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Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter
Open accessAbstract
Fluoride (F − ), the anionic form of fluorine and the 13th most abundant element in Earth’s crust, is toxic to most organisms above relatively low threshold concentrations. Environmental bacteria often tolerate elevated fluoride levels, and one of the known resistance mechanisms involves CrcB-mediated efflux. In the environmental bacterium Pseudomonas putida , CrcB export is the primary defense against fluoride stress. However, spontaneous NaF-tolerant mutants emerge even without this channel, suggesting the existence of additional pathways. To uncover new mechanisms of fluoride tolerance, we performed a genome-wide screen of over 141,000 transposon mutants. We identified PP_3125, a Cro/cI-type transcriptional regulator, as essential for high fluoride tolerance in a Δ crcB background. Transcriptomic and proteomic analyses revealed PP_3125-regulated genes, including a gene encoding the benzoate transporter BenE-I, which contributes to fluoride tolerance. These findings demonstrate that bacterial transporters can acquire broader functions beyond their canonical roles and reveal previously unrecognized fluoride tolerance strategies in P. putida . Together, our results expand the understanding of microbial adaptation to toxic ions and provide new targets for engineering stress-resilient strains for environmental and industrial applications. IMPORTANCE: Our work identifies a new fluoride tolerance mechanism in Pseudomonas putida that functions independently of the well-characterized CrcB efflux system. We show that inactivation of transcriptional regulator PP_3125 activates a transporter with an unexpected moonlighting role in fluoride tolerance, highlighting how bacteria can repurpose existing functions to survive environmental stress. This discovery deepens our understanding of microbial stress responses and suggests strategies to engineer robust microbial strains capable of thriving in fluoride-contaminated settings. Such strains could be valuable for bioremediation, sustainable bioprocessing, and other biotechnological applications where fluoride exposure limits microbial performance.
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TL;DR
In a ΔcrcB background of *Pseudomonas putida* KT2440, inactivation of the Cro/cI-type regulator PP_3125 increases NaF tolerance, and this phenotype is linked to derepression of the benzoate transporter BenE-I. BenE-I overexpression improves fluoride tolerance without lowering intracellular fluoride, suggesting an indirect, moonlighting-like role.
Key findings
- A genome-wide screen of ~141,000 mini-Tn5 mutants in ΔcrcB identified 48 NaF-tolerant mutants; 24 were mapped, and 16 had insertions in PP_3125. Spontaneous NaF-tolerant ΔcrcB mutants arose at a median of 5 CFU mL−1 on 5 mM NaF and all carried large deletions (18 kb–340 kb; 18–345 genes) encompassing PP_3125.
- ΔcrcBΔ3125 grew without restriction at 0.5 mM NaF while ΔcrcB was restricted; in liquid medium at 0.5 mM NaF, lag phase was 1.6 h vs. 10.7 h (adjusted P = 0.0293). At 2.5 mM NaF, ΔcrcB lag phase was 18.9 h, while spontaneous mutants and ΔcrcBΔ3125 were sevenfold shorter, though not significantly different.
- Proteomics and transcriptomics showed PP_3125 deletion derepressed five adjacent genes, PP_2033–PP_2037; proteomics detected overproduction of PP_2036, PP_2037, and BenE-I (PP_2035). BenE-I overexpression in ΔcrcB or ΔcrcBΔ5 enabled growth at 20 mM NaF, whereas ΔcrcBΔ5 lacking all five genes could not grow at 2.5 mM NaF.
- The fluoride-responsive riboswitch biosensor showed that benE-I deletion elevated intracellular fluoride, but benE-I overexpression did not lower it; PI-permeable cells increased with NaF, suggesting BenE-I contributes indirectly rather than as a fluoride exporter.
Methods
- Spontaneous NaF-tolerant mutant selection, whole-genome sequencing of four mutants, mini-Tn5 transposon mutagenesis (~141,000 mutants) in ΔcrcB, arbitrary PCR mapping, and targeted deletions/complementation in *P. putida* KT2440.
- Growth assays in liquid medium (OD600 every 10 min for 24 h; 0–2.5 mM NaF) and on solid LB plates (0–30 mM NaF, 20 h at 30°C); proteomics by label-free LC-MS/MS with MaxQuant/Perseus and RNA-seq analyzed with DESeq2 under NaF conditions matched for a twofold lag-phase increase.
- Intracellular fluoride measured with an FRS-msfGFP biosensor and flow cytometry with SYTO9/PI; statistics used Kruskal-Wallis with Dunn’s post-hoc test and Benjamini-Hochberg FDR 0.05.
Limitations
- Stated: proteomic and transcriptomic snapshots were limited to early growth phases, and PP_3125 may regulate additional gene sets later; the precise function of BenE-I, the broader PP_3125 regulon, and the molecular triggers of stress-induced deletions remain unresolved.
- Stated/evident: large spontaneous deletions (18–345 genes) made it difficult to pinpoint causal genes, prompting the transposon screen; BenE-I’s mechanism appears indirect because overexpression did not reduce intracellular fluoride.
- Evident: some key comparisons were not statistically significant (e.g., spontaneous mutants/ΔcrcBΔ3125 vs. ΔcrcB at 2.5 mM NaF; IPTG effect on lag phase), and leaky Ptac expression plus IPTG effects complicate interpretation of overexpression experiments.
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- Findingown data
In P. putida, deletion of benE-I elevates the intracellular fluoride concentration.
“As presented in Fig. 8a, the deletion of benE-I elevates the intracellular fluoride concentration, but the overexpression of this gene does not lower it.”
Quote in the paperSubstantial claim - Findingown data
Deletion of PP_2037, aldolase, lengthened the lag phase.
“Although deletion of PP_2037, aldolase, did lengthen the lag phase, the overexpression of the gene did not have any effect on the growth.”
Quote in the paperNot a substantial claim - Findingown data
Inactivation of PP_3125 derepresses five genes, including the gene encoding the benzoate transporter BenE-I.
“We further demonstrated that inactivation of PP_3125 derepresses five genes, including the gene encoding the benzoate transporter BenE-I”
- Also stated in: § Abstract · plain
“Transcriptomic and proteomic analyses revealed PP_3125-regulated genes, including a gene encoding the benzoate transporter BenE-I”
Quote in the paperSubstantial claim - Also stated in: § Abstract · plain
- Findingown data
Among 24 transposon mutants with identified mini-Tn5 locations, 16 contained insertions in PP_3125.
“from those 24 mutants analyzed, 16 contained the transposon insertions into the PP_3125 gene”
Quote in the paperSubstantial claim - Findingown data
All four isolated spontaneous NaF-tolerant ΔcrcB mutants (crcB1, crcB4, crcB12, crcB18) carried large genomic deletions ranging from 18 kb to 340 kb.
“Comparative genomic analysis revealed that all four Na-tolerant ΔcrcB mutants carried large deletions in their genomes, ranging from 18 kb to 340 kb (corresponding to 18–345 genes), as illustrated in Fig. S1.”
Quote in the paperSubstantial claim
- Findingreasoning
The ΔcrcBΔ3125-tac3125 strain showed similar growth to the ΔcrcB strain in NaF-containing media even without IPTG induction.
“Due to leaky expression from the Ptac promoter, the ΔcrcBΔ3125-tac3125 strain showed similar growth to the ΔcrcB strain in NaF-containing media, even without IPTG induction (Fig. 2 and 3).”
Quote in the paperNot a substantial claim - Findingown data
When NaF was added, the growth patterns of spontaneous NaF-tolerant ΔcrcB mutants closely resembled the growth of the ΔcrcBΔ3125 strain.
“However, when NaF was added, their growth patterns closely resembled the growth of the ΔcrcBΔ3125 strain.”
Quote in the paperSubstantial claim - Findingown data
The addition of IPTG into the growth medium further amplified the impact of the five-gene deletion, leading to more pronounced growth inhibition by NaF.
“The addition of IPTG into the growth medium of bacteria further amplified the impact of the five-gene deletion, leading to even more pronounced growth inhibition by NaF.”
Quote in the paperNot a substantial claim - Findingown data
When PP_3125 proficient strains were compared to the strain lacking PP_3125 in the presence of fluoride, only five genes were confirmed to be differentially expressed.
“When PP_3125 proficient strains were compared to the strain lacking the regulator PP_3125 in the presence of fluoride in the growth medium, we were able to confirm only the differential expression of five genes.”
Quote in the paperSubstantial claim - Findingown data
PP_3125 was always included in the DNA region deleted in the spontaneous NaF-tolerant ΔcrcB mutants crcB1, crcB4, crcB12, and crcB18.
“It is worth noting that the PP_3125 gene was always included in the DNA region that was deleted in the spontaneous NaF-tolerant ΔcrcB mutants crcB1, crcB4, crcB12, and crcB18, as shown in Fig. S1.”
Quote in the paperNot a substantial claim - Findingown data
BenE-I contributes to fluoride tolerance in P. putida.
“a gene encoding the benzoate transporter BenE-I, which contributes to fluoride tolerance”
- Also stated in: § INTRODUCTION · plain
“the gene encoding the benzoate transporter BenE-I, which contributed to elevated fluoride tolerance”
Quote in the paperSubstantial claim - Also stated in: § INTRODUCTION · plain
- Findingown data
Introducing the BenE-I transporter significantly improved the fluoride tolerance of the ΔcrcBΔ5 strain, allowing growth at 2.5 mM NaF.
“In contrast, introducing the BenE-I transporter significantly improved the fluoride tolerance of the ΔcrcBΔ5 strain, allowing growth at 2.5 mM NaF.”
Quote in the paperSubstantial claim - Findingown data
Overexpression of benE-I does not lower the intracellular fluoride concentration.
“As presented in Fig. 8a, the deletion of benE-I elevates the intracellular fluoride concentration, but the overexpression of this gene does not lower it.”
Quote in the paperSubstantial claim - Findingown data
The lag phase of the BenE-I complementation strain was shorter than that of the deletion strain.
“An increase in fluoride tolerance in ΔcrcB strains occurred through the overexpression of this gene, both in the solid medium (Fig. 7) and in the liquid medium (Fig. 6) when we observed that the lag phase of the BenE-I complementation strain was shorter than that of the deletion strain (Fig. 6).”
Quote in the paperSubstantial claim - Findingown data
In the presence of NaF, only three proteins exhibited significantly differential expression when comparing the PP_3125 deletion strain to the WT strain.
“As shown in Fig. 5a, in the presence of NaF, only three proteins exhibited significantly differential expression when comparing the PP_3125 deletion strain to the WT strain in the presence of fluoride.”
Quote in the paperSubstantial claim - Findingown data
The fluoride tolerance levels of spontaneous mutants and the ΔcrcBΔ3125 deletion strain were comparable.
“Naturally occurring NaF-tolerant ΔcrcB mutants appeared to gain their tolerance through deletion of a genomic region containing PP_3125, as this gene was absent in all sequenced spontaneous mutants, and the fluoride tolerance levels of spontaneous mutants and the ΔcrcBΔ3125 deletion strain were comparable.”
Quote in the paperSubstantial claim - Findingreasoning
We did not observe a direct effect of aldolase on fluoride tolerance.
“Therefore, we did not observe a direct effect of aldolase on fluoride tolerance.”
Quote in the paperSubstantial claim - Findingown data
Deletion of PP_3125 in the ΔcrcB background led to a marked increase in fluoride tolerance.
“Deletion of PP_3125 in the ΔcrcB background led to a marked increase in fluoride tolerance (Fig. 3), mirroring the phenotype observed in spontaneous fluoride-tolerant mutants with large genomic deletions encompassing this gene (Fig. 4).”
Quote in the paperSubstantial claim - Findingown data
The ΔcrcBΔ5 strain lacking all five genes (PP_2033-PP_2037) could not grow in the presence of 2.5 mM NaF even after 24 h.
“As shown in Fig. 6, the ΔcrcBΔ5 strain lacking all five genes identified in the transcriptomics study (PP_2033-PP_2037) could not grow in the presence of 2.5 mM NaF, even after 24 h.”
Quote in the paperSubstantial claim - Findingown data
Five genes overexpressed in the absence of PP_3125 are located in the same genomic region, adjacent to each other.
“Five genes overexpressed in the absence of PP_3125 are located in the same genomic region, adjacent to each other.”
Quote in the paperSubstantial claim - Findingown data
Growing all strains under different NaF concentrations resulted in similar growth effects, specifically a doubling of the lag phase compared to growth without NaF.
“All strains were grown under different NaF concentrations, resulting in similar growth effects, specifically a doubling of the lag phase compared to the growth without NaF.”
Quote in the paperNot a substantial claim - Findingown data
Overexpression of PP_2037 did not have any effect on growth.
“Although deletion of PP_2037, aldolase, did lengthen the lag phase, the overexpression of the gene did not have any effect on the growth.”
Quote in the paperNot a substantial claim - Findingown data
In the absence of NaF, P. putida WT, ΔcrcB, Δ3125, ΔcrcBΔ3125, and ΔcrcBΔ3125-tac3125 strains exhibited similar growth patterns.
“in the absence of NaF, all strains exhibited similar growth patterns”
Quote in the paperSubstantial claim - Findingown data
BenE-I enhances fluoride tolerance when overexpressed.
“BenE-I, a benzoate transporter regulated by PP_3125, enhances fluoride tolerance when overexpressed.”
- Also stated in: § DISCUSSION · plain
“Among these genes, PP_2035, encoding for BenE-I transporter, a member of the benzoate/H+ symporter family, emerged as a key factor in promoting fluoride tolerance when overexpressed.”
Quote in the paperSubstantial claim - Also stated in: § DISCUSSION · plain
- Findingown data
The transposon insertions were located throughout the genome with no significant positional bias.
“Mutations were located all over the genome, as shown in Fig. 1a, which indicates that there was no significant positional bias in the assay.”
Quote in the paperNot a substantial claim - Findingown data
Introducing the PP_2034 transporter into the ΔcrcBΔ5 strain resulted in minimal growth toward the end of the experiment.
“Introducing the PP_2034 transporter into the ΔcrcBΔ5 strain resulted in minimal growth toward the end of the experiment; however, this growth was minimal.”
Quote in the paperNot a substantial claim - Findingown data
All compared strains grew equally well on LB media without NaF.
“The results shown in Fig. 7 demonstrated that all strains grew equally well on LB media without NaF.”
Quote in the paperNot a substantial claim - Findingown data
Spontaneous NaF-tolerant ΔcrcB mutants exhibited growth comparable to the wild-type strain in the absence of NaF.
“As shown in Fig. 4, the spontaneous mutants exhibited growth comparable to the wild-type (WT) strain in the absence of NaF.”
Quote in the paperNot a substantial claim - Findingown data
Inactivation of PP_3125, encoding a Cro/CI-type transcriptional regulator, increases fluoride tolerance of the P. putida ΔcrcB strain.
“Our results indicated that inactivation of PP_3125 encoding Cro/CI type transcriptional regulator increases the fluoride tolerance of the ΔcrcB strain.”
- Also stated in: § Abstract · plain
“We identified PP_3125, a Cro/cI-type transcriptional regulator, as essential for high fluoride tolerance in a ΔcrcB background.”
Quote in the paperSubstantial claim - Also stated in: § Abstract · plain
- Findingown data
The overproduced proteins in the PP_3125 deletion strain compared to WT in the presence of NaF were PP_2036, PP_2037, and BenE-I.
“The overproduced proteins were PP_2036 (4-hydroxy-tetrahydrodipicolinate synthase), PP_2037 (aldolase), and BenE-I (PP_2035, benzoate transport protein).”
Quote in the paperSubstantial claim - Findingreasoning
BenE-I is regulated by PP_3125.
“BenE-I, a benzoate transporter regulated by PP_3125, enhances fluoride tolerance when overexpressed.”
Quote in the paperSubstantial claim - Findingown data
The effect on membrane permeability is only visible in the presence of NaF.
“The effect on membrane permeability is only visible in the presence of NaF.”
Quote in the paperNot a substantial claim - Findingown data
In liquid media at 0.5 mM NaF, the ΔcrcB strain had a longer lag phase than the ΔcrcBΔ3125 strain (10.7 h vs. 1.6 h, adjusted P-value = 0.0293).
“in liquid media, at 0.5 mM NaF, the ΔcrcB strain showed a longer lag phase than ΔcrcBΔ3125 (10.7 h vs. 1.6 h, adjusted P-value = 0.0293).”
Quote in the paperSubstantial claim - Findingown data
Wild-type P. putida KT2440 cells can tolerate fluoride concentrations higher than 30 mM on LB agar plates.
“On LB agar plates, wild-type KT2440 cells can tolerate fluoride concentrations even higher than 30 mM (Fig. 7)”
Quote in the paperNot a substantial claim - Findingown data
Using transposon mutagenesis, PP_3125 was identified as the most frequently disrupted gene in fluoride-tolerant ΔcrcB mutants.
“Using transposon mutagenesis, we identified PP_3125 as the most frequently disrupted gene in fluoride-tolerant ΔcrcB mutants, indicating its central role as a negative regulator of the fluoride stress response.”
Quote in the paperSubstantial claim - Findingown data
When the BenE-I transporter-encoding gene under Ptac promoter control was introduced into the ΔcrcB strain, a shorter lag phase was observed compared to the ΔcrcB strain without the BenE-I transporter.
“Additionally, when the BenE-I transporter-encoding gene under the Ptac promoter control was introduced into the ΔcrcB strain, a shorter lag phase was observed, compared to the ΔcrcB strain without the BenE-I transporter.”
Quote in the paperNot a substantial claim - Findingown data
PP_3125 negatively regulates PP_2033 and an adjacent operon of four genes, PP_2034-PP_2037, including transporters and metabolic enzymes.
“Proteomic and transcriptomic analyses further revealed that PP_3125 negatively regulates PP_2033 and an adjacent operon of four genes (PP_2034–PP_2037), including transporters and metabolic enzymes.”
Quote in the paperSubstantial claim - Findingown data
Among the five identified genes, PP_2036, PP_2037, and benE-I were also overexpressed in the proteomic analysis.
“Importantly, among the five identified genes, PP_2036, PP_2037, and benE-I (PP_2035) were also overexpressed in the proteomic analysis.”
Quote in the paperNot a substantial claim - Findingown data
The genes for PP_2036, PP_2037, and BenE-I were also overexpressed in the ΔcrcBΔ3125 strain compared to both the WT and the ΔcrcB strains in the presence of NaF.
“As these same protein genes were also overexpressed in the ΔcrcBΔ3125 strain compared to both the WT (Fig. S2) and the ΔcrcB strains in the presence of NaF (Fig. S3)”
Quote in the paperSubstantial claim - Findingown data
Transcriptome analysis revealed distinct effects of fluoride, crcB deletion, and PP_3125 deletion.
“Transcriptome analysis revealed the distinct effects of fluoride, crcB deletion, and PP_3125 deletion (Fig. 5b).”
Quote in the paperSubstantial claim - Findingown data
Overexpression of benE-I increased fluoride tolerance in ΔcrcB strains in both solid and liquid media.
“An increase in fluoride tolerance in ΔcrcB strains occurred through the overexpression of this gene, both in the solid medium (Fig. 7) and in the liquid medium (Fig. 6) when we observed that the lag phase of the BenE-I complementation strain was shorter than that of the deletion strain (Fig. 6).”
Quote in the paperSubstantial claim - Findingown data
When the BenE-I transporter was artificially overproduced under the Ptac promoter in the ΔcrcB strain or the ΔcrcBΔ5 strain, NaF tolerance increased further, allowing growth at 20 mM NaF.
“Moreover, we observed that when the BenE-I transporter was artificially overproduced under the control of the Ptac promoter in the ΔcrcB strain or in the ΔcrcBΔ5 strain, the NaF tolerance increased further, allowing bacterial growth even at 20 mM NaF (Fig. 7).”
Quote in the paperSubstantial claim - Findingown data
Out of approximately 141,000 transposon mutants of the ΔcrcB strain, 48 had higher fluoride tolerance.
“Out of 141,000 mutants, 48 had developed higher fluoride tolerance.”
Quote in the paperSubstantial claim - Findingown data
The median number of spontaneous NaF-tolerant mutants of the ΔcrcB strain emerging at 5 mM NaF was 5 CFUmL−1.
“We observed that the median number of NaF-tolerant mutants that emerged at 5 mM NaF was 5 CFUmL−1.”
Quote in the paperNot a substantial claim - Findingown data
Spontaneous NaF-tolerant mutants arise in a ΔcrcB derivative of P. putida.
“spontaneous NaF-tolerant mutants emerge even without this channel”
- Also stated in: § INTRODUCTION · plain
“the evolution of a ΔcrcB derivative of P. putida, lacking the fluoride efflux pump, can give rise to spontaneous NaF-tolerant mutants”
- Also stated in: § Genomic changes in spontaneous NaF+ mutants in the ΔcrcB strain · plain
“we observed the appearance of single colonies when the ΔcrcB strain was incubated on LB plates containing 5 mM NaF.”
Quote in the paperSubstantial claim - Also stated in: § INTRODUCTION · plain
- Findingown data
The PP_3125 gene was absent in all sequenced spontaneous NaF-tolerant mutants.
“Naturally occurring NaF-tolerant ΔcrcB mutants appeared to gain their tolerance through deletion of a genomic region containing PP_3125, as this gene was absent in all sequenced spontaneous mutants, and the fluoride tolerance levels of spontaneous mutants and the ΔcrcBΔ3125 deletion strain were comparable.”
Quote in the paperSubstantial claim - Findingown data
These deletions varied in size, from 18 to 345 genes.
“These deletions varied in size, from 18 to 345 genes, and included ECF family sigma factors, DNA polymerases (e.g., dnaEB, imuB), transporters, regulators, hydrolases, and multiple catabolic pathways such as benzoate and phenylacetate degradation operons (Table S1).”
Quote in the paperNot a substantial claim - Findingown data
Spontaneous fluoride-tolerant mutants showed large genomic deletions encompassing PP_3125 and up to 344 other genes.
“Interestingly, spontaneous fluoride-tolerant mutants showed large genomic deletions encompassing PP_3125 and up to 344 other genes.”
Quote in the paperSubstantial claim - Findingown data
The mutants exhibited no significant growth defects under standard laboratory conditions.
“Despite these extensive deletions, the mutants exhibited no significant growth defects under standard laboratory conditions and showed improved growth under fluoride stress (Fig. 4), suggesting that the deleted regions may include nonessential genes under those conditions or that the deletions conferred adaptive benefits.”
Quote in the paperSubstantial claim - Findingown data
These deletions included ECF family sigma factors, DNA polymerases, transporters, regulators, hydrolases, and multiple catabolic pathways such as benzoate and phenylacetate degradation operons.
“These deletions varied in size, from 18 to 345 genes, and included ECF family sigma factors, DNA polymerases (e.g., dnaEB, imuB), transporters, regulators, hydrolases, and multiple catabolic pathways such as benzoate and phenylacetate degradation operons (Table S1).”
Quote in the paperNot a substantial claim - Findingown data
The population of propidium iodide-permeable cells was remarkably increased in the presence of NaF.
“Our results also revealed that elevated fluoride concentration inside cells was associated with compromised membranes, as the population of cells with propidium iodide-permeable cells was remarkably increased in the presence of NaF (Fig. 8).”
Quote in the paperSubstantial claim - Findingown data
Elevated fluoride concentration inside cells was associated with compromised membranes.
“Our results also revealed that elevated fluoride concentration inside cells was associated with compromised membranes, as the population of cells with propidium iodide-permeable cells was remarkably increased in the presence of NaF (Fig. 8).”
Quote in the paperSubstantial claim - Findingown data
The mutants showed improved growth under fluoride stress.
“Despite these extensive deletions, the mutants exhibited no significant growth defects under standard laboratory conditions and showed improved growth under fluoride stress (Fig. 4), suggesting that the deleted regions may include nonessential genes under those conditions or that the deletions conferred adaptive benefits.”
Quote in the paperSubstantial claim - Findingown data
Complementation of the double mutant with the Ptac-benE-I expression cassette did not reduce the fluorescence signal.
“However, the complementation of this double mutant with the Ptac-benE-I expression cassette did not reduce the fluorescence signal.”
Quote in the paperSubstantial claim - Findingown data
The experiments performed by us did not support the possibility that BenE-I is involved in fluoride transport.
“The experiments performed by us did not support this possibility.”
Quote in the paperSubstantial claim - Findingown data
The fluorescence signal was increased in the CrcB-deficient strain when BenE-I was also deleted.
“We used the GFP-based fluoride biosensor to monitor intracellular fluoride concentration in P. putida cells and observed that the fluorescence signal was increased in the CrcB-deficient strain when BenE-I was also deleted.”
Quote in the paperSubstantial claim - Interpretationown data
The findings reveal previously unrecognized fluoride tolerance strategies in P. putida.
“and reveal previously unrecognized fluoride tolerance strategies in P. putida.”
Quote in the paperSubstantial claim - Interpretationreasoning
The five genes overexpressed in the absence of PP_3125 could be co-regulated.
“This indicated that these genes could be co-regulated.”
Quote in the paperNot a substantial claim - Interpretationown data
PP_3125 could be the negative regulator of BenE-I.
“As indicated by the growth in liquid media (Fig. 6), PP_3125 could be the negative regulator of BenE-I.”
Quote in the paperSubstantial claim- Rests on an assumption: PP_3125 functions as a transcription regulator.
- Interpretationnone given
Due to leaky expression from the Ptac promoter, the effect of the transporters was observed even in the absence of IPTG.
“Due to leaky expression from the Ptac promoter, the effect of the transporters was observed even in the absence of IPTG.”
Quote in the paperNot a substantial claim - Interpretationown data
Inactivation of PP_3125 activates a transporter with an unexpected moonlighting role in fluoride tolerance.
“We show that inactivation of transcriptional regulator PP_3125 activates a transporter with an unexpected moonlighting role in fluoride tolerance”
Quote in the paperSubstantial claim - Interpretationown data
PP_3125 functions as a negative regulator of a gene influencing fluoride tolerance in P. putida.
“Overall, these results demonstrate that PP_3125 functions as a negative regulator of a gene influencing fluoride tolerance in P. putida.”
Quote in the paperSubstantial claim - Interpretationown data
Other differentially expressed genes resulted from fluoride stress, as they were also differentially expressed in the crcB deletion strain.
“Other differently expressed genes resulted from the fluoride stress, as these genes were also differently expressed in the crcB deletion strain (Fig. S1).”
Quote in the paperSubstantial claim - Interpretationown data
Naturally occurring NaF-tolerant ΔcrcB mutants gained their tolerance through deletion of a genomic region containing PP_3125.
“Naturally occurring NaF-tolerant ΔcrcB mutants appeared to gain their tolerance through deletion of a genomic region containing PP_3125, as this gene was absent in all sequenced spontaneous mutants, and the fluoride tolerance levels of spontaneous mutants and the ΔcrcBΔ3125 deletion strain were comparable.”
Quote in the paperSubstantial claim - Premisecitation
PP_2035 encodes BenE-I, a member of the benzoate/H+ symporter family.
“Among these genes, PP_2035, encoding for BenE-I transporter, a member of the benzoate/H+ symporter family, emerged as a key factor in promoting fluoride tolerance when overexpressed.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [28] Choudhary A, Purohit H, Phale PS. 2017. Benzoate transport in Pseudomonas putida CSV86. FEMS Microbiol Lett 364. doi: 10.1093/femsle/fnx118 28591829 source
Checked before — its earlier verdict is held back until a new check runs.
- cites [29] Nishikawa Y, Yasumi Y, Noguchi S, Sakamoto H, Nikawa J. 2008. Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae. Biosci Biotechnol Biochem 72: 2034– 2038. doi: 10.1271/bbb.80156 18685209 source
- Interpretationown data
This work identifies a new fluoride tolerance mechanism in P. putida that functions independently of the CrcB efflux system.
“Our work identifies a new fluoride tolerance mechanism in Pseudomonas putida that functions independently of the well-characterized CrcB efflux system.”
Quote in the paperSubstantial claim - Interpretationown data
The findings demonstrate that bacterial transporters can acquire broader functions beyond their canonical roles.
“These findings demonstrate that bacterial transporters can acquire broader functions beyond their canonical roles”
Quote in the paperSubstantial claim - Premisecitation
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).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [14] 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
A check is scheduled.
- Premisecitation
The lag phase is the growth parameter most strongly affected by fluoride stress.
“The lag phase is the growth parameter most strongly affected by the fluoride stress (27).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27] Dodge AG, Thoma CJ, O’Connor MR, Wackett LP. 2024. Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion. mBio 15: e0278523. doi: 10.1128/mbio.02785-23 38063407 PMC10790756 source
A check is scheduled.
- Interpretationown data
NaF may regulate PP_3125 expression.
“this suggests that NaF may regulate PP_3125 expression.”
Quote in the paperNot a substantial claim - Premisenone given
In Pseudomonas putida, CrcB-mediated export is the primary mechanism of fluoride tolerance.
“In the environmental bacterium Pseudomonas putida, CrcB export is the primary defense against fluoride stress.”
- Also stated in: § INTRODUCTION · boosted
“As of yet, CrcB-mediated export is known to be the primary mechanism of fluoride tolerance in P. putida.”
Quote in the paperSubstantial claim - Also stated in: § INTRODUCTION · boosted
- Interpretationreasoning
Deletion of the aldolase gene could negatively affect the expression of benE-I because it belongs to the same operon and is located downstream.
“As the aldolase gene belongs to the same operon as benE-I, its deletion could negatively affect the expression of benE-I, which is located downstream of the aldolase gene.”
Quote in the paperSubstantial claim - Interpretationown data
The similar growth of the ΔcrcBΔ3125-tac3125 strain to the ΔcrcB strain in NaF-containing media without IPTG is due to leaky expression from the Ptac promoter.
“Due to leaky expression from the Ptac promoter, the ΔcrcBΔ3125-tac3125 strain showed similar growth to the ΔcrcB strain in NaF-containing media, even without IPTG induction (Fig. 2 and 3).”
Quote in the paperSubstantial claim - Interpretationown data
This work uncovers an additional, CrcB-independent pathway involving PP_3125.
“This work uncovers an additional, CrcB-independent pathway involving the transcriptional regulator PP_3125.”
Quote in the paperSubstantial claim - Interpretationown data
The emergence of spontaneous NaF-tolerant mutants in the ΔcrcB strain suggests the existence of secondary or compensatory fluoride tolerance mechanisms.
“suggesting the existence of additional pathways”
- Also stated in: § INTRODUCTION · hedged
“suggesting the existence of secondary or compensatory tolerance mechanisms”
Quote in the paperSubstantial claim - Also stated in: § INTRODUCTION · hedged
- Interpretationown data
BenE-I has a role in fluoride tolerance.
“The benzoate transporter BenE-I has a role in fluoride tolerance, as its overexpression enables strains with lower fluoride tolerance to tolerate higher fluoride concentrations but does not decrease intracellular fluoride concentrations.”
Quote in the paperSubstantial claim - Interpretationown data
The NaF tolerance observed in the spontaneous mutants is most probably due to the absence of PP_3125.
“However, the results still indicate that the NaF tolerance observed in the spontaneous mutants is most probably due to the absence of PP_3125.”
Quote in the paperSubstantial claim - Interpretationown data
PP_3125 functions as a central negative regulator of fluoride tolerance, suppressing the expression of transporter genes like benE-I that mitigate fluoride toxicity.
“Taken together, our data support a model in which PP_3125 functions as a central negative regulator of fluoride tolerance, suppressing the expression of transporter genes like benE-I that mitigate fluoride toxicity.”
Quote in the paperSubstantial claim - Premisecitation
Many of the actions of fluoride are related to those of organic weak acids, including metabolic acids, fatty acids, and benzoic acid, which de-energize the cell membrane.
“Importantly, many of the actions of fluoride are related to those of organic weak acids, including metabolic acids, fatty acids, and benzoic acid—all of which de-energize the cell membrane (26).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26] 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
- Study design and conductown data
The intracellular F− concentration in the strains carrying pS441·FRSv1 correlated with msfGFP fluorescence.
“The intracellular F− concentration in these strains correlated with msfGFP fluorescence.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [7] 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
- Interpretationreasoning
BenE-I could influence both benzoate and fluoride stress responses.
“Given that BenE-I is characterized as one of the benzoate transporters in P. putida, this protein could somehow influence both benzoate and fluoride stress responses.”
Quote in the paperSubstantial claim - Interpretationreasoning
Deletion of PP_3125, whether through spontaneous genomic rearrangement or targeted mutagenesis, leads to upregulation of protective pathways.
“Deletion of PP_3125, whether through spontaneous genomic rearrangement or targeted mutagenesis, leads to upregulation of these protective pathways.”
Quote in the paperSubstantial claim - Interpretationown data
BenE-I contributes to fluoride tolerance through an indirect mechanism.
“Thus, our results rather imply that BenE-I contributes to fluoride tolerance through an indirect mechanism.”
Quote in the paperSubstantial claim - Interpretationnone given
PP_3125 is a key node in the regulation of fluoride tolerance in P. putida.
“this study identifies PP_3125 as a key node in the regulation of fluoride tolerance in P. putida”
Quote in the paperSubstantial claim - Premisecitation
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.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [14] 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
- It may state two separate claims as one. 24
- Probably not a substantial claim. 48
It may state two separate claims as one.
Findingown dataExpressing benE-I did not restore fluoride tolerance to wild-type levels, but the tolerance was notably higher than that of the ΔcrcB strain.
“Although expressing benE-I did not restore the fluoride tolerance to the wild-type levels, the tolerance was notably higher than that of the ΔcrcB strain.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataRemoving transcription regulator PP_3125 increased fluoride tolerance of the ΔcrcB strain, as the ΔcrcBΔ3125 strain was able to grow on 5 mM NaF as well as the wild-type strain.
“Removing transcription regulator PP_3125 increased fluoride tolerance of the ΔcrcB strain, as the strain missing both CrcB and PP_3125 was able to grow on 5 mM NaF, as well as the wild-type strain.”
Quote in the paperSubstantial claim- Rests on an assumption: PP_3125 functions as a transcription regulator.
It may state two separate claims as one.
Findingown dataThe deleted genomic regions in the four spontaneous NaF-tolerant ΔcrcB mutants overlapped but had variable start and end points.
“While the deleted genomic regions overlapped, their start and end points varied.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataExposure of the ΔcrcB and ΔcrcBΔ3125 strains to fluoride affects the length of the lag phase but not the maximum growth rate.
“The difference in the maximum growth rate was not significant, which shows that exposure of these strains to fluoride affects the length of the lag phase but not the growth rate.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataDeletion of PP_2037 (aldolase gene) reduced fluoride tolerance, but overexpression did not restore tolerance.
“The deletion of PP_2037, the aldolase gene, reduced the fluoride tolerance, but the overexpression did not restore the tolerance (Fig. S4).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataIPTG itself affected bacterial growth, as the ΔcrcB strain with an empty plasmid exhibited a longer lag phase with IPTG (19.2 h) compared to without IPTG (14.7 h), but the difference was not statistically significant.
“However, we observed that IPTG itself affected bacterial growth, as the ΔcrcB strain with an empty plasmid exhibited a longer lag phase in the presence of IPTG compared to growth without IPTG supplementation (14.7 h without IPTG vs. 19.2 h with IPTG; however, no statistically significant difference was observed).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataStrains lacking the CrcB channel did not grow on 5 mM NaF, whereas the WT strain tolerated over 30 mM NaF.
“Strains lacking the CrcB channel did not grow on 5 mM NaF, whereas the WT strain tolerated over 30 mM NaF.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Findingown dataThe population with a permeable membrane is larger in benE-I deletion strains than in controls.
“The population with a permeable membrane is larger in the benE-I deletion strains and slightly smaller in the benE-I overexpression strains, but the difference is not statistically significant.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Findingown dataThe other two genes, PP_2034 and PP_2033, were clustered in the same region.
“The other two genes, PP_2034 (a hypothetical transporter) and PP_2033 (of unknown function), were clustered in the same region.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataAt 0.5 mM NaF on LB agar plates, the ΔcrcB strain showed reduced growth whereas the ΔcrcBΔ3125 strain showed no growth restrictions.
“in the presence of 0.5 mM NaF, the ΔcrcB strain showed reduced growth on LB agar plates, whereas the ΔcrcBΔ3125 strain did not show any growth restrictions (Fig. 2).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe population with a permeable membrane is slightly smaller in benE-I overexpression strains, but the difference is not statistically significant.
“The population with a permeable membrane is larger in the benE-I deletion strains and slightly smaller in the benE-I overexpression strains, but the difference is not statistically significant.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataWith 2.5 mM NaF, the lag phase of the ΔcrcB strain was 18.9 h, while the lag phase of the spontaneous mutants and the ΔcrcBΔ3125 strain was seven times shorter, but the differences were not significant.
“When the lag phase of the ΔcrcB strain with 2.5 mM NaF was 18.9 h long, the lag phase of the spontaneous mutants and the ΔcrcBΔ3125 strain was seven times shorter, but the differences were not significant.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAbout 141,000 transposon mutants were obtained from three independent transposon mutagenesis experiments, out of which 48 had a fluoride-tolerant phenotype.
“About 141,000 transposon mutants from three independent transposon mutagenesis experiments were obtained, out of which 48 had a fluoride-tolerant phenotype.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationBenF operates as a benzoate-specific porin and efflux pump.
“Key transporters involved in benzoate transport are BenK, BenE, and BenF. BenK and BenE function as aromatic acid-H+ symporters, while BenF operates as a benzoate-specific porin and efflux pump (28, 29).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [28] Choudhary A, Purohit H, Phale PS. 2017. Benzoate transport in Pseudomonas putida CSV86. FEMS Microbiol Lett 364. doi: 10.1093/femsle/fnx118 28591829 source
Checked before — its earlier verdict is held back until a new check runs.
- cites [29] Nishikawa Y, Yasumi Y, Noguchi S, Sakamoto H, Nikawa J. 2008. Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae. Biosci Biotechnol Biochem 72: 2034– 2038. doi: 10.1271/bbb.80156 18685209 source
It may state two separate claims as one.
Interpretationown dataWhen PP_3125 is absent, the expression of the benE-I transporter is higher, and bacteria can tolerate higher concentrations of NaF.
“When this regulator is absent, the expression of the benE-I transporter is higher, and bacteria can tolerate higher concentrations of NaF.”
Quote in the paperSubstantial claim- Rests on an assumption: PP_3125 functions as a transcription regulator.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationOther 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).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [13] Mitsuhata C, Puteri MM, Ohara Y, Tatsukawa N, Kozai K. 2014. Possible involvement of enolase in fluoride resistance in Streptococcus mutans. Pediatric Dental Journal 24: 12– 16. doi: 10.1016/j.pdj.2013.10.002 source
Checked before — its earlier verdict is held back until a new check runs.
- cites [14] 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.
PremisecitationThere are two distinct classes of fluoride export proteins: ClCF and CrcB (Fluc) channels.
“There are two distinct classes of fluoride export proteins: ClCF and CrcB (Fluc) channels (5, 6, 22).”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [5] Stockbridge RB, Wackett LP. 2024. The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis. Nat Commun 15: 4593. doi: 10.1038/s41467-024-49018-1 38816380 PMC11139923 source
- cites [6] 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 [22] Stockbridge Randy B, Lim H-H, Otten R, Williams C, Shane T, Weinberg Z, Miller C. 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci USA 109: 15289– 15294. doi: 10.1073/pnas.1210896109 22949689 PMC3458365 source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Interpretationown dataFor the ΔcrcBΔ5 strain carrying an empty plasmid on 2.5 mM NaF without IPTG, the slight variation in optical density at the beginning of the experiment was interpreted as the start of growth, and thus the lag phase is not shown to be short, although there is no real growth.
“In Fig. 6, for the ΔcrcBΔ5 strain carrying an empty plasmid on 2.5 mM NaF without IPTG, there was a slight variation in the optical density measurements at the beginning of the experiment, which was interpreted as the start of growth, and thus, the lag phase is not shown to be short, although there is no real growth.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
InterpretationreasoningThe lack of significance in the difference in lag phase with 2.5 mM NaF might be due to a very high variability in the growth pattern of the ΔcrcB strain.
“This might be due to a very high variability in the growth pattern of the ΔcrcB.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
InterpretationreasoningDeletion of aldolase may affect the expression of benE-I because the aldolase gene is located upstream of the benzoate transporter gene.
“As the aldolase gene is located upstream of the benzoate transporter gene, it is possible that this deletion affected the expression of benE-I (Fig. S4).”
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.
PremisecitationCrcB functions as a passive ion channel and is considered a primary defense against fluoride toxicity in many bacteria, including Escherichia coli, Bacillus subtilis, and Pseudomonas species.
“CrcB functions as a passive ion channel and is considered a primary defense mechanism against fluoride toxicity in many bacteria, including Escherichia coli, Bacillus subtilis, and Pseudomonas species (5–7).”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [5] Stockbridge RB, Wackett LP. 2024. The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis. Nat Commun 15: 4593. doi: 10.1038/s41467-024-49018-1 38816380 PMC11139923 source
- cites [6] 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 [7] 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
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationCrcB export proteins function as passive fluoride-specific ion channels that require Na+ ions for transport activity.
“In contrast, CrcB export proteins function as passive fluoride-specific ion channels that require Na+ ions for transport activity (4, 5, 23–26).”
Quote in the paperNot a substantial claim0 of 6 sources checked- cites [4] Breaker RR. 2012. New insight on the response of bacteria to fluoride. Caries Res 46: 78– 81. doi: 10.1159/000336397 22327376 PMC3331882 source
- cites [5] Stockbridge RB, Wackett LP. 2024. The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis. Nat Commun 15: 4593. doi: 10.1038/s41467-024-49018-1 38816380 PMC11139923 source
- cites [23] McIlwain BC, Martin K, Hayter EA, Stockbridge RB. 2020. An interfacial sodium ion is an essential structural feature of fluc family fluoride channels. J Mol Biol 432: 1098– 1108. doi: 10.1016/j.jmb.2020.01.007 31945374 PMC7054162 source
- cites [24] Li S, Smith KD, Davis JH, Gordon PB, Breaker RR, Strobel SA. 2013. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins. Proc Natl Acad Sci USA 110: 19018– 19023. doi: 10.1073/pnas.1310439110 24173035 PMC3839697 source
- cites [25] Stockbridge R.B, Robertson JL, Kolmakova-Partensky L, Miller C. 2013 A family of fluoride-specific ion channels with dual-topology architecture. elife 2: e01084. doi: 10.7554/eLife.01084 23991286 PMC3755343 source
- cites [26] 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
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationAt sublethal concentrations, fluoride disrupts microbial metabolic processes by inhibiting key enzymes such as enolase and pyrophosphatases, often by forming complexes with essential metal cofactors.
“At sublethal concentrations, fluoride disrupts metabolic processes by inhibiting key enzymes, such as enolase and pyrophosphatases, often by forming complexes with essential metal cofactors (4, 5).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [4] Breaker RR. 2012. New insight on the response of bacteria to fluoride. Caries Res 46: 78– 81. doi: 10.1159/000336397 22327376 PMC3331882 source
- cites [5] Stockbridge RB, Wackett LP. 2024. The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis. Nat Commun 15: 4593. doi: 10.1038/s41467-024-49018-1 38816380 PMC11139923 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationBenE-I is a benzoate transporter.
“The functioning of BenE1-I as a benzoate transporter has been reported in previous studies (28, 29).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [28] Choudhary A, Purohit H, Phale PS. 2017. Benzoate transport in Pseudomonas putida CSV86. FEMS Microbiol Lett 364. doi: 10.1093/femsle/fnx118 28591829 source
Checked before — its earlier verdict is held back until a new check runs.
- cites [29] Nishikawa Y, Yasumi Y, Noguchi S, Sakamoto H, Nikawa J. 2008. Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae. Biosci Biotechnol Biochem 72: 2034– 2038. doi: 10.1271/bbb.80156 18685209 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationKey transporters involved in benzoate transport in P. putida are BenK, BenE, and BenF.
“Key transporters involved in benzoate transport are BenK, BenE, and BenF. BenK and BenE function as aromatic acid-H+ symporters, while BenF operates as a benzoate-specific porin and efflux pump (28, 29).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [28] Choudhary A, Purohit H, Phale PS. 2017. Benzoate transport in Pseudomonas putida CSV86. FEMS Microbiol Lett 364. doi: 10.1093/femsle/fnx118 28591829 source
Checked before — its earlier verdict is held back until a new check runs.
- cites [29] Nishikawa Y, Yasumi Y, Noguchi S, Sakamoto H, Nikawa J. 2008. Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae. Biosci Biotechnol Biochem 72: 2034– 2038. doi: 10.1271/bbb.80156 18685209 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationClCF transporters function as proton-fluoride antiporters, facilitating active extrusion of fluoride from the cell.
“ClCF transporters function as proton-fluoride antiporters, facilitating active extrusion of fluoride from the cell (22).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [22] Stockbridge Randy B, Lim H-H, Otten R, Williams C, Shane T, Weinberg Z, Miller C. 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci USA 109: 15289– 15294. doi: 10.1073/pnas.1210896109 22949689 PMC3458365 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationBenK and BenE function as aromatic acid-H+ symporters.
“Key transporters involved in benzoate transport are BenK, BenE, and BenF. BenK and BenE function as aromatic acid-H+ symporters, while BenF operates as a benzoate-specific porin and efflux pump (28, 29).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [28] Choudhary A, Purohit H, Phale PS. 2017. Benzoate transport in Pseudomonas putida CSV86. FEMS Microbiol Lett 364. doi: 10.1093/femsle/fnx118 28591829 source
Checked before — its earlier verdict is held back until a new check runs.
- cites [29] Nishikawa Y, Yasumi Y, Noguchi S, Sakamoto H, Nikawa J. 2008. Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae. Biosci Biotechnol Biochem 72: 2034– 2038. doi: 10.1271/bbb.80156 18685209 source
Probably not a substantial claim.
PremisecitationThe CrcB export protein, also known as Fluc, exports intracellular fluoride ions.
“Previous studies have highlighted the role of the CrcB export protein (also known as Fluc) in exporting intracellular fluoride ions (7).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [7] 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
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Interpretationown dataThe intracellular fluoride concentration is probably higher due to more permeable cell membranes.
“The intracellular fluoride is probably higher due to more permeable cell membranes, as seen in Fig. 8b.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationCrcB channels are highly selective for fluoride.
“While ClCF transporters can transport both chloride and fluoride ions or only fluoride, CrcB channels are highly selective for fluoride (6, 12).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [6] 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 [12] Brammer AE, Stockbridge RB, Miller C. 2014. F-/Cl- selectivity in CLCF-type F-/H+ antiporters. J Gen Physiol 144: 129– 136. doi: 10.1085/jgp.201411225 25070431 PMC4113899 source
Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
Interpretationnone givenThe high variability in growth curves of fluoride-sensitive strains in liquid media at the end of the experiment could be attributed to the emergence of spontaneous mutants capable of tolerating elevated fluoride concentrations.
“The high variability in growth curves of fluoride-sensitive strains in liquid media at the end of the experiment could be attributed to the emergence of spontaneous mutants capable of tolerating elevated fluoride concentrations.”
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.
PremisecitationClCF transporters can transport both chloride and fluoride ions or only fluoride.
“While ClCF transporters can transport both chloride and fluoride ions or only fluoride, CrcB channels are highly selective for fluoride (6, 12).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [6] 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 [12] Brammer AE, Stockbridge RB, Miller C. 2014. F-/Cl- selectivity in CLCF-type F-/H+ antiporters. J Gen Physiol 144: 129– 136. doi: 10.1085/jgp.201411225 25070431 PMC4113899 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenTwo biological replicas in two technical parallels were analyzed for flow cytometry.
“Two biological replicas in two technical parallels were analyzed.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataThe absence of pSW(I-SceI) was confirmed by PCR.
“The absence of pSW(I-SceI) was confirmed by PCR.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataThe PP_3125 deletion was verified by PCR and DNA sequencing.
“PCR and DNA sequencing verified the PP_3125 deletion.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Interpretationown dataThe diversity in the start and end points of observed deletions suggests the absence of a single deletion mechanism and highlights the stochastic nature of stress-induced genomic rearrangements.
“The diversity in the start and end points of observed deletions (Fig. S1) further suggests the absence of a single deletion mechanism and highlights the stochastic nature of stress-induced genomic rearrangements.”
Quote in the paperNot a substantial claimProbably not a substantial claim. It may be too vague to check against evidence.
Interpretationnone givenBenE-I is a promising target for engineering enhanced fluoride tolerance.
“highlights BenE-I as a promising target for engineering enhanced fluoride tolerance”
Quote in the paperNot a substantial claimProbably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.
InterpretationreasoningMultiple mechanisms may underlie these large-scale deletions.
“Multiple mechanisms may underlie these large-scale deletions.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Interpretationown dataThe regulatory landscape of PP_3125 may extend beyond what was captured in proteomic and transcriptomic snapshots limited to early growth phases.
“The regulatory landscape of PP_3125 may extend beyond what was captured in our proteomic and transcriptomic snapshots, which were limited to early growth phases.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationP. 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”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] 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.
InterpretationreasoningThe lack of clear functional linkage among genes in the PP_2034-PP_2037 operon raises the possibility that their roles in fluoride tolerance may not be confined to canonical metabolic pathways.
“The lack of clear functional linkage among genes in the PP_2034–PP_2037 operon raises the possibility that their roles in fluoride tolerance may not be confined to canonical metabolic pathways.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationBased on the KEGG database, aldolase PP_2037 is predicted to belong to six different pathways.
“Based on the KEGG database, aldolase PP_2037 is predicted to belong to six different pathways: monobactam biosynthesis, lysine biosynthesis, metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and biosynthesis of amino acids;”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [30] Kanehisa M, Furumichi M, Sato Y, Ishiguro-Watanabe M, Tanabe M. 2021. KEGG: integrating viruses and cellular organisms. Nucleic Acids Res 49: D545– D551. doi: 10.1093/nar/gkaa970 33125081 PMC7779016 source
Probably not a substantial claim.
Study design and conductnone givenTo be included in the analysis, a protein must be detected in all three replicates of one group.
“To be included in the analysis, a protein must be detected in all three replicates of one group.”
Quote in the paperSubstantial claimProbably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.
InterpretationreasoningIt is plausible that proteins encoded by PP_2034-PP_2037 may perform secondary functions relevant to ion homeostasis, membrane remodeling, or stress adaptation.
“Given the observed regulatory complexity and the lack of clear pathway associations for most genes in this operon, it is plausible that their encoded proteins may perform secondary functions relevant to ion homeostasis, membrane remodeling, or stress adaptation.”
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.
Study design and conductnone givenThe positive clones on LB Gm plates were identified by PCR and verified by sequencing.
“The positive clones on LB Gm plates were identified by PCR and verified by sequencing.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
InterpretationreasoningIt is likely that PP_3125 influences additional gene sets during later growth stages, potentially coordinating broader adaptive responses to prolonged fluoride exposure.
“It is likely that PP_3125 influences additional gene sets during later growth stages, potentially coordinating broader adaptive responses to prolonged fluoride exposure.”
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.
InterpretationreasoningEnvironmental stresses like fluoride exposure can cause DNA damage, and imprecise repair via the non-homologous end joining (NHEJ) pathway may result in random deletions, but other mechanisms must be involved because the deletions are 18-300 kb.
“It is possible that environmental stresses like fluoride exposure can cause DNA damage, and imprecise repair via the non-homologous end joining (NHEJ) pathway (37) may result in random deletions, but as the deletions are 18–300 kb, there needs to be other mechanisms involved too.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [37] Shen M, Zhang H, Shen W, Zou Z, Lu S, Li G, He X, Agnello M, Shi W, Hu F, Le S. 2018. Pseudomonas aeruginosa MutL promotes large chromosomal deletions through non-homologous end joining to prevent bacteriophage predation. Nucleic Acids Res 46: 4505– 4514. doi: 10.1093/nar/gky160 29514250 PMC5961081 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationMultifunctionality has been increasingly recognized in bacteria, where metabolic enzymes, structural proteins, and transporters adopt additional roles beyond their canonical functions.
“Such multifunctionality has been increasingly recognized in bacteria, where metabolic enzymes, structural proteins, and transporters adopt additional roles beyond their canonical functions (31, 32).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [31] Henderson B, Martin A. 2013. Bacterial moonlighting proteins and bacterial virulence, p 155– 213. In Dobrindt U, Hacker JH, Svanborg C (ed), Between pathogenicity and commensalism [Internet]. Springer, Berlin, Heidelberg.
- cites [32] Jeffery CJ. 1999. Moonlighting proteins. Trends Biochem Sci 24: 8– 11. doi: 10.1016/s0968-0004(98)01335-8 10087914 source
Checked before — its earlier verdict is held back until a new check runs.
It may state two separate claims as one.
InterpretationreasoningBenE-I could act not only as a substrate-specific transporter of benzoate but also as a structural and regulatory component of the membrane stress response by influencing fluoride tolerance independently of benzoate.
“Thus, BenE-I could act not only as a substrate-specific transporter of benzoate but also as a structural and regulatory component of the membrane stress response by influencing fluoride tolerance independently of benzoate.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataThe data did not follow a normal distribution.
“As the data did not follow a normal distribution, the non-parametric method was used to compare different data sets.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationEnolase and glyceraldehyde-3-phosphate dehydrogenase are well-known metabolic enzymes that also act as RNA-binding proteins or surface adhesins in diverse bacterial species.
“For example, enolase and glyceraldehyde-3-phosphate dehydrogenase are well-known metabolic enzymes that also act as RNA-binding proteins or surface adhesins in diverse bacterial species (33, 34).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [33] Huberts D, van der Klei IJ. 2010. Moonlighting proteins: an intriguing mode of multitasking. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1803: 520– 525. doi: 10.1016/j.bbamcr.2010.01.022 20144902 source
- cites [34] Pancholi V, Chhatwal GS. 2003. Housekeeping enzymes as virulence factors for pathogens. Int J Med Microbiol 293: 391– 401. doi: 10.1078/1438-4221-00283 14760970 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationGenome plasticity in P. putida is supported by the presence of mobile genetic elements such as transposons and prophages.
“Genome plasticity in P. putida is supported by the presence of mobile genetic elements such as transposons and prophages.”
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.
PremisecitationP. putida KT2440 contains prophages capable of spontaneous excision, which may promote deletion events even in the absence of phage infectivity.
“Moreover, P. putida KT2440 contains prophages capable of spontaneous excision, which may promote deletion events even in the absence of phage infectivity (36).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [36] Martínez-García E, Jatsenko T, Kivisaar M, de Lorenzo V. 2015. Freeing Pseudomonas putida KT2440 of its proviral load strengthens endurance to environmental stresses. Environ Microbiol 17: 76– 90. doi: 10.1111/1462-2920.12492 24762028
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductown dataFour biological replicates with at least 10 parallels were analyzed for the frequency of spontaneous NaF-tolerant mutants.
“Four biological replicates with at least 10 parallels were analyzed.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Interpretationnone givenThe evolutionary emergence of large deletions encompassing PP_3125 under stress conditions reflects the organism's remarkable genomic plasticity.
“The evolutionary emergence of large deletions encompassing PP_3125 under stress conditions reflects the organism’s remarkable genomic plasticity.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
InterpretationreasoningThe fluoride-responsive expression pattern of PP_3125 hints at a yet-to-be-characterized regulatory mechanism that senses and responds to fluoride stress.
“The fluoride-responsive expression pattern of PP_3125 hints at a yet-to-be-characterized regulatory mechanism that senses and responds to fluoride stress.”
Quote in the paperNot a substantial claimProbably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.
InterpretationreasoningThe possibility that BenE-I exhibits moonlighting activities cannot be excluded.
“In the context of fluoride tolerance, the possibility that BenE-I exhibits moonlighting activities cannot be excluded.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe bph-sal element in P. putida KF715 is known to be both deletable and transferable, reflecting high genomic fluidity.
“For instance, the bph-sal element in P. putida KF715 is known to be both deletable and transferable, reflecting high genomic fluidity (35).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [35] Suenaga H, Fujihara H, Kimura N, Hirose J, Watanabe T, Futagami T, Goto M, Shimodaira J, Furukawa K. 2017. Insights into the genomic plasticity of Pseudomonas putida KF715, a strain with unique biphenyl-utilizing activity and genome instability properties. Environ Microbiol Rep 9: 589– 598. doi: 10.1111/1758-2229.12561 28631340 source
Checked before — its earlier verdict is held back until a new check runs.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationBased on the KEGG database, 4-hydroxy-tetrahydrodipicolinate synthase PP_2036 is predicted to belong to four different pathways.
“4-hydroxy-tetrahydrodipicolinate synthase is predicted to belong to the following four different pathways: ppu00040 Pentose and glucuronate interconversions, fructose and mannose metabolism, metabolic pathways, and microbial metabolism in diverse environments (30; entry ppu:PP_2036: available from: https://www.kegg.jp/entry/ppu:PP_2036; retrieved 23 March 2026; entry ppu:PP_2037: available from:”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [30] Kanehisa M, Furumichi M, Sato Y, Ishiguro-Watanabe M, Tanabe M. 2021. KEGG: integrating viruses and cellular organisms. Nucleic Acids Res 49: D545– D551. doi: 10.1093/nar/gkaa970 33125081 PMC7779016 source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Interpretationown dataThe function of operon PP_2034-PP_2037 is unclear because its genes belong to different metabolic pathways and steps, and other genes have no pathway associations.
“What the operon PP_2034-PP_2037, where benE-I belongs, is unclear, as the aldolase and 4-hydroxy-tetrahydrodipicolinate synthase belong to different metabolic pathways and different steps, and all three other genes have no pathways associated with them.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenMeasurements were carried out every 10 min on three independent biological replicates for the biosensor experiment.
“Measurements were carried out every 10 min on three independent biological replicates”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
InterpretationreasoningGenes in the PP_2034-PP_2037 operon could participate in alternative processes through functional versatility.
“Instead, they could participate in alternative processes through functional versatility.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Interpretationown dataThe deleted regions may include nonessential genes under standard laboratory conditions or the deletions conferred adaptive benefits.
“Despite these extensive deletions, the mutants exhibited no significant growth defects under standard laboratory conditions and showed improved growth under fluoride stress (Fig. 4), suggesting that the deleted regions may include nonessential genes under those conditions or that the deletions conferred adaptive benefits.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
PP_3125 is located within the deleted regions of the spontaneously occurring NaF-tolerant ΔcrcB mutants.
“Since PP_3125 is located within the deleted regions of the spontaneously occurring NaF-tolerant ΔcrcB mutants”
The paper relies on this without support.
PP_3125 functions as a transcription regulator.
“Removing transcription regulator PP_3125 increased fluoride tolerance of the ΔcrcB strain”
The paper relies on this without support.
BenE-I is a benzoate transport protein.
“BenE-I (PP_2035, benzoate transport protein)”
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.
Transposon selection concentration is reported as both 5 and 15 mM
MajorThe results say transposon mutants were screened on 5 mM NaF plates, but the methods say they were replica-plated onto 15 mM NaF.
“Approximately 141,000 transposon mutants from three independent transposon mutagenesis experiments were obtained, and the fluoride tolerance of the mutants was analyzed on plates containing 5 mM NaF.”
“The total number of transposon mutant colonies was counted, and the colonies were transferred to LB plates containing 15 mM NaF using replica plating.”
Abstract reverses PP_3125's effect on fluoride tolerance
MajorThe abstract calls PP_3125 essential for high fluoride tolerance in the ΔcrcB background, whereas the results state that inactivating it increases tolerance in that background.
“We identified PP_3125, a Cro/cI-type transcriptional regulator, as essential for high fluoride tolerance in a ΔcrcB background.”
“Our results indicated that inactivation of PP_3125 encoding Cro/CI type transcriptional regulator increases the fluoride tolerance of the ΔcrcB strain.”
Spot-assay IPTG concentration differs between methods and Fig. 7
MajorThe growth-analysis methods specify 0.5 mM IPTG for dilution spot assays, whereas the Fig. 7 caption specifies 0.1 mM IPTG for its dilution spot assay.
“10-fold serial dilutions of the cultures were spotted as 5 µL drops onto LB-Km agar plates supplemented with 0.5 mM IPTG and different concentrations of NaF (0–30 mM NaF).”
“Figure: Fig 7 Dilution spot assay of P. putida WT, ΔcrcBΔ5, ΔcrcBΔ3125, ΔcrcB_tac_benE-I, and ΔcrcBΔ5_tac_benEI on LB-Km plates supplemented with 0–30 mM NaF and 0.1 mM IPTG as Ptac-promoter inducer.”
Stated NaF concentration range excludes a reported experiment
MinorThe materials section states that NaF was used at up to 30 mM, while the proteomic and transcriptomic methods report using 33 mM for the WT strain.
“Sodium fluoride (NaF) was purchased from Sigma-Aldrich (St. Louis, MO, USA; cat. #201154) and used at concentrations ranging from 0 to 30 mM, as specified in the text.”
“for the WT strain, 33 mM NaF was used.”
Proteomic culture count conflicts with stated biological duplicates
MinorThe proteomic and transcriptomic methods say three independent proteomic cultures were submitted, but later describe the cultures as grown in biological duplicates.
“For proteomic analysis, three, and for the transcriptomic analysis, two independent cultures of each P. putida strain were sent to be analyzed.”
“Cultures were grown in biological duplicates until the beginning of the exponential phase, with an OD580 of 0.1, and biomass was harvested and washed twice with 1× M9 buffer (42 mM KH2PO4, 24 mM Na2HPO4, 19 mM NH4Cl, and 9 mM NaCl).”
Complemented aldolase strain has incompatible genotype descriptions
MinorTable 1 names the complemented strain ΔcrcBΔ3125Δald_tac_ald, but describes it as derived from a ΔcrcBΔbenE-I strain rather than the named aldolase-deletion strain.
“ΔcrcBΔ3125Δald_tac_ald | KT2440 ΔcrcBΔbenE-I strain containing lacI-Ptac-ald in the intergenic region between glmS and PP5408 (Gmr) | This study”
“ΔcrcBΔ3125Δald | KT2440 ∆crcB Δald (PP_2037) | This study”
Fig. 8 lists a NaF concentration absent from the biosensor methods
MinorThe Fig. 8 biosensor caption includes a 15 mM NaF condition, but the corresponding methods list only 0, 0.25, and 1 mM.
“compared to the WT strain incubated in the presence of different NaF concentrations (0, 0.25, 1, and 15 mM).”
“Cells were grown for 3 h at 30°C with agitation at 282 rpm, after which NaF was added at 0, 0.25, and 1 mM concentrations.”
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CLC F , Not CrcB , Protects Pseudomonas putida ATCC 12633 During Intense Fluoride Stress
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Metadata
- Authors
- Ets, L., Ilves, H., Juhe, L., Ilmjärv, T., Puiggené, Ò., Nikel, P. I., & Kivisaar, M.
- Affiliations
- University of Tartu (EE)
- Novo Nordisk Foundation (DK)
- Corresponding author
- Ets, Lea
- Kivisaar, Maia
- Venue
- Journal of Bacteriology
- Publisher
- American Society for Microbiology
- Published
- 2026-04-23
- Volume
- 208
- Issue
- 4
- Pages
- e0047925-e0047925
- ISSN
- 0021-9193 (ISSN-L)
- 1098-5530
- PMC
- PMC13104614
- OpenAlex
- W7147536887
- Cited by
- 1
- References
- 64
- Citations by year
- 2026: 1
- FWCI
- 2.33
- Type
- journal article
- Language
- en
- Open access
- diamond
- doi.org
- Licence
- Locations
- Journal of Bacteriology (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (submittedVersion, cc-by-nd)
- PubMed (publishedVersion)
- PubMed Central (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Estonian Research Competency Council (PRG707, TARISTU24-TK14)
- European Union’s Horizon 2020 Research and Inovation Programme (No. 814418, No. 101082049)
- The Novo Nordisk Foundation (NNF20CC0035580, NNF18OC0034818, NNF21OC0067996, NNF24OC0091501, NNF23OC0083631)
- 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
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Keywords
- fluoride tolerance, Pseudomonas putida, fluoride stress, transcription factors, adaptive evolution, transposon mutant screen, bioremediation
- MeSH terms
- Adaptation, Physiological, Bacterial Proteins, Benzoates, Fluorides, Gene Expression Regulation, Bacterial, Pseudomonas putida, Membrane Transport Proteins
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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