Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels

The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.


Comments to the Author(s)
General: Wording in the introduction could be improved to improve readability and provide more details. Overall descriptions of the methodology seem very thorough but the authors should consider moving mesh size calculations to the methods. It seems that the only time period assessed in the paper was three hours which is fine for assessing initial adhesion but doesn't necessarily cover any potential changes over a longer timescale; the justification for selecting 3 hours should be noted. Overall, presentation of the results is nice but could use a few minor improvements for either clarification or readability. In both presentation of results and the discussion of those results, there is a heavy statistical overtone. Clarification of some statistical terms would be appreciated and perhaps the usage of some of those statistics to reach conclusions is a bit overreaching. However, to the credit of the authors, they acknowledge this to some extent in certain sections of the paper.
Specific: • P3, Line 21-22-Technically speaking biofilms aren't responsible for infection, maybe rephrase to "biofilms are found in" or "biofilms comprise of" • P3, Line 46-48-Maybe here it might be worth mentioning the coatings that have been designed to release antibacterial agents/etc. While they may technically address pre-attachment and attachment itself, they can technically still be released during (or after) maturation. • P4, Line 22-Is c-di-GMP is important for biological things besides biofilm formation. Could the selection and relevance of c-di-GMP be further explained? • P4, Line 31-Please provide a better justification/explanation for why PEGDA is an ideal material. The reviewer assumes because it is transparent but this (or other rationales) should be stated • Why was DMA used to measure the compressive modulus? Was rheology used as a comparison? How does the compressive modulus compare to an Young's modulus? Could you please put your mechanical data into context with published literature. • P6, Line 20-Could the authors please clarify if the PAO1 pseudomonas is that a clinical strain or more of a "lab strain" • P9 Line 53-54-Why were two different PEG precursors used in this work? Could this be further clarified please. What was the effect on hydrogel mesh size, crosslinking ratio, surface/bulk chemistry. It is unclear why the same MW was not used to minimize potential differences between gels. The reviewer notes that the authors discuss mesh size later on in the manuscript, but it would be helpful to introduce this concept earlier. • It is unclear why the methods for calculating mesh size aren't provided in the methods section. There is no need to detail this in the results section. • The authors should compare their calculated mesh size to that in literature. • Figure 1 would be improved if both samples were provided on the same axis scale (log scale perhaps) to show the relationship between the two samples. • Figure 2-Part b could be improved via a better caption to clarify the difference between WT reporter and WT Control and why they are on opposite sides of the graph. • Figure 3-An explanation of kurtosis could prove useful to readers that aren't as versed in the jargon of statistics. • P14 Line 29-30-"To examine how heterogeneous response might interplay with • substrate stiffness, we measured the skewness and kurtosis of all attenuation-and • metabolism-corrected brightness distributions -skewness to determine • asymmetry and kurtosis to determine the preponderance of outliers (58). We • found that, for all timepoints, the skewness and kurtosis were higher for • populations attached to the softer hydrogel than for those attached to the stiffer • hydrogel (Fig. 3 B and C). This showed that attachment to the softer gel was • associated with more outliers and therefore, a more distinct subpopulation of • "strong responders". This statement is hard to understand/justify. The reviewer feels like a definitive conclusion to this issue can only be reached by looking at the genetics and not statistics alone. Could the authors please explain this further?

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
In this manuscript, Blacutt et al. quantitatively characterized mechanobiological responses of P. aeruginosa on the stiffness of substrates for biofilm formation, using PEGDA hydrogels of higher and lower stiffness and confocal imaging. For rigorous fluorescent imaging-based evaluation, they calibrated attenuation of emitted fluorescent light through hydrogels and baseline metabolism of the tested cells. The authors' results clearly show differences in the fluorescence signal between the hard and soft hydrogel cases. They repeated their experiment using the bacteria strain not producing the envelope protein, and discussed a possibility for the bacteria cell could sense differences in pore size in addition to stiffness. The manuscript is well organized, and results are well presented. The conclusion is well supported by the presented data. I suggest minor revision with the following questions and suggestions.
1. Line 39-41 in Page 1: It would be more rigorous to indicate that E values are approximate values by using ~.
2. What is the gel thickness for bacteria culture? The thickness of hydrogel samples for swelling and stiffness measurement is shown clearly, but the gel thickness for bacteria culture is not easily found. It would be informative to clearly show the gel thickness of this case, because cells are known to sense the stiffness of the substrate or container below the gel (i.e., effective stiffness of hydrogel).
3. For the compressive modulus measurement part, it is recommended to include examples of the measured stress-strain curves in the SI or in Figure 1 (as insets) to indicate the strain range used for the modulus measurement. 4. One hour was allowed for bacteria to adhere to hydrogel, and I wonder how this time duration was determined. Could imaging be done 0.5 hour after the inoculation of hydrogels? 5. The last line in Page 8: How many cells were typically imaged at each time point? Since probability distribution curves are shown in the manuscript, providing this number (rough estimate or range) seems required for supporting the reliability of the curves. 2A & 4A: It would be informative to provide the time point and gel type (hard or soft) for the shown image. 7. Figure 2C: I just wonder why the curves are not symmetric and why they do not collapse even after correction. Are these due to any possible limitations of the authors' method? Then, don't they have to be considered in analyzing images of bacteria? 8. How was the attenuation factor of 0.662 shown in Page 12 determined? Is it just a ratio of the mean values? 9. Figure 3A: The brightness level of the soft gel case became higher than that of the hard gel between ~140 min and 200 min. What does this mean?

Figures
10. Line 38-45 in Page 14: What are "strong responders" exactly? Are they related to increased cdi-GMP concentration ("strong" suggests this correlation)? If so, is more strong responders on the soft gel contradict to the higher population of bacteria with increased c-di-GMP concentration? I felt this part is confusing. In this sense, I wonder whether the attenuation factor shown in Page 12 was measured repeatedly and whether the authors got similar values as 0.662. This is also important because similar attenuation factor values obtained from repeated experiments would show that the authors' method is repeatable.
Also, I wonder how repeatable the average fluorescence intensity of control bacteria was. If this were not repeatable (similar between repeated experiments), the authors' way of removing the baseline metabolism level could be problematic.

Decision letter (RSOS-201453.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Gordon
On behalf of the Editors, we are pleased to inform you that your Manuscript RSOS-201453 "Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signaling by bacteria on biomedical hydrogels" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referees' reports. Please find the referees' comments along with any feedback from the Editors below my signature.
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Thank you for submitting your manuscript to Royal Society Open Science and we look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Wording in the introduction could be improved to improve readability and provide more details. Overall descriptions of the methodology seem very thorough but the authors should consider moving mesh size calculations to the methods. It seems that the only time period assessed in the paper was three hours which is fine for assessing initial adhesion but doesn't necessarily cover any potential changes over a longer timescale; the justification for selecting 3 hours should be noted. Overall, presentation of the results is nice but could use a few minor improvements for either clarification or readability. In both presentation of results and the discussion of those results, there is a heavy statistical overtone. Clarification of some statistical terms would be appreciated and perhaps the usage of some of those statistics to reach conclusions is a bit overreaching. However, to the credit of the authors, they acknowledge this to some extent in certain sections of the paper.
Specific: • P3, Line 21-22-Technically speaking biofilms aren't responsible for infection, maybe rephrase to "biofilms are found in" or "biofilms comprise of" • P3, Line 46-48-Maybe here it might be worth mentioning the coatings that have been designed to release antibacterial agents/etc. While they may technically address pre-attachment and attachment itself, they can technically still be released during (or after) maturation. • P4, Line 22-Is c-di-GMP is important for biological things besides biofilm formation. Could the selection and relevance of c-di-GMP be further explained? • P4, Line 31-Please provide a better justification/explanation for why PEGDA is an ideal material. The reviewer assumes because it is transparent but this (or other rationales) should be stated • Why was DMA used to measure the compressive modulus? Was rheology used as a comparison? How does the compressive modulus compare to an Young's modulus? Could you please put your mechanical data into context with published literature.
• P6, Line 20-Could the authors please clarify if the PAO1 pseudomonas is that a clinical strain or more of a "lab strain" • P9 Line 53-54-Why were two different PEG precursors used in this work? Could this be further clarified please. What was the effect on hydrogel mesh size, crosslinking ratio, surface/bulk chemistry. It is unclear why the same MW was not used to minimize potential differences between gels. The reviewer notes that the authors discuss mesh size later on in the manuscript, but it would be helpful to introduce this concept earlier.
• It is unclear why the methods for calculating mesh size aren't provided in the methods section. There is no need to detail this in the results section.
• The authors should compare their calculated mesh size to that in literature.
• Figure 1 would be improved if both samples were provided on the same axis scale (log scale perhaps) to show the relationship between the two samples. • Figure 2-Part b could be improved via a better caption to clarify the difference between WT reporter and WT Control and why they are on opposite sides of the graph. • Figure 3-An explanation of kurtosis could prove useful to readers that aren't as versed in the jargon of statistics. • P14 Line 29-30-"To examine how heterogeneous response might interplay with • substrate stiffness, we measured the skewness and kurtosis of all attenuation-and • metabolism-corrected brightness distributions -skewness to determine • asymmetry and kurtosis to determine the preponderance of outliers (58). We • found that, for all timepoints, the skewness and kurtosis were higher for • populations attached to the softer hydrogel than for those attached to the stiffer • hydrogel (Fig. 3 B and C). This showed that attachment to the softer gel was • associated with more outliers and therefore, a more distinct subpopulation of • "strong responders". This statement is hard to understand/justify. The reviewer feels like a definitive conclusion to this issue can only be reached by looking at the genetics and not statistics alone. Could the authors please explain this further?
Reviewer: 2 Comments to the Author(s) In this manuscript, Blacutt et al. quantitatively characterized mechanobiological responses of P. aeruginosa on the stiffness of substrates for biofilm formation, using PEGDA hydrogels of higher and lower stiffness and confocal imaging. For rigorous fluorescent imaging-based evaluation, they calibrated attenuation of emitted fluorescent light through hydrogels and baseline metabolism of the tested cells. The authors' results clearly show differences in the fluorescence signal between the hard and soft hydrogel cases. They repeated their experiment using the bacteria strain not producing the envelope protein, and discussed a possibility for the bacteria cell could sense differences in pore size in addition to stiffness. The manuscript is well organized, and results are well presented. The conclusion is well supported by the presented data. I suggest minor revision with the following questions and suggestions.
1. Line 39-41 in Page 1: It would be more rigorous to indicate that E values are approximate values by using ~.
2. What is the gel thickness for bacteria culture? The thickness of hydrogel samples for swelling and stiffness measurement is shown clearly, but the gel thickness for bacteria culture is not easily found. It would be informative to clearly show the gel thickness of this case, because cells are known to sense the stiffness of the substrate or container below the gel (i.e., effective stiffness of hydrogel). 4. One hour was allowed for bacteria to adhere to hydrogel, and I wonder how this time duration was determined. Could imaging be done 0.5 hour after the inoculation of hydrogels? 5. The last line in Page 8: How many cells were typically imaged at each time point? Since probability distribution curves are shown in the manuscript, providing this number (rough estimate or range) seems required for supporting the reliability of the curves. 6. Figures 2A & 4A: It would be informative to provide the time point and gel type (hard or soft) for the shown image. 7. Figure 2C: I just wonder why the curves are not symmetric and why they do not collapse even after correction. Are these due to any possible limitations of the authors' method? Then, don't they have to be considered in analyzing images of bacteria?
8. How was the attenuation factor of 0.662 shown in Page 12 determined? Is it just a ratio of the mean values? 9. Figure 3A: The brightness level of the soft gel case became higher than that of the hard gel between ~140 min and 200 min. What does this mean?
10. Line 38-45 in Page 14: What are "strong responders" exactly? Are they related to increased cdi-GMP concentration ("strong" suggests this correlation)? If so, is more strong responders on the soft gel contradict to the higher population of bacteria with increased c-di-GMP concentration? I felt this part is confusing. In this sense, I wonder whether the attenuation factor shown in Page 12 was measured repeatedly and whether the authors got similar values as 0.662. This is also important because similar attenuation factor values obtained from repeated experiments would show that the authors' method is repeatable.
Also, I wonder how repeatable the average fluorescence intensity of control bacteria was. If this were not repeatable (similar between repeated experiments), the authors' way of removing the baseline metabolism level could be problematic.

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Decision letter (RSOS-201453.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Gordon,
It is a pleasure to accept your manuscript entitled "Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signaling by bacteria on biomedical hydrogels" in its current form for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org) and the production office (openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a co-author (if available) to manage the proofing process, and ensure they are copied into your email to the journal. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. We thank the editor and both reviewers for their time and attention to our paper. In response to their comments, we have made extensive revisions, detailed below.

Reviewer: 1 Comments to the Author(s)
General: Wording in the introduction could be improved to improve readability and provide more details.
Response: Thank you.

Changes:
We have made changes to the text of the Introduction, indicated by highlighting in the revised manuscript. We have also broken long paragraphs up into shorter paragraphs, to improve readability.

Response: Thank you and done.
Changes: As requested, we have moved the mesh size calculations to the Methods section.
It seems that the only time period assessed in the paper was three hours which is fine for assessing initial adhesion but doesn't necessarily cover any potential changes over a longer timescale; the justification for selecting 3 hours should be noted.
Response: Thank you and done. We have clarified the justification for this timescale by adding the highlighted statement below.
Changes: "This timescale was chosen because not long after 240 minutes postattachment the local bacterial density can be too high to allow single-cell brightness to be confidently measured; this time-span is comparable to that covered in related prior work (1, 2)." Here, the reference numbers correspond to references at the end of this Response. Different reference numbers are found in the manuscript.
Overall, presentation of the results is nice but could use a few minor improvements for either clarification or readability. In both presentation of results and the discussion of those results, there is a heavy statistical overtone. Clarification of some statistical terms would be appreciated and perhaps the usage of some of those statistics to reach conclusions is a bit overreaching. However, to the credit of the authors, they acknowledge this to some extent in certain sections of the paper.
Response: Thank you and done. We have added a further brief explanation of skewness and kurtosis in the main manuscript text, before the callout to Figure 4 B and C.
Changes: "The skewness values we measured were all positive, in agreement with our observation that measured distributions had a "tail" on the right (brighter) side of the mean; differences in the size of the skewness measure differences in how much of the distributions is found on the right (brighter) side of the mean. Kurtosis measures how much a distribution lies in the tail(s) and high kurtosis values corresponds to "heavy" tails, or having more of the distribution farther from the mean. "

Specific: • P3, Line 21-22-Technically speaking biofilms aren't responsible for infection, maybe rephrase to "biofilms are found in" or "biofilms comprise of"
Response: Thank you and done. We have rephrased this.
Changes: "As a result, biofilms are a large and growing problem in the healthcare industry, estimated to be found in 80% of all microbial infections" • P3, Line 46-48-Maybe here it might be worth mentioning the coatings that have been designed to release antibacterial agents/etc. While they may technically address preattachment and attachment itself, they can technically still be released during (or after) maturation.
Response: Thank you and done. We have added a parenthetical note to this effect.
Changes: "Although antifouling coatings are effective in slowing bacterial colonization of the surface, their effectiveness is limited since they only target the attachment stage of biofilm development (in contrast, coatings can release antimicrobial agents across a range of timescales could that include biofilm maturation)." Response: Thank you and done. We have added the following text and references (numbering is for the references at the end of this Response; different numbers are found in the manuscript).
Changes: "C-di-GMP is the best-studied of the cyclic dinucleotide signaling molecules and the most widespread among bacterial species (3). C-di-GMP coordinates both flagella-and pilus-driven motility (and can, in turn, be impacted by active motility elements such as flagellar stators (4) and a pilus motor (1)), virulence, and progression through the cell cycle, as well as biofilm formation and the production and secretion of proteins and polysaccharides such as biofilm matrix materials (3,5). C-di-GMP has also been associated with promoting antibiotic tolerance (6)(7)(8)."

• P4, Line 31-Please provide a better justification/explanation for why PEGDA is an ideal material. The reviewer assumes because it is transparent but this (or other rationales) should be stated
Response: Thank you for your feedback. In addition to the tunable mechanical properties and general biocompatibility, the transparency of PEGDA facilitated the fluorescence imaging of bacterial cells. We have added this rationale to the revised manuscript with mechanical tunability discussed in the next paragraph in detail (numbering is for the references at the end of this Response; different numbers are found in the manuscript).
Changes: "PEGDA hydrogels are biocompatible and have been used as 3D constructs for tissue engineering (9, 10) and matrices for controlled release of drugs (11,12). In addition to tunable matrix stiffness, the transparency of PEGDA hydrogels facilitates the confocal fluorescence imaging for the quantification of adhered bacterial cells. This makes PEGDA an ideal test material for a pilot study to develop a method for measuring the effects of real-world biomedical materials on c-di-GMP signaling by P. aeruginosa." • Why was DMA used to measure the compressive modulus? Was rheology used as a comparison? How does the compressive modulus compare to an Young's modulus? Could you please put your mechanical data into context with published literature.

Response:
The reviewer's note is well appreciated. The dynamic mechanical analyzer (DMA) has been utilized as a standard tool to characterize the hydrogel mechanical properties in many previous studies. DMA exerts oscillatory compressive or tensile force to evaluate soft or stiff materials. In determining the storage and loss moduli of the material, historical studies have displayed comparable DMA and rheometer results. For a linear isotropic Hookean material, Young's modulus of elasticity is considered equivalent to the compressive (or tensile) modulus.
Although the polymeric hydrogel is not a perfectly linear isotropic Hookean material, the linear, elastic region was first identified using a strain sweep in compression test and the storage modulus under oscillatory compression was determined in this linear region. We used this compressive modulus to represent the hydrogel stiffness in this study. As suggested, we have included literature hydrogel mechanical properties published previously for comparison.
Changes: "This provides control over the hydrogel mechanical properties, such as compressive modulus. Browning et al. reported this compositional control of PEGDA hydrogel stiffness with a compressive modulus range from 50 to 2500 kPa (13). Previous researchers have found that fewer bacteria adhere to soft PEGDA hydrogels than to stiff PEGDA hydrogels (14,15)."

• P6, Line 20-Could the authors please clarify if the PAO1 pseudomonas is that a clinical strain or more of a "lab strain"
Response: Thank you and done. We have clarified that PAO1 is a lab strain.
Changes: "We used wild-type (WT) P. aeruginosa strain PAO1 and the mutant ∆pilY1, also in the PAO1 background, in our experiments (16). PAO1 is a widelyused lab strain." • P9 Line 53-54-Why were two different PEG precursors used in this work? Could this be further clarified please. What was the effect on hydrogel mesh size, crosslinking ratio, surface/bulk chemistry. It is unclear why the same MW was not used to minimize potential differences between gels. The reviewer notes that the authors discuss mesh size later on in the manuscript, but it would be helpful to introduce this concept earlier.
Response: As mentioned in the introduction section, the molecular weight of the PEGDA macromer and the concentration can be used to modulate the hydrogel mechanical properties. In general, macromer molecular weight (e.g. 2kDa vs 10kDa) has a larger effect on stiffness than macromer concentration (e.g. 10% vs 20%). To obtain a substantial mechanical difference between the soft and stiff hydrogels, we used both macromer molecular weight and concentration. We have added the highlighted statement below to make this more clear. A smaller molecular weight PEGDA macromer typically results in higher crosslink density and smaller mesh size of the resultant hydrogel. Similarly, a higher macromer concentration typically increases the hydrogel crosslink density with a reduced hydrogel mesh size. As a result, the 50 wt% 2 kDa PEGDA hydrogels showed a much higher compressive modulus and smaller mesh size than the 10 wt% 10 kDa PEDA hydrogels. As noted by the reviewer, the authors recognized the potential effect of hydrogel mesh size on the nutrient and growth substrate transportation, and eventually, on the bacterial adhesion. We have previously decoupled these two variables with Response: Panel B of this figure (which is now Figure 3) looked like that because we had formatted it clumsily -these are actually two different plots, using the same y-axis. We were trying to save space but it made the figure too difficult to read. We have split the graphs so that it is more clear that they are two graphs (because now there is a gap between them) and split this panel into two different panels, B and C. We have also relabeled the other panels and adjusted the figure caption accordingly. We made additional minor formatting changes to this figure to try to make it read more easily. We made similar formatting changes to Figure 5.

Changes: "
The skewness values we measured were all positive, in agreement with our observation that measured distributions had a "tail" on the right (brighter) side of the mean; differences in the size of the skewness measure differences in how much of the distributions is found on the right (brighter) side of the mean. Kurtosis measures how much a distribution lies in the tail(s) and high kurtosis values corresponds to "heavy" tails, or having more of the distribution farther from the mean. " • P14 Line 29-30-"To examine how heterogeneous response might interplay with • substrate stiffness, we measured the skewness and kurtosis of all attenuation-and • metabolism-corrected brightness distributions -skewness to determine • asymmetry and kurtosis to determine the preponderance of outliers (58). We • found that, for all timepoints, the skewness and kurtosis were higher for • populations attached to the softer hydrogel than for those attached to the stiffer • hydrogel (Fig. 3 B and C). This showed that attachment to the softer gel was • associated with more outliers and therefore, a more distinct subpopulation of • "strong responders". This statement is hard to understand/justify. The reviewer feels like a definitive conclusion to this issue can only be reached by looking at the genetics and not statistics alone. Could the authors please explain this further?
Response: Thank you and done. We have added a clarification that "strong responder" here refers to the brightness of the GFP reporter which we use as our proxy measure for c-di-GMP concentration -i.e., this is meant to be a phenotypic, not a genotypic, description.
Changes: "This showed that attachment to the softer gel was associated with more outliers and therefore, a more distinct subpopulation of "strong responders" as measured by the brightness of the GFP reporter which we use as our proxy measure for c-di-GMP concentration. This does not indicate that the population on the soft gel has a stronger overall response to surface attachment, as shown by the average values in Figure 4A; rather, it indicates that a smaller fraction of the population responds strongly to attaching to a soft gel than responds strongly to attaching to a stiff gel. In contrast, attachment to the stiff gel was associated with fewer "strong responder" outliers and therefore a higher proportion of the population responding with increased c-di-GMP concentration."

Reviewer: 2
Comments to the Author(s) In this manuscript, Blacutt et al. quantitatively characterized mechanobiological responses of P. aeruginosa on the stiffness of substrates for biofilm formation, using PEGDA hydrogels of higher and lower stiffness and confocal imaging. For rigorous fluorescent imaging-based evaluation, they calibrated attenuation of emitted fluorescent light through hydrogels and baseline metabolism of the tested cells. The authors' results clearly show differences in the fluorescence signal between the hard and soft hydrogel cases. They repeated their experiment using the bacteria strain not producing the envelope protein, and discussed a possibility for the bacteria cell could sense differences in pore size in addition to stiffness. The manuscript is well organized, and results are well presented. The conclusion is well supported by the presented data. I suggest minor revision with the following questions and suggestions. Changes: "It is well-established that P. aeruginosa cells increase intracellular levels of c-di-GMP after attaching to rigid solids such as glass, which has an elastic modulus of about ~20 GPa." 2. What is the gel thickness for bacteria culture? The thickness of hydrogel samples for swelling and stiffness measurement is shown clearly, but the gel thickness for bacteria culture is not easily found. It would be informative to clearly show the gel thickness of this case, because cells are known to sense the stiffness of the substrate or container below the gel (i.e., effective stiffness of hydrogel).
Response: Thank you for this. We have added additional information about how the gels were formed for imaging and how thick they were.

Changes: "Hydrogel Fabrication and Characterization
Hydrogels were prepared by first dissolving PEGDA in deionized water at a concentration of 10 wt% 10 kDa PEGDA or 50 wt% 2 kDa PEGDA. A photoinitiator solution (Irgacure 2959, 10 wt% in 70% ethanol) was then added at 1 vol% of the precursor solution. Imaging specimens were prepared by pipetting 4 µL of the PEGDA solution into curing molds. The mold consists of an imaging spacer liner (Grace Bio-Labs SecureSeal™ Imaging Spacers) placed on a coverslip and sealed against a glass plate. Hydrogels were crosslinked by a 12-minute exposure to long wave UV light (Ultraviolet Products High Performance UV Transilluminator, 365 nm, 4mW/cm 2 , Analytik Jena). The imaging spacers used each had a single well of diameter 13 mm and the liner, which was used as the mold for casting PEGDA gels, has a thickness of about 0.05 mm (Grace Bio-Labs, personal communication). Thus, the pre-swelling thickness of PEGDA gels used for imaging was about 0.03 mm. The adhesive spacers themselves were attached to the coverslip to enclose the gel after it was cast; these spacers have a thickness of 0.12 mm. Gels were then swollen to their equilibrium height by adding liquid medium. At the start of each imaging session the microscope objective was first focused on the coverslip bottom and then focused on the bacteria on the top of the gel. The height difference between these positions, read off the control software, gave an approximate measurement of gel thickness. Gels ranged from 0.1 mm to 0.13 mm in thickness." Figure 1 (as insets) to indicate the strain range used for the modulus measurement.

Response:
The authors appreciate this comment. We agree that examples of stress-strain curves would help the readers understanding the meaning of the mechanical data. Stress-strain curves of both hydrogels have been included as a new Figure 1, and all subsequent figures renumbered accordingly.

Changes:
We show stress-strain curves as a new Figure 1 (see below).  Response: It took a little less than an hour to identify the 10-15 sites containing adhered bacteria that would be subsequently monitored by timelapse microscopy. To keep the timestep between introducing bacteria to the substrate and beginning the timelapse microscopy the same across all experiments, we chose 1 hour as the time to allow adhesion.

Changes:
We have clarified this by adjusting the highlighted text: "For all experiments, we used an Olympus FV1000 motorized inverted IX81 microscope suite, with instrument computer running FV10-ASW version 4.2b software, to image attached bacteria using laser-scanning confocal microscopy. To prepare the bacteria, we first diluted 40 µL of an overnight culture into 5 mL of fresh LB media containing gentamicin. We then placed an imaging spacer (Grace Bio-Labs SecureSeal™ Imaging Spacers) on both the microscope slide and coverslip around the PEGDA hydrogel. 25 µL of the bacterial dilution was inoculated onto the PEGDA hydrogel substrate on a glass coverslip and sealed to a microscope slide. The slide was then placed on the microscope stage and bacteria were allowed to adhere to the hydrogel for an hour prior to imaging. During this hour 10-15 locations containing adhered bacteria were identified for subsequent time-series imaging. Imaging was done using a 60x oil-immersion objective, a 488-nm laser with a 405/488 excitation filter, and an emission filter of 505/605. For each day's worth of experiments, 10-15 sites were imaged every 30 minutes for a total of 3 hours. This process was repeated on three different days for each condition. To image each site a confocal Z-stack was taken with a depth of 6 µm and an interslice size of 750 nm."