Different lanthanide elements induce strong gene expression changes in a lanthanide-accumulating methylotroph

ABSTRACT Lanthanides (Ln) are the most recently described life metals and are central to methylotrophy (type of metabolism in which organic substrates without carbon-carbon bonds serve as carbon and energy source) in diverse taxa. We recently characterized a novel, Ln-dependent, and Ln-accumulating methylotroph, Beijerinckiaceae bacterium RH AL1, which requires lighter Ln (La, Ce, Nd) for methanol oxidation. Starting from two sets of incubations, one with different La concentrations (50 nM and 1 µM) and one with different Ln elements [La, Nd, or an Ln cocktail (containing Ce, Nd, Dy, Ho, Er, Yb)], we could show that La concentration and different Ln elements strongly affect gene expression and intracellular Ln accumulation. Differential gene expression analysis revealed that up to 41% of the encoded genes were differentially expressed. The effects of La concentration and Ln elements were not limited to Ln-dependent methanol oxidation but reached into many aspects of metabolism. We observed that Ln influence the flagellar and chemotactic machinery and that they affect polyhydroxyalkanoate biosynthesis. The most differentially expressed genes included lanM, coding for the well-characterized lanthanide-binding protein lanmodulin, and a glucose dehydrogenase gene linked to the conversion of β-D-glucose to D-glucono-1,5-lactone, a known potential metal chelator. Electron microscopy, together with RNAseq, suggested that Beijerinckiaceae bacterium RH AL1 can discriminate between Ln elements and that they are differently taken up and accumulated. The discrimination of Ln and links between Ln and various aspects of metabolism underline a broader physiological role for Ln in Beijerinckiaceae bacterium RH AL1. IMPORTANCE Since its discovery, Ln-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding Ln recycling and circular economy. The physiological role of Ln is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) Ln affect metabolism have rarely been done. We noticed unexpectedly pronounced changes in gene expression caused by different Ln supplementation. Our research suggests that strain RH AL1 distinguishes different Ln elements and that the effect of Ln reaches into many aspects of metabolism, for instance, chemotaxis, motility, and polyhydroxyalkanoate metabolism. Our findings regarding Ln accumulation suggest a distinction between individual Ln elements and provide insights relating to intracellular Ln homeostasis. Understanding comprehensively how microbes distinguish and handle different Ln elements is key for turning knowledge into application regarding Ln-centered biometallurgy.


Figures
General: 1. Overall: Great in appearance.However, the manuscript needs more comprehensive descriptions in the figure legends.
2. It would be helpful to implement a schematic figure explaining how the experiments were set up and carried out for growth and transcriptomics, because the written explanations often don't completely clarify the details of it.

Specific:
-Figure 1: a) It would be much more accurate to target a timeframe within the exponential phase to get maximum td values.b) Why are the doubling times in La rather different between the left and right panels?c) The legend is highly insufficient and doesn't include 1B.d) The redundancy in 1uM La in both panels is confusing.Where did the 1uM La data come from?The cultures from the left panel, or right?-Figure 2: This figure had a number of aspects about it that made it difficult to follow.From a statistical standpoint, for comparisons like this it makes more sense to have more stringent cutoffs for statistical significance to be assessed.A more common FDR value to use as a significance cutoff is FDR < 0.01.In the figure legend, I think it makes better sense to have A-C labels at the beginning rather than the ends of statements.For panels B and C, it would be clearer to represent the comparisons with word descriptions rather than numbering 1-4.In Figure 2C, it does not make sense to have single comparisons rather than intersections between comparisons.In the case the single comparisons (2 and 3) are represented, why are the unique genes to 1 and 4 also not shown?-Figure 3: I am unsure that this is accomplishing what it is seeking to.I would potentially separate the central cell illustration from the surrounding data panels into separate figures.Alternatively, it might make sense to provide more color-coding to the central illustrations to have processes align with the figure legend on the side.
-Figure 4 is not referred to directly anywhere in the text.Please adjust and insert appropriate reference points.
-Figure 5 refers to PHB, but the text does not explain that PHB is a specific type of PHA molecule until later in the discussion section.

Minor comments
General: 1. Report methanol and pyruvate concentrations as molar values, for better relevance and consistency with the lanthanide concentrations.
2. At least once, report the concentration of each single lanthanide compound within the combined cocktail treatment.
3. The authors refer to their focal strain in different ways (e.g., "Beijerinckiaceae bacterium RH AL1", "RH AL1", "strain AL1", etc.) throughout the paper.After initial introduction, choose one in order to maintain consistency throughout.
4. In some of the results, the authors report the trend of a comparison, but not the quantitative measure.I would suggest reporting log2 fold changes throughout for consistency and added information to the reader.5. Unless the authors plan to refer to terms throughout, it might be confusing to define new terms like the lanthanome.Specific: -Line 28-29 -This sentence is vaguely worded, rephrase.
-Line 30 -Emphasizing the unknown or hypothetical genes feels like something to bring up later, but not in the abstract.
-Line 58 -The authors should define methylotrophy upon first usage.
-Line 113-114 -different time (t) designations are confusing -Line 121 -What are the associated ODs with this?Can it really be considered mid-exponential phase?-Line 122 -Confused about the cutoff of |0.58|, it feels a bit arbitrary.Is this explained anywhere?-Lines 163-166 -These two sentences feel a bit uninformative and out-of-place for a results section.Are they really necessary?I would consider rephrasing or even cutting these statements from the manuscript.-Line 196 -Add "log2FC" -also check throughout to make sure of its presence.
-Line 198-199 -Wording makes it unclear which one of the two is lower.
-Line 225 -Say more about the nature of the type of t-test performed.
-Line 212 -Why is only one number reported for RHAL1_02672 when 2 numbers are reported for the other genes listed here?-Line 219 -Replace the "+" sign here.
-Line 234 -How do you attribute distinct signals to specific lanthanides here?It is confusing.
-Line 236 -Use elemental abbreviations to be consistent throughout.
-Line 237 -Confused about why P is invoked here, there is no verbiage to preface why.
-Line 240-241 -Confusing way to start discussion section... -Line 251 -Suggested change to start the sentence: "Based on these data..." -Line 254 -A supplemental table of lanthanide names, since you are using so many of them, would be helpful to someone who does not regularly work with all these compounds.
-Line 257 -Insert text "in this study" in the sentence to reorient reader.
-Line 261 -do not define metalation anywhere in the text.A little hint to the reader would be helpful.
-Line 274 -"in a non-methylotrophic background" alone is quite vague -could you use text to expand more on this a bit? -Line 283-284 -Is this sentence important/necessary? -Line 286 -Is there something particularly interesting about pyruvate that makes it interesting/worth reporting on?Similarly, is the whole long following section focused upon Calcium completely necessary?The way the discussion is written, going back and forth between Ca and Ln is actually more confusing rather than informative.
-Line 300 -What type of sensitivity are you talking about?-Line 313 -First mention of the term lanthanide phosphates.Should be broached earlier when P is first mentioned.
-Line 348 -Is sulfur metabolism being invoked or not really?Seems like really soft language considering a lot of space in text and figures elsewhere have already been devoted to sulfur.
-Line 355 -This is not necessary to be quoted or cited, in my opinion.
-Line 376 -Unclear if all samples were acclimated on 1) homologous conditions into which they were diluted, 2) if all were acclimated in identical condition, or 3) some hybrid of 1) and 2).
-Line 719 -this might be the only place in the paper where the lanthanome is directly defined.It should be defined in the text as well.
Reviewer #2 (Comments for the Author): This paper by Gorniak et al., investigates how different lanthanides affect gene expression and metabolism using lanthanidedependent, and lanthanide-accumulating methylotroph, Beijerinckiaceae bacterium RH AL1.Lanthanide accumulation in RH AL1 was previously shown by this group and in this work the authors looked at different lanthanides and concentrations of lanthanum, to identify the differentially expressed genes.Overall the work is exciting and the conclusions are valid.It is quite interesting to see the differences and commonalities in response to different Ln elements.The effects of differential gene expression and their physiological significance are yet to be understood.I have minor comments to further improve this manuscript.1. Please include full forms of all abbreviations and details to the figure legends to help the reader understand the figures presented without referring back to the text in the results section.For instance in Fig. 1 -please mention La, Nd, cocktail stand for, panel A to the left shows lanthanum concentrations?Panel B is missing from the legend.In Panel B, what do copies refer to?Also, mention the number of replicates for the growth assays and experiments.
2. Please mention the number of replicates performed for the RNA-seq experiment for the 4 different conditions (preferably in the main text and figure legend) 3. What do the terms "overrepresentation" and "gene enrichment" mean in terms of biology?Are all the genes that are identified from the gene enrichment analysis also identified as overrepresented in the overrepresentation analysis?4. It is interesting to see hits in folate biosynthesis, secondary metabolism, two-component systems, etc but the gene expression appears different under La versus cocktail or pairwise comparisons.The authors could elaborate/discuss a bit more about the potential physiological implications of these changes in different pathways with respect to separate conditions in the discussion.

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Microbiology Spectrum Review, June 2023 Article Title: Different lanthanide elements induce strong gene expression changes in a lanthanideaccumulating methylotroph
Article Authors: Linda Gorniak , Julia Bechwar, Martin Westermann, Frank Steiniger, Carl-Eric Wegner

Summary
The manuscript documents the differential impacts of alternative lanthanide rare earth metals upon gene expression within the recently characterized methylotrophic bacterium Beijerinckiaceae bacterium RH AL1.Lanthanides have recently garnered much interest as an alternative metal enabling methylotrophic metabolism, primarily by serving as a cofactor for XoxF-like dehydrogenase enzymes.The authors observe strong changes in gene expression depending on what lanthanides cells are grown in the presence of, including diverse metabolic processes such as chemotaxis and PHA synthesis.The study is valuable to the growing field of lanthanide-focused biology, as it takes a more in-depth view of the impacts of alternative and single vs. combinatorial lanthanides upon gene expression.In particular, comparisons of how different (utilizable) lanthanides affect metabolism have rarely been done, and strain RH AL1 appears to distinguish between different lanthanide elements.

Major comments
1.The manuscript needs significant rewriting to make it clearer and more impactful.As an example, text descriptions of the groups being compared are confusing at times.The authors should also preface sections in the discussion section.They often dive right into paragraphs with detailed statements without providing the appropriate context, rationale, and bigger picture themes, and need to set these up more carefully.For example, in viewing the paragraph focused on calcium (Lines 289-296), the first sentence is not required, and the paragraph would read better if it started from the second sentence.This section and others ask the reader to connect too many dots rather than stating them more explicitly (or testing them directly in the study).Other paragraphs are rather unfocused: e.g., Lines 348-353 read as just a sequence of facts about sulfur metabolism rather than a cohesive discussion of the potential impacts that lanthanides have upon sulfur metabolism.
2. The authors overstate the impact of the findings at times, such as suggesting the presence of different uptake mechanisms for different lanthanides in the abstract, something that is not convincingly broached at all with the experiments performed in this study.On Lines 337-341, the authors mention the upregulation of "heavy metal efflux mechanisms" (should be "genes").This is a trend of expression and sets up a testable hypothesis over the specificity of these chelators, but is not conclusive.There are a few other places where the term "mechanism" is invoked but does not seem to be done so accurately.
3. The text has a lot of soft, "maybe so", kind of language, most noticeably in the discussion section, which leaves it unclear to the reader how seriously to take some of the statements being made.1.Why do the authors start growth on pyruvate?Are lanthanides required for growth under this condition?This also constitutes a switch (pyruvate-to methanol-based growth) to methylotrophy, which could include related transcriptomic changes that will get lost in this analysis.

Figures look nice but
2. The setup for the transcriptomics is a bit confusing (and is why I suggest including an accompanying figure below).It seems that for this, all treatments should either be a) pregrown in the same lanthanide condition, or b) pregrown in a condition identical to that which it will be transferred into.If neither of these conditions is satisfied, then it feels like the cross-comparison between differently treated samples is problematic.
3. It would be informative for the authors to speak to the concentrations of lanthanides used in the study, as compared to measured or expected concentrations available in typical environmental conditions.

General:
1. Overall: Great in appearance.However, the manuscript needs more comprehensive descriptions in the figure legends.
2. It would be helpful to implement a schematic figure explaining how the experiments were set up and carried out for growth and transcriptomics, because the written explanations often don't completely clarify the details of it.

Specific:
-Figure 1: a) It would be much more accurate to target a timeframe within the exponential phase to get maximum td values.b) Why are the doubling times in La rather different between the left and right panels?c) The legend is highly insufficient and doesn't include 1B.d) The redundancy in 1uM La in both panels is confusing.Where did the 1uM La data come from?The cultures from the left panel, or right?-Figure 2: This figure had a number of aspects about it that made it difficult to follow.From a statistical standpoint, for comparisons like this it makes more sense to have more stringent cutoffs for statistical significance to be assessed.A more common FDR value to use as a significance cutoff is FDR < 0.01.In the figure legend, I think it makes better sense to have A-C labels at the beginning rather than the ends of statements.For panels B and C, it would be clearer to represent the comparisons with word descriptions rather than numbering 1-4.In Figure 2C, it does not make sense to have single comparisons rather than intersections between comparisons.In the case the single comparisons (2 and 3) are represented, why are the unique genes to 1 and 4 also not shown?-Figure 3: I am unsure that this is accomplishing what it is seeking to.I would potentially separate the central cell illustration from the surrounding data panels into separate figures.Alternatively, it might make sense to provide more color-coding to the central illustrations to have processes align with the figure legend on the side.
-Figure 4 is not referred to directly anywhere in the text.Please adjust and insert appropriate reference points.
-Figure 5 refers to PHB, but the text does not explain that PHB is a specific type of PHA molecule until later in the discussion section.

General:
1. Report methanol and pyruvate concentrations as molar values, for better relevance and consistency with the lanthanide concentrations.
2. At least once, report the concentration of each single lanthanide compound within the combined cocktail treatment.
3. The authors refer to their focal strain in different ways (e.g., "Beijerinckiaceae bacterium RH AL1", "RH AL1", "strain AL1", etc.) throughout the paper.After initial introduction, choose one in order to maintain consistency throughout.
4. In some of the results, the authors report the trend of a comparison, but not the quantitative measure.I would suggest reporting log2 fold changes throughout for consistency and added information to the reader.5. Unless the authors plan to refer to terms throughout, it might be confusing to define new terms like the lanthanome.
-Line 30 -Emphasizing the unknown or hypothetical genes feels like something to bring up later, but not in the abstract.
-Line 58 -The authors should define methylotrophy upon first usage.
-Line 113-114 -different time (t) designations are confusing -Line 121 -What are the associated ODs with this?Can it really be considered mid-exponential phase?-Line 122 -Confused about the cutoff of |0.58|, it feels a bit arbitrary.Is this explained anywhere?-Lines 163-166 -These two sentences feel a bit uninformative and out-of-place for a results section.Are they really necessary?I would consider rephrasing or even cutting these statements from the manuscript.
-Line 196 -Add "log2FC" -also check throughout to make sure of its presence.
-Line 198-199 -Wording makes it unclear which one of the two is lower.
-Line 225 -Say more about the nature of the type of t-test performed.
-Line 212 -Why is only one number reported for RHAL1_02672 when 2 numbers are reported for the other genes listed here?-Line 219 -Replace the "+" sign here.
-Line 234 -How do you attribute distinct signals to specific lanthanides here?It is confusing.
-Line 236 -Use elemental abbreviations to be consistent throughout.
-Line 237 -Confused about why P is invoked here, there is no verbiage to preface why.
-Line 240-241 -Confusing way to start discussion section… -Line 251 -Suggested change to start the sentence: "Based on these data…" -Line 254 -A supplemental table of lanthanide names, since you are using so many of them, would be helpful to someone who does not regularly work with all these compounds.-Line 257 -Insert text "in this study" in the sentence to reorient reader.
-Line 261 -do not define metalation anywhere in the text.A little hint to the reader would be helpful.
-Line 274 -"in a non-methylotrophic background" alone is quite vague -could you use text to expand more on this a bit? -Line 283-284 -Is this sentence important/necessary? -Line 286 -Is there something particularly interesting about pyruvate that makes it interesting/worth reporting on?Similarly, is the whole long following section focused upon Calcium completely necessary?The way the discussion is written, going back and forth between Ca and Ln is actually more confusing rather than informative.
-Line 300 -What type of sensitivity are you talking about?-Line 313 -First mention of the term lanthanide phosphates.Should be broached earlier when P is first mentioned.
-Line 348 -Is sulfur metabolism being invoked or not really?Seems like really soft language considering a lot of space in text and figures elsewhere have already been devoted to sulfur.
-Line 355 -This is not necessary to be quoted or cited, in my opinion.
-Line 376 -Unclear if all samples were acclimated on 1) homologous conditions into which they were diluted, 2) if all were acclimated in identical condition, or 3) some hybrid of 1) and 2).
-Line 719 -this might be the only place in the paper where the lanthanome is directly defined.It should be defined in the text as well.

General remarks:
Additional data: Carried electron microscopy combined with elemental analysis by means of energy-dispersive X-ray spectroscopy suggested that different lanthanide (Ln) elements are preferentially taken up when Beijerinckiaceae bacterium RH AL1 is cultivated with a cocktail of light and heavy lanthanides.We repeated the incubations to strengthen this point.We took samples during the late-exponential growth phase and subjected cell-free supernatant samples to elemental analysis through ICP-MS (inductively coupled plasma mass spectrometry).Obtained results revealed that the supernatant samples were depleted of lighter lanthanides (Ce, Nd), while heavier lanthanides (Dy, Ho, Er, Yb) were still detectable.We detected 3.02 ± 1.58% of the initially present Er, and 75.93 ± 3.58% of the initially present Yb.These findings support our claim that Beijerinckiaceae bacterium RH AL1 selectively takes up lanthanides, and we added them to the manuscript, see lines 285-290, and Table S16.
The lack of La in intracellular inclusions, when strain RH AL1 was cultivated with our Ln cocktail, was puzzling and prompted us to determine the Ln content of our medium after adding the Ln cocktail.ICP-MS revealed that the Ln cocktail contained Ce, Nd, Dy, Ho, Er, Yb, but that we erroneously stated that it contains La.We corrected this in the main text, see lines e.g.124-125, describe the preparation of the cocktail in the methods section, see lines 461-470, and added the elemental analysis data, see Table S2.
Additional methods: We added details regarding the carried out ICP-MS analysis to the methods section, see lines 465-467.Regarding RNAseq data processing, we prepared a reproducible data processing workflow, which we reference in the methods part, see lines 571-572, and which is available online (https://github.com/wegnerce/smk_rnaseq).

Major comments:
The manuscript documents the differential impacts of alternative lanthanide rare earth metals upon gene expression within the recently characterized methylotrophic bacterium Beijerinckiaceae bacterium RH AL1.

Lanthanides have recently garnered much interest as an alternative metal enabling methylotrophic metabolism, primarily by serving as a cofactor for XoxF-like dehydrogenase enzymes. The authors observe strong changes in gene expression depending on what lanthanides cells are grown in the presence of, including diverse metabolic processes such as chemotaxis and PHA synthesis. The study is valuable to the growing field of lanthanide-focused biology, as it takes a more in-depth view of the impacts of alternative and single vs. combinatorial lanthanides upon gene expression. In particular, comparisons of how different (utilizable) lanthanides affect metabolism have rarely been done, and strain RH AL1 appears to distinguish between different lanthanide elements.
Thank you for your assessment of the manuscript.You can find our answers to your comments below.
1.The manuscript needs significant rewriting to make it clearer and more impactful.As an example, text descriptions of the groups being compared are confusing at times.The authors should also preface sections in the discussion section.They often dive right into paragraphs with detailed statements without providing the appropriate context, rationale, and bigger picture themes, and need to set these up more carefully.For example, in viewing the paragraph focused on calcium (Lines 289-296), the first sentence is not required, and the paragraph would read better if it started from the second sentence.This section and others ask the reader to connect too many dots rather than stating them more explicitly (or testing them directly in the study).Other paragraphs are rather unfocused: e.g., Lines 348-353 read as just a sequence of facts about sulfur metabolism rather than a cohesive discussion of the potential impacts that lanthanides have upon sulfur metabolism.
We agree that the manuscript lacked in parts clarity and readability, during the revisions we worked on improving both.Please see for instance the reworked sections dedicated to Ca and Ln (lines 350-380) and alkanesulfonates and Ln (lines 423-434) in the discussion, or the partially reworked introduction (lines 102-107).
2. The authors overstate the impact of the findings at times, such as suggesting the presence of different uptake mechanisms for different lanthanides in the abstract, something that is not convincingly broached at all with the experiments performed in this study.On Lines 337-341, the authors mention the upregulation of "heavy metal efflux mechanisms" (should be "genes").This is a trend of expression and sets up a testable hypothesis over the specificity of these chelators, but is not conclusive.There are a few other places where the term "mechanism" is invoked but does not seem to be done so accurately.
We have weakened our statements referring to mechanisms, see for instance lines 388-398, where we no longer state that different Ln elements are deposited by different mechanisms.

3
. The text has a lot of soft, "maybe so", kind of language, most noticeably in the discussion section, which leaves it unclear to the reader how seriously to take some of the statements being made.
You are absolutely right and we would love to have more definitive answers.We modified the text to make it less fuzzy, e.g. the sections about Ca and alkanesulfonates (lines 350-380 + lines 423-434).Our findings raise a lot of questions that we can not immediately answer, but that we are striving to address in follow-up work.One focus is, for instance, intracellular Ln storage and the potential link to PHA metabolism.Working with non-model microorganisms is challenging as a lot of resources and techniques are not easily available.Having, for instance, genetic tools would facilitate testing hypotheses in a (more) straightforward way.So far we lack them, but establishing them is one of our main interests right now.

Figures look nice but are not always referred to correctly/sufficiently. This includes both references in the text as well as the level of description provided in the accompanying figure legends.
We worked on the clarity of the figure legends (see for instance the modified legend of Figure 2) and tried to incorporate figures better in the main text (see for instance lines 298-301 + 350).

Methods:
1. Why do the authors start growth on pyruvate?Are lanthanides required for growth under this condition?This also constitutes a switch (pyruvate-to methanol-based growth) to methylotrophy, which could include related transcriptomic changes that will get lost in this analysis.
We previously reported that Beijerinckiaceae bacterium RH AL1 accumulates lanthanides intracellularly (Wegner et al., 2021; https://doi.org/10.1128/AEM.03144-20)during lanthanide-dependent growth with methanol as the carbon source.Pyruvate was used as the carbon source for the pre-cultures to prevent the carryover of lanthanides, which could impact gene expression.If pre-cultures were grown with methanol and La, our de facto standard conditions, and Nd or the Ln cocktail are added for subsequent incubations, residual La from the inoculum could mask effects caused by Nd or the Ln cocktail.
2. The setup for the transcriptomics is a bit confusing (and is why I suggest including an accompanying figure below).It seems that for this, all treatments should either be a) pregrown in the same lanthanide condition, or b) pregrown in a condition identical to that which it will be transferred into.If neither of these conditions is satisfied, then it feels like the cross-comparison between differently treated samples is problematic.
We have added a figure to the manuscript that explains the cultivation setup, see Figure 1.As outlined above, pyruvate was chosen as the carbon source for pre-cultures to prevent lanthanide carryover.During the incubations for downstream RNAseq analysis, all cultures were grown with methanol as the carbon source.The cultures only differed with respect to Ln supplementation (different La concentration, and different Ln elements added).This is a good point, we added information about environmental concentrations of lanthanides to the main text, see lines 310-315.Please note that it is not easy to link concentrations in the environment (usually given in ppm when dealing with terrestrial systems) with concentrations used in the lab.

Figures:
General: 1. Overall: Great in appearance.However, the manuscript needs more comprehensive descriptions in the figure legends.
The figure legends have been revised to increase readability, see for instance the rewritten legend of Figure 3.
2. It would be helpful to implement a schematic figure explaining how the experiments were set up and carried out for growth and transcriptomics, because the written explanations often don't completely clarify the details of it.
We have added a figure to the manuscript that explains the cultivation setup, see Figure 1.

Specific:
Figure 1: a) It would be much more accurate to target a timeframe within the exponential phase to get maximum td values.b) Why are the doubling times in La rather different between the left and right panels?c) The legend is highly insufficient and doesn't include 1B.d) The redundancy in 1uM La in both panels is confusing.Where did the 1uM La data come from?The cultures from the left panel, or right?a) We have targeted a timeframe within the exponential phase for the calculation of µ and t d .We used time intervals that span at least three measurements.We have revisited our calculations and the determined t d and µ values and corrected them.The growth curve data was added to the supplement, see Table S3.b) We have seen differences of around 10% regarding µ and t d in the past between different incubation runs.c) The legend of the former Figure 1, now Figure 2, was rewritten to make it more understandable.d) The carried out RNAseq-based gene expression analyses were based on two sets of incubations, one with two different La concentrations (50 nM and 1 µM), and one with different lanthanide supplementation.Both sets of incubations included cultures supplemented with 1 µM La.We are not sure what you are referring to when you say 1 µM La data.In Figures 3-5, the 1 µM data (considered for comparison 1) was generated based on samples from the incubation shown in the left panel of now Figure 2.
Figure 2: This figure had a number of aspects about it that made it difficult to follow.From a statistical standpoint, for comparisons like this it makes more sense to have more stringent cutoffs for statistical significance to be assessed.A more common FDR value to use as a significance cutoff is FDR < 0.01.In the figure legend, I think it makes better sense to have A-C labels at the beginning rather than the ends of statements.For panels B and C, it would be clearer to represent the comparisons with word descriptions rather than numbering 1-4.In Figure 2C, it does not make sense to have single comparisons rather than intersections between comparisons.In the case the single comparisons (2 and 3) are represented, why are the unique genes to 1 and 4 also not shown?
We agree, that more stringent FDR cutoffs lead to more robust results.However, an FDR cutoff of < 0.05 is commonly used for RNAseq-based differential gene expression analysis (exemplary studies that look at differential gene expression analysis from a more technical view: https://doi.org/10.1186/s13059-019-1716-1;https://doi.org/10.1002/cpmb.68).One of the few RNAseq studies addressing Ln-dependent gene expression changes (https://doi.org/10.1038/s41598-019-41043-1) in M. extorquens AM1 used an FDR cutoff of 0.15.A-C labels are now placed at the beginning of statements throughout the manuscript.For panels B and C word descriptions have been added in addition to the numbering.Panel C only shows sets of genes > 100, the number of unique genes for 1 and 4 was lower.We clarified this in the revised legend.We agree, the cell illustration provided only little additional value.In the revised manuscript, the former Figure 3 was split into Figure 4 and Figure 8. Figure 4 highlights changes in gene expression with respect to certain functional aspects using ridge plots, Figure 8 uses the cell illustration to summarize our understanding of the impact of Ln on cellular physiology in Beijerinckiaceae bacterium RH AL1.PHB is now first defined in the main text in the context of gene expression changes linked to PHA metabolism, see lines 204-206.

Minor comments:
General: 1. Report methanol and pyruvate concentrations as molar values, for better relevance and consistency with the lanthanide concentrations.
We now report the concentrations as molar values, see e.g.line 122 and lines 451 + 456.

At least once, report the concentration of each single lanthanide compound within the combined cocktail treatment.
We added this information to the manuscript text (lines 467-469) and explain the preparation of the cocktail in the materials and methods section, see lines 461-470.The composition of the cocktail is also given in Table S2.
3. The authors refer to their focal strain in different ways (e.g., "Beijerinckiaceae bacterium RH AL1", "RH AL1", "strain AL1", etc.) throughout the paper.After initial introduction, choose one in order to maintain consistency throughout.
In the revised manuscript we consistently refer to Beijerinckiaceae bacterium RH AL1 or strain RH AL1. 4. In some of the results, the authors report the trend of a comparison, but not the quantitative measure.I would suggest reporting log2 fold changes throughout for consistency and added information to the reader.
We checked that log 2 FC values are given wherever we explicitly refer to gene expression changes.

Unless the authors plan to refer to terms throughout, it might be confusing to define new terms like the lanthanome.
The term "lanthanome" (http://dx.doi.org/10.1021/jacs.8b12155) is not really new anymore, it is meanwhile commonly used in literature.We define it upon the first usage in the revised manuscript, see lines 218-224.
We omitted this sentence in the revised manuscript.
Line 30 -Emphasizing the unknown or hypothetical genes feels like something to bring up later, but not in the abstract.
Figure 3 (formerly Figure 2).Two-component systems are also a rather broad KEGG pathway that overlaps with flagellar assembly and bacterial chemotaxis and includes genes from both.Genes can generally be assigned to multiple pathways.We see that this is misleading and clarify this in the results section, see lines 158-159.We mention now genes coding for the sensor kinases CheA and PleC as examples for distinct two-component system genes (lines 159-161) Chemotaxis and motility are covered in in the discussion, see lines 350-359.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred are not always referred to correctly/sufficiently.This includes both references in the text as well as the level of description provided in the accompanying figure legends.

Figure 3 :
Figure 3: I am unsure that this is accomplishing what it is seeking to.I would potentially separate the central cell illustration from the surrounding data panels into separate figures.Alternatively, it might make sense to provide more color-coding to the central illustrations to have processes align with the figure legend on the side.

Figure 4
Figure4is not referred to directly anywhere in the text.Please adjust and insert appropriate reference points.

Figure 5
Figure5refers to PHB, but the text does not explain that PHB is a specific type of PHA molecule until later in the discussion section.