An Advanced Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity

Reviewer #1 (Public Review):

Summary:

In this manuscript, Yan and colleagues introduce a modification to the previously published PETRI-seq bacterial single-cell protocol to include a ribosomal depletion step based on a DNA probe set that selectively hybridizes with ribosome-derived (rRNA) cDNA fragments. They show that their modification of the PETRI-seq protocol increases the fraction of informative non-rRNA reads from ~4-10% to 54-92%. The authors apply their protocol to investigating heterogeneity in a biofilm model of E. coli, and convincingly show how their technology can detect minority subpopulations within a complex community.

Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single-cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single-cell RNA-seq.

Weaknesses:

The manuscript is written in a very compressed style and many technical details of the evaluations conducted are unclear and processed data has not been made available for evaluation, limiting the ability of the reader to independently judge the merits of the method.

Reviewer #2 (Public Review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. Given that PdeI is a phosphodiesterase, which is supposed to promote hydrolysis of c-di-GMP, this finding is unexpected.

Weaknesses:

With the descriptions and writing of the manuscript, it is hard to place the findings about the PdeI into existing context (i.e. it is well known that c-di-GMP is involved in biofilm development and is heterogeneously distributed in several species' biofilms; it is also known that E.coli diesterases regulate this second messenger, i.e. https://journals.asm.org/doi/full/10.1128/jb.00604-15).
There is also no explanation for the apparently contradictory upregulation of c-di-GMP in cells expressing higher PdeI levels. Perhaps the examination of the rest of the genes in cluster 2 of the biofilm sample could be useful to explain the observed association.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

[...] Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single-cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single-cell RNA-seq.

Weaknesses:

The manuscript is written in a very compressed style and many technical details of the evaluations conducted are unclear and processed data has not been made available for evaluation, limiting the ability of the reader to independently judge the merits of the method.

Thank you for your thoughtful and constructive review of our manuscript. We appreciate your recognition of the strengths of our work and the potential impact of our modified PETRI-seq protocol on the field of bacterial single-cell RNA-seq. We are grateful for the opportunity to address your concerns and improve the clarity and accessibility of our manuscript.

We acknowledge your feedback regarding the compressed writing style and lack of technical details，which are constrained by the requirements of the Short Report format in eLife. We will addresse these issues in our revised manuscript as follows:

(1) Expanded methodology section: We will provide a more comprehensive description of our experimental procedures, including detailed protocols for the ribosomal depletion step and data analysis pipeline. This will enable readers to better understand and potentially replicate our methods.

(2) Clarification of technical evaluations: We will elaborate on the specifics of our evaluations, including the criteria used for assessing the efficiency of ribosomal depletion and the methods employed for identifying and characterizing subpopulations within the E. coli biofilm model.

(3) Data availability: We apologize for the oversight in not making our processed data readily available. We have deposited all relevant datasets, including raw and source data, in appropriate public repositories (GEO number: GSE260458) and provide clear instructions for accessing this data in the revised manuscript.

(4) Supplementary information: To maintain the concise nature of the main text while providing necessary details, we will inculde additional supplementary information. This will cover extended methodology, detailed statistical analyses, and comprehensive data tables to support our findings.

(5) Discussion of limitations: We will include a more thorough discussion of the potential limitations of our modified protocol and areas for future improvement.

​We believe these changes will significantly improve the clarity and reproducibility of our work, allowing readers to better evaluate the merits of our method.

Reviewer #2 (Public Review):

[...] Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. Given that PdeI is a phosphodiesterase, which is supposed to promote hydrolysis of c-di-GMP, this finding is unexpected.

Weaknesses:

With the descriptions and writing of the manuscript, it is hard to place the findings about the PdeI into existing context (i.e. it is well known that c-di-GMP is involved in biofilm development and is heterogeneously distributed in several species' biofilms; it is also known that E.coli diesterases regulate this second messenger, i.e. https://journals.asm.org/doi/full/10.1128/jb.00604-15).
There is also no explanation for the apparently contradictory upregulation of c-di-GMP in cells expressing higher PdeI levels. Perhaps the examination of the rest of the genes in cluster 2 of the biofilm sample could be useful to explain the observed association.

Thank you for your thoughtful and constructive review of our manuscript. We are pleased that the reviewer recognizes the value and efficiency of our rRNA depletion method for PETRI-seq, as well as its potential impact on the field. We would like to address the points raised by the reviewer and provide additional context and clarification regarding the function of PdeI in c-di-GMP regulation.

We acknowledge that c-di-GMP’s role in biofilm development and its heterogeneous distribution in bacterial biofilms are well studied. We appreciate the reviewer's observation regarding the seemingly contradictory relationship between increased PdeI expression and elevated c-di-GMP levels. This is indeed an intriguing finding that warrants further explanation.

PdeI was predicted to be a phosphodiesterase responsible for c-di-GMP degradation. This prediction is based on sequence analysis where PdeI contains an intact EAL domain known for degrading c-di-GMP. However, it is noteworthy that PdeI also contains a divergent GGDEF domain, which is typically associated with c-di-GMP synthesis. This dual-domain architecture suggests a potential for complex regulatory roles. As reported, the knockout of the major phosphodiesterase PdeH in E. coli leads to the accumulation of c-di-GMP. Further, a point mutation on PdeI's divergent GGDEF domain (G412S) in this PdeH knockout strain resulted in decreased c-di-GMP levels, implying that the wild-type GGDEF domain in PdeI has a role in maintaining or increasing c-di-GMP levels in the cell. Additionally, PdeI contains a CHASE (cyclases/histidine kinase-associated sensory) domain. Combined with our experimental results demonstrating that PdeI is a membrane-associated protein, we predict that PdeI functions as a sensor that integrates environmental signals with c-di-GMP production under complex regulatory mechanisms. The experimental evidence, along with domain analysis, suggests that PdeI could contribute to c-di-GMP synthesis, rebutting the notion that it solely functions as a phosphodiesterase. Furthermore, our single-cell experiments showed a positive correlation between PdeI expression levels and c-di-GMP levels (Fig. 2J). HPLC LC-MS/MS analysis further confirmed that PdeI overexpression (induced by arabinose) upregulated c-di-GMP levels (Fig. 2K). Importantly, in our HPLC LC-MS/MS analysis, we compared the PdeI overexpression strain with the wild-type MG1655 strain, thereby excluding the influence of other genes in cluster 2. In summary, while PdeI is predicted to be a phosphodiesterase based on its sequence and the presence of an EAL domain, the additional presence of a divergent GGDEF domain and experimental evidence suggests that PdeI has a function in upregulating c-di-GMP levels. These findings support the hypothesis that PdeI may have both synthetic and regulatory roles in c-di-GMP metabolism.