exerts a multi‐level effect on P. putida, unique and different when compared to that brought about by other halides, which leads to a significant restructuring of central carbon metabolism towards counteracting the toxic effects of fluoride salts.”
Quote in the paperSubstantial claim- Also stated in: § INTRODUCTION · hedged
Probably not a substantial claim.
Study design and conductcitationIn-frame, scar-less deletion mutants circumvent potential polar effects brought about by transposon insertion.
“we constructed in‐frame, scar‐less deletion mutants of P. putida KT2440—to circumvent any potential polar effect brought about by transposon insertion (Hutchison et al., 2019).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Hutchison et al., 2019 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenThree independent biological replicates were carried out for each condition in the Tn5 transposon mutant library screening by growth-based selection.
“Three independent biological replicates were carried out for each condition.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationLapA seems to be also involved in the tolerance of P. putida KT2440 towards urea.
“This protein seems to be also involved in the tolerance of P. putida KT2440 towards urea (Reva et al., 2006).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Reva et al., 2006 source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisereasoningThe EDEMP cycle regenerates hexose phosphates and favours catabolic NADPH formation.
“Thus, the architecture of the so‐called EDEMP cycle regenerates hexose phosphates and favours catabolic NADPH formation [Figure 5(A)].”
Quote in the paperNot a substantial claimProbably not a substantial claim.
Study design and conductcitationThe intracellular pH was measured using a genetically encoded, non-invasive pH biosensor (PHP) previously tailored for Pseudomonas species and other Gram-negative bacteria.
“we used a genetically encoded, non‐invasive pH biosensor that we have previously tailored for Pseudomonas species and other Gram‐negative bacteria (Arce‐Rodríguez et al., 2019).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Arce‐Rodríguez et al., 2019 source
It may state two separate claims as one.
InterpretationcitationThe depletion of NADPH and NADH could be explained by partial inhibition of redox-active dehydrogenases or by higher consumption of NAD(P)H to counter oxidative stress imposed by NaF.
“This observation could be explained either by the partial inhibition of redox‐active dehydrogenases, e.g. G6P dehydrogenase or the aforementioned isocitrate dehydrogenase, or because of higher rates of consumption of NAD(P)H to counterbalance the oxidative stress imposed by NaF (Johnston & Strobel, 2019).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Johnston & Strobel, 2019 source
It may state two separate claims as one.
PremisecitationThe deletion of fluoride transporter in bacterial cells leads to a higher concentration of F− in the cytoplasm than outside the cell (weak acid accumulation or ion trapping effect), which affects the ability of cells lacking the transporter to grow in the presence of NaF under acidic pH conditions.
“the deletion of fluoride transporter in bacterial cells leads to a higher concentration of F− in the cytoplasm than outside of the cell—the so‐called weak acid accumulation or ion trapping effect, which affects the ability of cells lacking the transporter to grow in the presence of NaF under acidic pH conditions (Ji et al., 2014).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Ji et al., 2014
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
It may state two separate claims as one.
Interpretationown dataHF is the active, toxic form of fluorine that affects bacterial cells probably by direct permeation through the cell membrane.
“highlighting that HF is the active, toxic form of fluorine that affects bacterial cells probably by direct permeation through the cell membrane.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe expression of metH was found to be upregulated in P. putida KT2440 under other stress conditions, such as toluene and o-xylene addition.
“Importantly, the expression of metH was also found to be upregulated in P. putida KT2440 cultivated under other stress conditions, e.g. addition of toluene and o‐xylene (Domínguez‐Cuevas et al., 2006).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Domínguez‐Cuevas et al., 2006 source
It may state two separate claims as one.
InterpretationreasoningA potential collapse of the H+ gradient due to increased intracellular F− could rob the cell of its capacity to couple energy metabolism to ATP synthesis, directly impacting growth patterns.
“It is worth noticing that the potential collapse of the H+ gradient due to an increase in the intracellular F− concentration could rob the cell of its capacity to couple energy metabolism to ATP synthesis—as indicated by the imbalance in the catabolic redox potential—which would directly impact growth patterns.”
Quote in the paperNot a substantial claimProbably not a substantial claim.
PremisecitationEfficient defluorination activities, either native or engineered, will call for high levels of tolerance to increased intracellular F− concentrations by the microbial host.
“As summarized by Wackett (2022) in a recent report, efficient defluorination activities (either native or engineered) will call for high levels of tolerance to increased intracellular F− concentrations by the microbial host.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Wackett (2022)
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
It may state two separate claims as one.
PremisecitationSeveral glycolytic enzymes require Mg2+ and Mn2+ for activity, and F− sequesters these cofactors and impairs enzyme activity in a rather unspecific fashion.
“Several of these enzymes require Mg2+ and Mn2+ for their activity, and it has been shown that F− sequesters the cofactors and impairs enzyme activity in a rather unspecific fashion (Adamek et al., 2005; Johnston & Strobel, 2020).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites Adamek et al., 2005
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Johnston & Strobel, 2020 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationL-methionine has been demonstrated to exert a protective role in rat tissues in the presence of F−, probably mediated by activation of anti-oxidative enzymes.
“Moreover, and relevant for the conditions analysed herein, l‐methionine has been demonstrated to exert a protective role in rat tissues in the presence of F−, probably mediated by an activation of anti‐oxidative enzymes (Błaszczyk et al., 2010).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Błaszczyk et al., 2010
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationMicrobial organisms naturally exposed to toxic F− levels have developed fluoride-sensing RNA devices, such as riboswitches, to control expression of genes encoding proteins that relieve the deleterious effects of fluoride.
“Furthermore, microbial organisms naturally exposed to toxic F− levels have developed fluoride‐sensing RNA devices (e.g. riboswitches, harnessed as biosensors in this study) to control the expression of genes encoding proteins that relieve the deleterious effects of this anion (Baker et al., 2012).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Baker et al., 2012 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationPseudomonas putida KT2440 runs a cyclic glycolytic metabolism (EDEMP cycle) composed of Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways.
“Pseudomonas putida KT2440 runs a cyclic glycolytic metabolism, composed by catabolic and anabolic activities of the Entner–Doudoroff pathway and the Embden–Meyerhof–Parnas (EMP) pathway (in a gluconeogenic fashion), together with the pentose phosphate (PP) pathway (Nikel et al., 2015b; Volke et al., 2021).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Nikel et al., 2015b
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Volke et al., 2021 source
It may state two separate claims as one.
Interpretationown dataAlthough F− affected the intracellular pH in both bacterial strains, the transporter mutant suffered acidification of the cytoplasm even at low salt concentrations.
“This observation suggests that, although F− affected the intracellular pH in both bacterial strains, the transporter mutant suffered acidification of the cytoplasm even at low salt concentrations [Figure 4(F)].”
Quote in the paperSubstantial claimIt may state two separate claims as one.
PremisecitationIf the HF ↔ H+ + F− equilibrium is displaced towards HF dissociation, this would partially collapse the H+ gradient and uncouple energy metabolism and probably other ion homeostatic processes.
“If the equilibrium is displaced towards HF dissociation, this phenomenon would partially collapse the H+ gradient and uncouple energy metabolism—and probably other ion homeostatic processes as well (Danchin & Nikel, 2019).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Danchin & Nikel, 2019 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationL-methionine biosynthesis is regulated as part of the general stress response in E. coli.
“l‐Methionine biosynthesis is also regulated as part of the general stress response in E. coli (Hondorp & Matthews, 2004).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Hondorp & Matthews, 2004 source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationPhenol red is a pH indicator that transitions from yellow to red over pH 6.8 to 8.2 and turns fuchsia above pH 8.2.
“a pH indicator that undergoes a gradual transition from yellow (λ emission = 443 nm) to red (λ excitation = 570 nm) over a pH range from 6.8 to 8.2 (Yamaguchi et al., 1997). Above pH = 8.2, phenol red turns a bright pink (fuchsia) colour.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Yamaguchi et al., 1997 source
It may state two separate claims as one.
PremisecitationMany actions of F− are connected to its weak-acid (or strong-base) character, making its mechanism of action comparable to that of organic weak acids, which de-energize the cell membrane by dissipating ΔpH.
“Since many of the actions of F− are connected to its weak‐acid (or strong‐base) character, the mechanism of action of this anion is comparable to that of organic weak acids, e.g. metabolic acids (acetate), food preservatives, non‐steroidal anti‐inflammatory agents and fatty acids—all of which act to de‐energize the cell membrane by dissipating ΔpH (Marquis et al., 2003).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Marquis et al., 2003 source
The paper relies on this without support.
NaF at 50 mM imposes stressful conditions on P. putida KT2440.
“A Tn5 mutant library derived from P. putida KT2440::mini‐Tn7(Gm), i.e. P. putida KT2440::mini‐Tn7(Gm)::mini‐Tn5(Km)::GFP (Calero et al., 2018), was used in growth‐based, Tn‐Seq selection experiments under NaF stressful conditions.”
“A fraction of this overnight LB medium culture was used to inoculate 50 ml of M9 minimal medium with adequate antibiotics to a starting OD600 of 0.1 after washing the cells with M9 minimal medium or M9 minimal medium supplemented with NaF at 50 mM.”
The paper relies on this without support.
CrcB functions as a fluoride efflux transporter in P. putida KT2440, so that loss of CrcB leads to intracellular F− accumulation.
“P. putida KT2440 and its ΔcrcB knock‐out derivative were used as negative and positive controls of intracellular F− accumulation, respectively, and the fluorescence readout was normalized to that of the wild‐type strain under each culture condition.”
The paper relies on this without support.
The main mechanism by which bacteria counteract fluoride toxicity is active export.
“the main mechanism to counteract toxicity seems to be active export.”
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?
The induction of the membrane export protein CrcB is highly protective against fluoride.
“The induction of the membrane export protein-designated CrcB is shown to be highly protective against fluoride (9, 12, 13).”
From Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion (2024) · cites this paper as [9]
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).”
From Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion (2024) · cites this paper as [9]
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.”
From Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion (2024) · cites this paper as [9]
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).”
From Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion (2024) · cites this paper as [9]
Earlier studies strongly indicate that fluoride anion toxicity is a major impediment to progress in both organofluorine synthesis and degradation.
“Earlier studies on biological organofluorine synthesis and degradation strongly indicate that fluoride anion toxicity is a major impediment to progress in both fields of study21,55,63,64.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Fluoride is highly toxic to the bacteria carrying out biosynthetic or biodegradation reactions.
“Though NaF is much safer for humans than HF and fluorine gas, fluoride is highly toxic to the bacteria carrying out biosynthetic or biodegradation reactions21,22.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Fluoride powerfully inhibits metabolism.
“Both efforts are undermined by the substantial toxicity of fluoride, an anion that powerfully inhibits metabolism.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Stress-response pathways, especially oxidative stress responses, are activated in a broad cross-section of bacterial species upon fluoride challenge.
“Stress-response pathways, especially oxidative stress responses, are activated in a broad cross-section of bacterial species upon fluoride challenge21,88,95,97,101.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Extreme fluoride tolerant strains have been identified in the context of organofluorine biosynthesis, biodegradation, and fluoride sequestration.
“Such strains have been identified in the context of organofluorine biosynthesis21, biodegradation63 and fluoride sequestration113.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Fluoride-stressed P. putida has elevated levels of methionine and tyrosine.
“For example, fluoride-stressed P. putida has elevated levels of methionine and tyrosine21.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
In P. putida, fluoride causes accumulation of metabolites in upper glycolysis and depletion of PEP and other downstream products, including TCA cycle intermediates.
“In P. putida, this is reflected by the accumulation of metabolites in upper glycolysis, and the depletion of PEP and other downstream products, including TCA cycle intermediates21.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Genes associated with adhesion, biofilm formation, cell membrane structure and integrity, and polysaccharide export are upregulated in response to fluoride by diverse bacteria, including P. putida, S. mutans, S. sobrinus, and A. ferrooxidans.
“Genes associated with adhesion, biofilm formation, cell membrane structure and integrity, and polysaccharide export are upregulated in response to fluoride by diverse bacteria, including P. putida, S. mutans, S. sobrinus, and A. ferrooxidans21,96,101.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Membrane permeation of the weak acid HF is the major route to fluoride accumulation in the cytosol, and fluoride exposure is correlated with cytoplasmic acidification.
“Since membrane permeation of the weak acid HF is the major route to fluoride accumulation in the cytosol92, fluoride exposure is correlated with cytoplasmic acidification21.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [21]
Other less-studied fluoride tolerance mechanisms include mutated enolase enzymes in some fluoride-tolerant S. mutans strains and rarA, lapA, and lapD in P. putida.
“Other, less-studied fluoride tolerance mechanisms include mutated enolase enzymes in some fluoride-tolerant S. mutans strains (13) and rarA, lapA, and lapD in Pseudomonas putida (14).”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [14]
The P. putida ΔcrcB strain shows growth restrictions at 0.25 mM NaF.
“the strain lacking the CrcB channel (ΔcrcB strain) shows growth restrictions already at 0.25 mM of NaF (14).”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [14]
Fluoride exerts a multi-level stress response on P. putida, affecting central carbon metabolism and energy metabolism.
“This study also indicated that fluoride exerts a multi-level stress response on P. putida, which affects central carbon metabolism and energy metabolism.”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [14]
P. putida CrcB-deficient mutant suffers acidification of the cytoplasm when exposed to fluoride.
“Previous studies (14) demonstrated that P. putida CrcB-deficient mutant suffers acidification of”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [14]
The fluoride retention in biofilms with ∆CLCF S. mutans mirrors intracellular fluoride accumulation measured in other microbes that lack fluoride exporters.
“This effect mirrors the intracellular fluoride accumulation that has been measured in other microbes that lack fluoride exporters (17, 20, 52).”
From Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models (2024) · cites this paper as [52]
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.
Tn-Seq harvest is described as both late exponential and growth end-point
MinorThe experimental procedure says cultures were harvested in late exponential phase. Figure 2B instead says they were harvested when OD600 no longer changed.
“When the cultures reached late exponential phase (at an OD600 of approximately 2), the cells were harvested”
“the biomass was harvested for gDNA extraction and processing at the end‐point (i.e. no further changes in OD600) for each culture condition”
The flhA deletion plasmid has a different locus number
MinorTable 1 assigns flhA the locus PP_4344 in the deletion strain but PP_434 in the plasmid intended to delete that gene.
“| P. putida ΔflhA | P. putida KT2440 derivative, in‐frame deletion of flhA (PP_4344) | This study |”
“| pGNW2·ΔflhA | Derivative of vector pGNW2 for deletion of flhA (PP_434) in P. putida KT2440 | This study |”
Figure 3B gives a different incubation time
MinorThe results text says the final cell density plotted in Figure 3B was measured after 20 h, but the figure caption says it was measured after 15 h.
“Specific growth rates [Figure 3(A)] and the cell density after 20 h of incubation [Figure 3(B)] were compared”
“(B) Final cell density (estimated as the optical density measured at 600 nm, OD600) of the WT strain and the individual P. putida mutants after 15 h of incubation”
pH-biosensor methods misidentify an excitation wavelength as emission
MinorThe methods describe 485 and 405 nm as excitation and emission wavelengths, respectively. The results instead identify both 405 and 485 nm as excitation peaks used for the ratio.
“ratiometric fluorescence determinations, using wavelengths of excitation and emission of 485 and 405 nm, respectively”
“the ratio between its two excitation peaks (λ excitation = 405 and 485 nm)”
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Metadata
- Authors
- Calero, P., Gurdo, N., & Nikel, P. I.
- Affiliations
- Novo Nordisk Foundation (DK)
- Corresponding author
- Nikel, Pablo I.
- Venue
- Environmental Microbiology
- Publisher
- Wiley
- Published
- 2022-07
- Volume
- 24
- Issue
- 11
- Pages
- 5082-5104
- ISSN
- 1462-2912 (ISSN-L)
- 1462-2920
- PMC
- PMC9796867
- OpenAlex
- W4283215829
- Cited by
- 56
- References
- 117
- Citations by year
- 2026: 15 · 2025: 15 · 2024: 17 · 2023: 8 · 2022: 1
- FWCI
- 5.03
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Environmental Microbiology (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, other-oa)
- Funders
- H2020 Environment (814418)
- Novo Nordisk Fonden (NNF18OC0034818, NNF20CC0035580, NNF21OC0067996)
- Natur og Univers, Det Frie Forskningsråd (8021‐00039B)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Physical Sciences › Environmental Science › Water Science and Technology › Fluoride Effects and Removal
- Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Chromium effects and bioremediation
- Keywords
- fluoride toxicity, sodium fluoride, Pseudomonas putida KT2440, bacterial stress response, central carbon metabolism, intracellular pH, extracellular pH, soil bacteria
- MeSH terms
- Fluorides, Minerals, Soil, Pseudomonas putida, Genomics
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
A language model drew the claims from the paper on 2026-11-10, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-11-10 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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