Bacterial multispecies interaction mechanisms dictate biogeographic arrangement between the oral commensals Corynebacterium matruchotii and Streptococcus mitis

ABSTRACT Polymicrobial biofilms are present in many environments particularly in the human oral cavity where they can prevent or facilitate the onset of disease. While recent advances have provided a clear picture of both the constituents and their biogeographic arrangement, it is still unclear what mechanisms of interaction occur between individual species in close proximity within these communities. In this study, we investigated two mechanisms of interaction between the highly abundant supragingival plaque (SUPP) commensal Corynebacterium matruchotii and Streptococcus mitis which are directly adjacent/attached in vivo. We discovered that C. matruchotii enhanced the fitness of streptococci dependent on its ability to detoxify streptococcal-produced hydrogen peroxide and its ability to oxidize lactate also produced by streptococci. We demonstrate that the fitness of adjacent streptococci was linked to that of C. matruchotii and that these mechanisms support the previously described “corncob” arrangement between these species but that this is favorable only in aerobic conditions. Furthermore, we utilized scanning electrochemical microscopy to quantify lactate production and consumption between individual bacterial cells for the first time, revealing that lactate oxidation provides a fitness benefit to S. mitis not due to pH mitigation. This study describes mechanistic interactions between two highly abundant human commensals that can explain their observed in vivo spatial arrangements and suggest a way by which they may help preserve a healthy oral bacterial community. IMPORTANCE As the microbiome era matures, the need for mechanistic interaction data between species is crucial to understand how stable microbiomes are preserved, especially in healthy conditions where the microbiota could help resist opportunistic or exogenous pathogens. Here we reveal multiple mechanisms of interaction between two commensals that dictate their biogeographic relationship to each other in previously described structures in human supragingival plaque. Using a novel variation for chemical detection, we observed metabolite exchange between individual bacterial cells in real time validating the ability of these organisms to carry out metabolic crossfeeding at distal and temporal scales observed in vivo. These findings reveal one way by which these interactions are both favorable to the interacting commensals and potentially the host.

The manuscript describes a dual species interaction between two prominent and relevant members of the oral biofilm, S. mitis and C. matruchotii.The authors demonstrate that the metabolic exchange between both species is a benefitting factor in this dual species community.S. mitis is a producer of lactate and hydrogen peroxide, and C. matruchotti is able to metabolize lactate and detoxify hydrogen peroxide, thus counteracting the self inhibitory effect of hydrogen peroxide on S. mitis.Overall this manuscript is well written and highly relevant to oral biofilm development and provides a mechanistic explanation of species distribution.The usage of SECM to demonstrate metabolite exchange is highly innovative.

I only have minor comments:
For Fig. 5, it is not clear how representative this image is.Was this done multiple times?Is there some kind of quantification available?
It would be nice to include a bigger picture in the discussion, including some of the other species that are known to metabolize lactic acid or detoxify hydrogen peroxide.In this manuscript, Almeida and Puri et al. were interesting in characterizing metabolite-mediated interactions between the common oral commensal bacteria Corynebacterium matruchotii and Streptococcus mitis.To do so, the authors use a combination of traditional bacterial coculture and genetics, as well as a novel method for directly visualizing metabolic exchange between cells that they call scanning electrochemical microscopy (SCEM).Together, Almeida and Puri determine that C. matruchotii is able to support to growth of S. mitis by detoxifying produced hydrogen peroxide and consuming produced lactate.The culture experiments are sound, but I have questions regarding the genetics and SCEM methods.
Major Comments 1.The authors generate numerous mutants in both C. matruchotii and S. mitis throughout this manuscript, but they never complement the mutations in trans to confirm that their deletions are specifically responsible for the effects that they observe.2. How are the authors sure that their SCEM method is specifically measuring L-lactate concentrations?For instance, have the authors repeated their SCEM experiment using a S. mitis Δldh mutant?What about addition of an LDH inhibitor like oxamic acid?Alternatively, what happens if the authors image a coculture of S. mitis and a C. matruchotii mutant deleted lutABC and the two potentially reversible lactate dehydrogenase genes?3. Much of the Supplemental Results could be incorporated into the main manuscript text and would better inform readers as to how the SCEM method works Minor Comments L136: Why were these cutoffs chosen for the RNA seq experiment?Can the authors provide the corresponding P-values in Table S2 and S3? L161, 174, 196-7: The authors state "data not shown" in several instances.L242-63: Much of this text belongs in the methods section L317-9: If the transcriptional responses of S. mitis to C. matruchotii are part of a separate study, why are they included as Table S3?
Figure 2, 3: Include details in the legends indicating that these experiments were under aerobic conditions Figure 5: Can the authors report the data in panels B and C with respect to measured L-lactate concentration? Figure S2: The legend indicates that this experiment was not replicated (i.e., N=1).The authors should replicate this experiment before reporting the results at L170.Line Comments L36-7: What is meant by "directly adjacent in vivo"?L44,157,272: Replace "1st" with "first" L51: Be more clear about what "they" refers to in this sentence L77,81: Avoid opening sentences with "It".Be more clear was to what "it" is referring to L80: replace "with the" with "where the" L80: replace "being" with "it" L86-7: What is the "hedgehog" model?Why is it relevant for this work?L91: Spell out hydrogen peroxide fully during its first use L93: Sentence has two instances of "which can" L98, 136, 141, 171, 181, 391, 321, 332, 376, 393, 415: If a species name starts a sentence, then the genus should be written out fully L100: add "-mediated" after Corynebacterium L102,247-8: Enclose the i.e. statement in parentheses L105-8: Recommend splitting into two sentences L122: Would suggest including a preamble sentence before jumping into results L130-1: Would move this sentence to the start of the following paragraph L137-41: Would suggest splitting into two sentences L184: Replace "data anaerobically" with "anaerobic data" L195: Would suggest referencing Table S2 here L203: What is "it" referring to?L217: add "hypothesis" after "to test this..." L218,219,221,222,224: The "WT" abbreviation has been defined L310: add "led to" before "upregulation" L361: What is "(KIM REF+)"?L384-6: This sentence is repeated from L376-9 P411: Several instances of "will be" in this section To the editor and reviewers: Please note that all responses to the reviewers are here in red and original review comments in black.We thank both reviewers for their requests and comments and we appreciate their time in reviewing and helping to improve this manuscript.We outline below our attempts to address all issues raised by writing and further experimentation.We would like to note that our companion manuscript regarding more of the operational details of the SECM technology used here has been accepted and now in press (DOI: 10.1021/acs.analchem.3c01498).Also, all line numbers used in the response below refer to the original main draft "marked up" copy which is a track changes copy showing where text has been modified in response to reviewer comments.We again thank the reviewers and apologize if we were unable to completely satisfy every request given.We have done our best to explain any circumstances as to why we chose to present some information in the format here.

Reviewer #1 (Comments for the Author):
The manuscript describes a dual species interaction between two prominent and relevant members of the oral biofilm, S. mitis and C. matruchotii.The authors demonstrate that the metabolic exchange between both species is a benefitting factor in this dual species community.S. mitis is a producer of lactate and hydrogen peroxide, and C. matruchotti is able to metabolize lactate and detoxify hydrogen peroxide, thus counteracting the self inhibitory effect of hydrogen peroxide on S. mitis.Overall this manuscript is well written and highly relevant to oral biofilm development and provides a mechanistic explanation of species distribution.The usage of SECM to demonstrate metabolite exchange is highly innovative.

I only have minor comments:
For Fig. 5, it is not clear how representative this image is.Was this done multiple times?Is there some kind of quantification available?Similar SECM images were reproducibly obtained over >3 coculture samples, where higher current over S. mitis and lower current over C. matruchotii than the background current indicate in situ production and consumption of lactate, respectively.In the main text (in page 7, line 254~275) and supporting information (in page S4 and Fig S3 in page S7), we mentioned that single S. mitis cells produce lactate up to 0.5 mM estimated by current in Fig 5C, theoretically simulated current-distance curves, and theoretically simulated concentration profile in Fig S3E and S3F.At the same time, C. matruchotii rapidly consumes lactate produced by S. mitis at a rate of ≥ 5 × 10 6 s -1 , thereby decreasing the local concentration of lactate up to 0.1 mM over C. matruchotii.More detailed information and rigorous discussion about the theoretical simulation and the quantitative analysis should be found in the companion manuscript now accepted in Analytical Chemistry (10.1021/acs.analchem.3c01498)(submission copies provided to Editor).In the companion manuscript, we proved that only S. mitis produces lactate, while C. matruchotii doesn't produce any lactate in each monoculture study.Single cells of S. mitis in monoculture produce similar level of lactate to coculture sample in below Fig S2B and S2C (companion manuscript).In contrast, a single cell of C. matruchotii in monoculture does not produce any lactate, thus nothing appeared in the SECM image based on lactate ion transfer (Fig S3B,companion manuscript).Herein, we share the additional SECM data (Fig S2 and S3) from the submitted companion manuscript as below for review purposes.It would be nice to include a bigger picture in the discussion, including some of the other species that are known to metabolize lactic acid or detoxify hydrogen peroxide.
We thank the reviewer for this suggestion.Further discussion of peroxide production and lactate metabolism are included in lines 307-319.

Supplemental files:
page 4 line 147: should it read probe instead of problem?
We thank the reviewer for their careful attention!It should be a two phase "problem" and we have corrected this error.Using COMSOL Multiphysics, we solved a diffusion problem of lactate between two phases of aqueous bulk phase and a single bacterium with laterally homogeneous membrane having a uniform permeability.

Fig. S2...growth curve should be in log-scale
We thank the reviewer for this correction, as part of other reviewer comments these data have now been replaced by table S1 in the supplement.

Reviewer #2 (Comments for the Author):
In this manuscript, Almeida and Puri et al. were interesting in characterizing metabolite-mediated interactions between the common oral commensal bacteria Corynebacterium matruchotii and Streptococcus mitis.To do so, the authors use a combination of traditional bacterial coculture and genetics, as well as a novel method for directly visualizing metabolic exchange between cells that they call scanning electrochemical microscopy (SCEM).Together, Almeida and Puri determine that C. matruchotii is able to support to growth of S. mitis by detoxifying produced hydrogen peroxide and consuming produced lactate.The culture experiments are sound, but I have questions regarding the genetics and SCEM methods.

Major Comments
1.The authors generate numerous mutants in both C. matruchotii and S. mitis throughout this manuscript, but they never complement the mutations in trans to confirm that their deletions are specifically responsible for the effects that they observe.
We have now generated and tested an in trans complement of lutABC to the original ΔlutABC C. matruchotii mutant.We verified here that the loss of growth rate in the lutABC mutant was complemented by addition of the lutABC operon in trans vs an empty vector ΔlutABC C. matruchotii.See lines 178-9, Materials and Methods and table S1.Also, please note the differences in growth rates from previous experiments were due to performing the new rate measurements in a different culture vessel type than previously.
The role of SpxB in H2O2 generation in S. mitis and other species has been previously well described which we discuss and provide further citations in response to reviewer 1 comments on lines 307-319.Likewise, complementation of spxB and restoration of H2O2 production in S. mitis has been previously described (https://www.nature.com/articles/s41598-021-04562-4#Sec2).We demonstrate here that removal of peroxide by exogenous catalase yielded a phenotypically similar result to that of the ΔspxB S. mitis (Fig. 3).
2. How are the authors sure that their SCEM method is specifically measuring L-lactate concentrations?For instance, have the authors repeated their SCEM experiment using a S. mitis Δldh mutant?What about addition of an LDH inhibitor like oxamic acid?Alternatively, what happens if the authors image a coculture of S. mitis and a C. matruchotii mutant deleted lutABC and the two potentially reversible lactate dehydrogenase genes?
We independently measured the lactate ion transfer in aqueous bulk solution containing 2 mM lactate in Fig S3B , where lactate ion transfer is induced at ~0.42 V more positive than E 1/2 of TEA+ ion transfer.The limiting currents for lactate ion transfer respond to the lactate concentration linearly as well.This bulk electrochemistry confirmed the selective potential for lactate ion transfer across the pipet tip interface and confirms that we are detecting lactate.
Once we defined the potential for lactate ion transfer under nearly diffusion-limited condition in the bulk solution, we applied the same potential to the tip during raster-scanning above co-cultured bacteria.In the companion manuscript now in print at Analytical Chemistry (DOI 10.1021/acs.analchem.3c01498)(and provided to the editor), we demonstrated that only S. mitis produces lactate, while C. matruchotii does not.Single cells of S. mitis in monoculture produce similar level of lactate to coculture sample in Fig S2B and S2C (companion manuscript).In contrast, single cells of C. matruchotii in monoculture do not produce any lactate, thus nothing appeared in the SECM image based on lactate ion transfer (Fig S3B,companion manuscript).The relevant companion manuscript figures have been included above in replies to reviewer 1 above.
Regarding an ldh mutant given that we had already verified our ability to detect lactate specifically in sterile medium with and without lactate added, we did not see a rationale for inhibiting ldh after the fact.Further, given that Streptococcal metabolism is highly dependent on glycolysis and that they do not respire, the likelihood of this mutant surviving much less behaving reproducibly in our SECM settings was unlikely thus it was not attempted.Lastly, we wish to note that we cannot differentiate between L or D lactate with SECM but that C. matruchotii does not appear to possess a lactate racemase nor Dlactate dehydrogenase.
3. Much of the Supplemental Results could be incorporated into the main manuscript text and would better inform readers as to how the SCEM method works We thank the reviewer and agree with this comment.However, given the extensive nature of SECM work included it was already necessary to publish this in a second manuscript given the size constrains and technical nature of the SECM developments utilized.We have modified some of the included text on SECM results to hopefully help be more informative.

Minor Comments
L136: Why were these cutoffs chosen for the RNA seq experiment?Can the authors provide the corresponding P-values in Table S2 and S3?The 2-fold cutoff is arbitrary but few genes are significantly differentially regulated below this value.Also, any phenotypic results for <2-fold changes in expression would be difficult to reproduce and outside of a few cases be of the lowest priority for further investigation.Our raw data and analysis methods are available for readers that would wish to investigate differentially expressed genes that fall below this cutoff.Table S3 has been removed based on reviewer comments below.L161, 174, 196-7: The authors state "data not shown" in several instances.
For L174 we have modified this section with new data.L161 refers to qualitative data regarding a visible color change (red to yellow) which in our opinion did not justify the space required for another figure.L196-7 was not shown because the anaerobic RNASeq dataset for C. matruchotii is part of a separate ongoing investigation and catalase expression in that dataset would be a singular "0" value only.We opted to leave this out due to space and scope constraints that have already led us to split the work into two separate manuscripts.This data is also supported by the fact that the catalase mutant could not even be generated or grown aerobically, yet was made in our hands in anaerobic conditions and can grow anaerobically.

L242-63: Much of this text belongs in the methods section
We thank the reviewer for this suggestion.However, this section describes observational data recorded during SECM data collection for TEA+ and lactate ion profiles.Also, given the above major suggestion (#3) by the reviewer we are a bit conflicted on removing this given that it helps explain the SECM technique and could be contrary to the reviewer's wishes above.We have edited some of the text within the indicated areas and in the SECM section in general to help with clarity.L317-9: If the transcriptional responses of S. mitis to C. matruchotii are part of a separate study, why are they included as Table S3?
We felt that these data would be of interest to the readers as it was acquired during the same RNASeq experiment as that from the C. matruchotii side.Given that we do not explore S. mitis responses to coculture conditions in this manuscript and to address this reviewer comment we have now removed these data.We appreciate the reviewers suggestion.We have updated the Fig. 5 legend to address this.
In the SECM image of Fig 5B, tip currents are dependent on not only TEA+ concentration in the bulk solution, but also a distance between a pipet tip and a glass substrate or a bacteria, and the permeability of bacterial membrane.The main reason for tip current changes over a bacteria is due to the hindered diffusion of TEA+ to the tip within a distance between a tip and nearly impermeable bacterial membrane, while TEA+ concentration exists almost uniformly outside of a bacteria.The conversion of tip current to TEA+ concentration is not feasible in this SECM image due to the non-linear relationship between the tip current and concentration of TEA+.Fig 5B is the SECM image probing only TEA+ not Llactate.
In the SECM image of Fig 5C, the tip current is dependent on not only lactate concentration in bulk solution, but also a distance between a pipet tip and a glass substrate or a bacteria, the lactate concentration produced by S. mitis, and the rate of lactate consumption at C. matruchotii.Due to these multi-determining factors for tip currents and the non-linear relationship between the tip current and lactate concentration, the current scale in the SECM image cannot be straightforwardly converted to lactate concentration.This is why we do Finite Element Methods using COMSOL Multiphysics to solve a diffusion problem of lactate near a bacterium, and theoretically simulate the tip current-distance between a tip and a bacteria, and the lactate concentration profile over a single bacteria to accurately extract the information of lactate concentration above a single bacteria (Fig S2E and S2F Figure S2: The legend indicates that this experiment was not replicated (i.e., N=1).The authors should replicate this experiment before reporting the results at L170.This experiment has been updated with the now complemented lutABC mutant strain and replaced by table S1.

Line Comments
We thank the reviewer for these beneficial comments, unless directly indicated below, all changes were addressed at the relevant lines and changes can be observed in the track-changes manuscript copy provided for the main draft.L36-7: What is meant by "directly adjacent in vivo"?L44,157,272: Replace "1st" with "first" L51: Be more clear about what "they" refers to in this sentence L77,81: Avoid opening sentences with "It".Be more clear was to what "it" is referring to L80: replace "with the" with "where the" L80: replace "being" with "it" L86-7: What is the "hedgehog" model?Why is it relevant for this work?
The citation for the model is provided upon its mention in the text.The model is the 1 st description of the biogeography of C. matruchotii in supragingival plaque and details how it has numerous streptococci attached directly to it which was the initial inspiration for studying these polymicrobial interactions further.
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Editor comments:
The work to complement the lutA-strain is appreciated.Please move table S1 to the main paper and revise accordingly.

To the editor and reviewers:
Please note that all responses to the reviewers are here in red and original review comments in black.We thank both reviewers for their requests and comments and we appreciate their time in reviewing and helping to improve this manuscript.We outline below our attempts to address all issues raised by writing and further experimentation.We would like to note that our companion manuscript regarding more of the operational details of the SECM technology used here has been accepted and now in press (DOI: 10.1021/acs.analchem.3c01498).Also, all line numbers used in the response below refer to the original main draft "marked up" copy which is a track changes copy showing where text has been modified in response to reviewer comments.We again thank the reviewers and apologize if we were unable to completely satisfy every request given.We have done our best to explain any circumstances as to why we chose to present some information in the format here.

Reviewer #1 (Comments for the Author):
The manuscript describes a dual species interaction between two prominent and relevant members of the oral biofilm, S. mitis and C. matruchotii.The authors demonstrate that the metabolic exchange between both species is a benefitting factor in this dual species community.S. mitis is a producer of lactate and hydrogen peroxide, and C. matruchotti is able to metabolize lactate and detoxify hydrogen peroxide, thus counteracting the self inhibitory effect of hydrogen peroxide on S. mitis.Overall this manuscript is well written and highly relevant to oral biofilm development and provides a mechanistic explanation of species distribution.The usage of SECM to demonstrate metabolite exchange is highly innovative.

I only have minor comments:
For Fig. 5, it is not clear how representative this image is.Was this done multiple times?Is there some kind of quantification available?Similar SECM images were reproducibly obtained over >3 coculture samples, where higher current over S. mitis and lower current over C. matruchotii than the background current indicate in situ production and consumption of lactate, respectively.In the main text (in page 7, line 254~275) and supporting information (in page S4 and Fig S3 in page S7), we mentioned that single S. mitis cells produce lactate up to 0.5 mM estimated by current in Fig 5C, theoretically simulated current-distance curves, and theoretically simulated concentration profile in Fig S3E and S3F.At the same time, C. matruchotii rapidly consumes lactate produced by S. mitis at a rate of ≥ 5 × 10 6 s -1 , thereby decreasing the local concentration of lactate up to 0.1 mM over C. matruchotii.More detailed information and rigorous discussion about the theoretical simulation and the quantitative analysis should be found in the companion manuscript now accepted in Analytical Chemistry (10.1021/acs.analchem.3c01498)(submission copies provided to Editor).In the companion manuscript, we proved that only S. mitis produces lactate, while C. matruchotii doesn't produce any lactate in each monoculture study.Single cells of S. mitis in monoculture produce similar level of lactate to coculture sample in below Fig S2B and S2C (companion manuscript).In contrast, a single cell of C. matruchotii in monoculture does not produce any lactate, thus nothing appeared in the SECM image based on lactate ion transfer (Fig S3B,companion manuscript).Herein, we share the additional SECM data (Fig S2 and S3) from the submitted companion manuscript as below for review purposes.
It would be nice to include a bigger picture in the discussion, including some of the other species that are known to metabolize lactic acid or detoxify hydrogen peroxide.
We thank the reviewer for this suggestion.Further discussion of peroxide production and lactate metabolism are included in lines 307-319.

Supplemental files:
page 4 line 147: should it read probe instead of problem?
We thank the reviewer for their careful attention!It should be a two phase "problem" and we have corrected this error.Using COMSOL Multiphysics, we solved a diffusion problem of lactate between two phases of aqueous bulk phase and a single bacterium with laterally homogeneous membrane having a uniform permeability.We thank the reviewer for this correction, as part of other reviewer comments these data have now been replaced by table S1 in the supplement.

Reviewer #2 (Comments for the Author):
In this manuscript, Almeida and Puri et al. were interesting in characterizing metabolite-mediated interactions between the common oral commensal bacteria Corynebacterium matruchotii and Streptococcus mitis.To do so, the authors use a combination of traditional bacterial coculture and genetics, as well as a novel method for directly visualizing metabolic exchange between cells that they call scanning electrochemical microscopy (SCEM).Together, Almeida and Puri determine that C. matruchotii is able to support to growth of S. mitis by detoxifying produced hydrogen peroxide and consuming produced lactate.The culture experiments are sound, but I have questions regarding the genetics and SCEM methods.

Major Comments
1.The authors generate numerous mutants in both C. matruchotii and S. mitis throughout this manuscript, but they never complement the mutations in trans to confirm that their deletions are specifically responsible for the effects that they observe.
We have now generated and tested an in trans complement of lutABC to the original ΔlutABC C. matruchotii mutant.We verified here that the loss of growth rate in the lutABC mutant was complemented by addition of the lutABC operon in trans vs an empty vector ΔlutABC C. matruchotii.See lines 178-9, Materials and Methods and table S1.Also, please note the differences in growth rates from previous experiments were due to performing the new rate measurements in a different culture vessel type than previously.
The role of SpxB in H2O2 generation in S. mitis and other species has been previously well described which we discuss and provide further citations in response to reviewer 1 comments on lines 307-319.Likewise, complementation of spxB and restoration of H2O2 production in S. mitis has been previously described (https://www.nature.com/articles/s41598-021-04562-4#Sec2).We demonstrate here that removal of peroxide by exogenous catalase yielded a phenotypically similar result to that of the ΔspxB S. mitis (Fig. 3).
2. How are the authors sure that their SCEM method is specifically measuring L-lactate concentrations?For instance, have the authors repeated their SCEM experiment using a S. mitis Δldh mutant?What about addition of an LDH inhibitor like oxamic acid?Alternatively, what happens if the authors image a coculture of S. mitis and a C. matruchotii mutant deleted lutABC and the two potentially reversible lactate dehydrogenase genes?
We independently measured the lactate ion transfer in aqueous bulk solution containing 2 mM lactate in Fig S3B , where lactate ion transfer is induced at ~0.42 V more positive than E 1/2 of TEA+ ion transfer.The limiting currents for lactate ion transfer respond to the lactate concentration linearly as well.This bulk electrochemistry confirmed the selective potential for lactate ion transfer across the pipet tip interface and confirms that we are detecting lactate.
Once we defined the potential for lactate ion transfer under nearly diffusion-limited condition in the bulk solution, we applied the same potential to the tip during raster-scanning above co-cultured bacteria.In the companion manuscript now in print at Analytical Chemistry (DOI 10.1021/acs.analchem.3c01498)(and provided to the editor), we demonstrated that only S. mitis produces lactate, while C. matruchotii does not.Single cells of S. mitis in monoculture produce similar level of lactate to coculture sample in Fig S2B and S2C (companion manuscript).In contrast, single cells of C. matruchotii in monoculture do not produce any lactate, thus nothing appeared in the SECM image based on lactate ion transfer (Fig S3B,companion manuscript).The relevant companion manuscript figures have been included above in replies to reviewer 1 above.
Regarding an ldh mutant given that we had already verified our ability to detect lactate specifically in sterile medium with and without lactate added, we did not see a rationale for inhibiting ldh after the fact.Further, given that Streptococcal metabolism is highly dependent on glycolysis and that they do not respire, the likelihood of this mutant surviving much less behaving reproducibly in our SECM settings was unlikely thus it was not attempted.Lastly, we wish to note that we cannot differentiate between L or D lactate with SECM but that C. matruchotii does not appear to possess a lactate racemase nor Dlactate dehydrogenase.
3. Much of the Supplemental Results could be incorporated into the main manuscript text and would better inform readers as to how the SCEM method works We thank the reviewer and agree with this comment.However, given the extensive nature of SECM work included it was already necessary to publish this in a second manuscript given the size constrains and technical nature of the SECM developments utilized.We have modified some of the included text on SECM results to hopefully help be more informative.

Minor Comments
L136: Why were these cutoffs chosen for the RNA seq experiment?Can the authors provide the corresponding P-values in Table S2 and S3?The 2-fold cutoff is arbitrary but few genes are significantly differentially regulated below this value.Also, any phenotypic results for <2-fold changes in expression would be difficult to reproduce and outside of a few cases be of the lowest priority for further investigation.Our raw data and analysis methods are available for readers that would wish to investigate differentially expressed genes that fall below this cutoff.Table S3 has been removed based on reviewer comments below.L161, 174, 196-7: The authors state "data not shown" in several instances.
For L174 we have modified this section with new data.L161 refers to qualitative data regarding a visible color change (red to yellow) which in our opinion did not justify the space required for another figure.L196-7 was not shown because the anaerobic RNASeq dataset for C. matruchotii is part of a separate ongoing investigation and catalase expression in that dataset would be a singular "0" value only.We opted to leave this out due to space and scope constraints that have already led us to split the work into two separate manuscripts.This data is also supported by the fact that the catalase mutant could not even be generated or grown aerobically, yet was made in our hands in anaerobic conditions and can grow anaerobically.

L242-63: Much of this text belongs in the methods section
We thank the reviewer for this suggestion.However, this section describes observational data recorded during SECM data collection for TEA+ and lactate ion profiles.Also, given the above major suggestion (#3) by the reviewer we are a bit conflicted on removing this given that it helps explain the SECM technique and could be contrary to the reviewer's wishes above.We have edited some of the text within the indicated areas and in the SECM section in general to help with clarity.L317-9: If the transcriptional responses of S. mitis to C. matruchotii are part of a separate study, why are they included as Table S3?
We felt that these data would be of interest to the readers as it was acquired during the same RNASeq experiment as that from the C. matruchotii side.Given that we do not explore S. mitis responses to coculture conditions in this manuscript and to address this reviewer comment we have now removed these data.We appreciate the reviewers suggestion.We have updated the Fig. 5 legend to address this.
In the SECM image of Fig 5B, tip currents are dependent on not only TEA+ concentration in the bulk solution, but also a distance between a pipet tip and a glass substrate or a bacteria, and the permeability of bacterial membrane.The main reason for tip current changes over a bacteria is due to the hindered diffusion of TEA+ to the tip within a distance between a tip and nearly impermeable bacterial membrane, while TEA+ concentration exists almost uniformly outside of a bacteria.The conversion of tip current to TEA+ concentration is not feasible in this SECM image due to the non-linear relationship between the tip current and concentration of TEA+.In the SECM image of Fig 5C, the tip current is dependent on not only lactate concentration in bulk solution, but also a distance between a pipet tip and a glass substrate or a bacteria, the lactate concentration produced by S. mitis, and the rate of lactate consumption at C. matruchotii.Due to these multi-determining factors for tip currents and the non-linear relationship between the tip current and lactate concentration, the current scale in the SECM image cannot be straightforwardly converted to lactate concentration.This is why we do Finite Element Methods using COMSOL Multiphysics to solve a diffusion problem of lactate near a bacterium, and theoretically simulate the tip current-distance between a tip and a bacteria, and the lactate concentration profile over a single bacteria to accurately extract the information of lactate concentration above a single bacteria (Fig S2E and S2F  This experiment has been updated with the now complemented lutABC mutant strain and replaced by table S1.

Figure S1 .
Figure S1.SECM images of S. mitis monoculture based on (A) TEA + IT for topography and (B) lactate IT for lactate mapping.A tip scan rate at 100 nm/100 ms during SECM imaging.(C) chronoamperometric responses based on lactate IT (raw data, cross sections of SECM images in (B)).The current polarity is set to negative for anionic current responses.

Figure S2 .
Figure S2.SECM images of C. matruchotii monoculture based on (A) TEA + IT for topography and (B) lactate IT for lactate mapping.A tip scan rate at 100 nm/100 ms during SECM imaging.

Figure 2 , 3 :
Figure 2, 3: Include details in the legends indicating that these experiments were under aerobic conditions Done, we thank the reviewer for this important clarification.

Figure 5 :
Figure 5: Can the authors report the data in panels B and C with respect to measured L-lactate concentration?
) the lactate ranges are reported in the Fig 5 legend as ~0.5mM above the S. mitis surface and ~0.1mM above the C. matruchotii surface.

Fig
Fig. S2...growth curve should be in log-scale

Figure 2 , 3 :
Figure 2, 3: Include details in the legends indicating that these experiments were under aerobic conditions Done, we thank the reviewer for this important clarification.

Figure 5 :
Figure 5: Can the authors report the data in panels B and C with respect to measured L-lactate concentration?
Fig 5B is the SECM image probing only TEA+ not Llactate.
) the lactate ranges are reported in the Fig 5 legend as ~0.5mM above the S. mitis surface and ~0.1mM above the C. matruchotii surface.

Figure S2 :
Figure S2: The legend indicates that this experiment was not replicated (i.e., N=1).The authors should replicate this experiment before reporting the results at L170.