PONE-D-20-06852

Development of an epigenetic tetracycline sensor system based on DNA methylation

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In the manuscript, Ullrich and co-authors develop a highly sensitive flow cytometery analysis and tetracycline biosensor in E. Coli. This new sensor platform was modified from a previous arabinose biosensor/memory system, applying a design of genetic circuit consisted of a triggering plasmid responding to tetracycline and a memory plasmid that establish memory after triggering signal was removed by controlling DNA methylation state on memory plasmid. Combining the signal enhancing effects from the circuit design and the high sensitivity of flow cytometery, this new system can push the detection limit of tetracycline to 0.1 ng/mL, which is much lower than previously reported systems. Overall, this manuscript was well written, the experiments carefully designed and performed, and the conclusions were supported by the data. This work should be of interest to the readership of PLOS ONE and I recommend to accept this manuscript for publication.

Reviewer #2: Suggestions and questions:

1. Tetracycline is degraded by by ultraviolet radiation, and also (less efficiently) by visible light. As a consequence, reliable monitoring of tetracycline in the environment should ideally detect chemical forms derived from tetracycline. Such forms have usually lost antibiotic activity. To mimic this situation, the authors might test whether the biosensor described in this manuscript is able to detect autoclaved chlortetracycline, which has inducing activity but not antibiotic activity.

2. Papers published in the 1980's (e. g., Moyed et al. 1983) showed that overexpression of tetracycline resistance proteins (e. g., from a multicopy plasmid) reduce tetracycline resistance. Should this phenomenon be taken into account? How important is plasmid copy number to make sure that the tetracycline sensor will work?

Comments about writing (line numbers are tentative and have been identified by the reviewer):

3. The introduction is very long. The description of the arabinose biosensor that provided the background for this study could be shortened without loss of clarity (especially considering that a figure describing the sensor is provided).

4. Page 3, line 6. Because epigenetic biosensors are rare in bacteria, you might give some more details about, for instance indicating that the previously described sensors (refs. 9 and 12) are based on DNA methylation by two model DNA methyltransferases, CcrM and Dam.

5. Page 2, line 14. Remove "the" before "CcrM".

6. Page 7, first paragraph. Flow cytometry has been widely used in eukaryotic epigenetic sensors and also in the prokaryotic sensor described in ref. 12. You might mention this fact to further support your strategy in this study (and also to be fair to the literature).

7. Page 11, Conclusions. Please revise the sentence that starts "This work...". The current sentence is difficult to read!

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Reviewer #1: No

Reviewer #2: Yes: Josep Casadesús

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Development of an epigenetic tetracycline sensor system based on DNA methylation

PONE-D-20-06852R1

Dear Dr. Jeltsch,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Nathaniel A. Hathaway, Ph.D.

Academic Editor

PLOS ONE