A toxin-mediated policing system in Bacillus optimizes division of labor via penalizing cheater-like nonproducers

Article activity feed

1. 

   Version published to 10.7554/elife.84743 on eLife

   Apr 25, 2023
2. 

   eLife

   Apr 12, 2023

   Author Response

   Reviewer #1 (Public Review):

   The manuscript by Huang et al. examines the potential "self-policing" of Bacillus cells within a biofilm. The authors first discover the co-regulation of lethal extracellular toxins (BAs) and the self-immunity mechanisms; the global regulator Spo0A controls both. The authors further show that a subpopulation of cells co-express these genes and speculate that these cells engage in preferential cooperation for biofilm formation (over cells that produce neither). Based on previous literature, the authors then evaluate the relative fitness of the wild-type strain compared to mutants locked into either constantly exporting the toxins or permanently immune to these poisons. The wild-type exhibited increased fitness (compared to the mutants) for the tested biofilm conditions. The manuscript …

   Author Response

   Reviewer #1 (Public Review):

   The manuscript by Huang et al. examines the potential "self-policing" of Bacillus cells within a biofilm. The authors first discover the co-regulation of lethal extracellular toxins (BAs) and the self-immunity mechanisms; the global regulator Spo0A controls both. The authors further show that a subpopulation of cells co-express these genes and speculate that these cells engage in preferential cooperation for biofilm formation (over cells that produce neither). Based on previous literature, the authors then evaluate the relative fitness of the wild-type strain compared to mutants locked into either constantly exporting the toxins or permanently immune to these poisons. The wild-type exhibited increased fitness (compared to the mutants) for the tested biofilm conditions. The manuscript raises interesting ideas and provides a potential model to probe questions of cooperatively in Bacillus biofilms.

   Strengths:

   • The authors use fluorescence-producing reporter strains to discern the spatial expression patterns within biofilms. This real-time imaging provides striking confirmation of their conclusions about shared co-regulation.

   • The authors also nicely deploy genetic constructs in microbiological assays to show how toxin production and immunity can influence biofilm phenotypes, including resilience to stress.

   Thank you very much for your positive comments. The detailed response to your comments and suggestions are as follows.

   Concerns:

   • My biggest concern is that the claim of policing on a single-cell level needs more quantitive microscopy, particularly of the xylose-induced strain. The data support a more tempered consideration of self-policing via BAs and self-resistance in this Bacillus species. It seems sufficient that this manuscript opens the door for a novel and readily examinable system for examining potential cooperation and its molecular controls (without making broader claims).

   Thank you very much for your comments. We demonstrated the policing system on a single-cell level by re-filming the progress of individual nonproducers from alive to death and even disappearance in a biofilm population (please see the pictures in Figure 2 and the statistical data in Figure 2-figure supplement 1 of the revised manuscript, as well as revised Figure 2-video 1-4). Alternatively, the xylose-induced strain (SQR9-Pxyl-accDA) was constructed to assess the involvement of AccDA expression (controlled by Spo0A~P in wild-type while induced by exogenous xylose here) in regulating BAs synthesis and immunity. The expression of AccDA is likely to be homogeneous in the colony with xylose addition, instead of a heterogeneous expression in the wild-type population.

   • The discussion is more speculative than the presented data warrants. For example, the speculation in lines 289 - 310 is not anchored in the results. It is hard for this reviewer to imagine how one would use the genetic framework and tools developed in this manuscript to address the ideas proposed in lines 289 - 310.

   Thank you for your comments. We have revised the discussion to ensure it is more related to data warrants than speculation. As a complement to the molecular mechanism of the policing system in the discussion, the hypothesis of the evolution of this system (Lines 289-310 in the original version) was included to give a possibility that how it raised, which is based on a couple of ecological theories with regards to division of labour and kin selection4-6; we have shortened this discussion in the revised manuscript.

   • Some conclusions (in the results section) are more decisive than the data supports. For example, the microscopy of the PI staining (as presented in Figure 2 and the supplemental movies) does not prove that only non-expressing cells die. Yet the conclusion in line 143 states that "ECM and BAs producers selectively punish the nonproducing siblings." Also, the presented data shows many non-labeled cells without PI; why do some nearby non-gfp-expressing cells remain alive?

   Thank you for your constructive comments. According to the reviewer's suggestion, an observation covering more complete biofilm forming process, as well as a more convinced data statistics, should be performed. We then re-conducted microscope observation lasting for 3 h during biofilm formation, and assess the source and location of dead cells for statistical analysis. The results showed that all dead cells were originated from the subpopulation that didn't express the gfp (the nonproducers), and the number of dead cells adjacent to the producers was significantly higher than that closed to the non-producers (please see the pictures in revised Figure 2 and Figure 2-figure supplement 1).

   In addition, regarding the survival of some non-gfp-expressing cells near the producers, based on several relevant literatures1-3 and the observation in the present study, we speculate that the coordination system for optimizing the division of labor is relatively temperate, thus only a part of the nonproducers (relative sensitive cells or facing higher concentrations of the toxin) are eliminated. We think this scene is a balance between restraining the cheater-like subpopulation and retaining the advantages of cell differentiation.

   Read the original source
3. 

   eLife

   Apr 10, 2023

   eLife assessment

   This manuscript reports important findings regarding the potential for self-policing and a division of labor among biofilm-inhabiting Bacillus cells. Overall, this work is robust in its use of various techniques and provides solid insights into the intersections of well-understood regulatory controls and the suppression of cheaters. Despite some concerns about the data, all reviewers were excited by the potential impact of this work. Colleagues interested in microbial social interactions should find this study's narrative about the internal mediation of cell differentiation particularly valuable.

   Read the original source
4. 

   eLife

   Apr 10, 2023

   Reviewer #1 (Public Review):

   The manuscript by Huang et al. examines the potential "self-policing" of Bacillus cells within a biofilm. The authors first discover the co-regulation of lethal extracellular toxins (BAs) and the self-immunity mechanisms; the global regulator spoA controls both. The authors further show that a subpopulation of cells co-express these genes and speculate that these cells engage in preferential cooperation for biofilm formation (over cells that produce neither). Based on previous literature, the authors then evaluate the relative fitness of the wild-type strain compared to mutants locked into either constantly exporting the toxins or permanently immune to these poisons. The wild-type exhibited increased fitness (compared to the mutants) for the tested biofilm conditions. The manuscript raises interesting ideas …

   Reviewer #1 (Public Review):

   The manuscript by Huang et al. examines the potential "self-policing" of Bacillus cells within a biofilm. The authors first discover the co-regulation of lethal extracellular toxins (BAs) and the self-immunity mechanisms; the global regulator spoA controls both. The authors further show that a subpopulation of cells co-express these genes and speculate that these cells engage in preferential cooperation for biofilm formation (over cells that produce neither). Based on previous literature, the authors then evaluate the relative fitness of the wild-type strain compared to mutants locked into either constantly exporting the toxins or permanently immune to these poisons. The wild-type exhibited increased fitness (compared to the mutants) for the tested biofilm conditions. The manuscript raises interesting ideas and provides a potential model to probe questions of cooperatively in Bacillus biofilms.

   Strengths:
   - The authors use fluorescence-producing reporter strains to discern the spatial expression patterns within biofilms. This real-time imaging provides striking confirmation of their conclusions about shared co-regulation.
   - The authors also nicely deploy genetic constructs in microbiological assays to show how toxin production and immunity can influence biofilm phenotypes, including resilience to stress.

   Concerns:
   - My biggest concern is that the claim of policing on a single-cell level needs more quantitive microscopy, particularly of the xylose-induced strain. The data support a more tempered consideration of self-policing via BAs and self-resistance in this Bacillus species. It seems sufficient that this manuscript opens the door for a novel and readily examinable system for examining potential cooperation and its molecular controls (without making broader claims).
   - The discussion is more speculative than the presented data warrants. For example, the speculation in lines 289 - 310 is not anchored in the results. It is hard for this reviewer to imagine how one would use the genetic framework and tools developed in this manuscript to address the ideas proposed in lines 289 - 310.
   - Some conclusions (in the results section) are more decisive than the data supports. For example, the microscopy of the PI staining (as presented in Figure 2 and the supplemental movies) does not prove that only non-expressing cells die. Yet the conclusion in line 143 states that "ECM and BAs producers selectively punish the nonproducing siblings." Also, the presented data shows many non-labeled cells without PI; why do some nearby non-gfp-expressing cells remain alive?

   Read the original source
5. 

   eLife

   Apr 10, 2023

   Reviewer #2 (Public Review):

   In this study, Huang et al. investigated Bacillus velezensis, a species that colonizes plant roots as part of the rhizosphere. They showed that clone of B. velezensis SQR9 retains a division of labor of motile, planktonic subpopulation that do not produce extracellular matrix (ECM) and biofilm-forming sessile subpopulation that do produce ECM. Specifically, the sessile subpopulation secret toxins named bacillunoic acids (BAs) to kill some, but not all, of the planktonic subpopulation. The killing mechanism is mediated by a global regulator Spo0A, which co-activates BAs production and immunity, as well as ECM production. A strain that has a disrupted policing system revealed reduced biofilm formation, lower resistance to environmental stresses and alleviated ability to colonize plant roots. Overall, the …

   Reviewer #2 (Public Review):

   In this study, Huang et al. investigated Bacillus velezensis, a species that colonizes plant roots as part of the rhizosphere. They showed that clone of B. velezensis SQR9 retains a division of labor of motile, planktonic subpopulation that do not produce extracellular matrix (ECM) and biofilm-forming sessile subpopulation that do produce ECM. Specifically, the sessile subpopulation secret toxins named bacillunoic acids (BAs) to kill some, but not all, of the planktonic subpopulation. The killing mechanism is mediated by a global regulator Spo0A, which co-activates BAs production and immunity, as well as ECM production. A strain that has a disrupted policing system revealed reduced biofilm formation, lower resistance to environmental stresses and alleviated ability to colonize plant roots. Overall, the toxin-mediated policing system is important for B. velezensis to mediate division of labor for enhancing population stability and ecological fitness when required (e.g., cell transition from a planktonic style to a multicellular style).

   Read the original source
6. 

   eLife

   Apr 10, 2023

   Reviewer #3 (Public Review):

   In this manuscript, Huang et al. use a variety of experimental approaches to investigate division of labor and cheater "policing" during biofilm formation in Bacillus velezensis SQR9. The authors show that SQR9 cells differentiate into two populations during biofilm development - one cell type produces extracellular matrix (ECM) and the other does not (referred to as "cheaters"). The authors go on to demonstrate that the ECM producing cells utilize a bacillunoic acid toxin system to selectively kill cheaters, keeping the cheater population in check which maintains the stability of the community. Further, the authors demonstrate that coordination of ECM production and synthesis of bacillunoic acid/immunity via acetyl-CoA carboxylase is mediated in part by Spo0A. I find the work as a whole to be compelling and …

   Reviewer #3 (Public Review):

   In this manuscript, Huang et al. use a variety of experimental approaches to investigate division of labor and cheater "policing" during biofilm formation in Bacillus velezensis SQR9. The authors show that SQR9 cells differentiate into two populations during biofilm development - one cell type produces extracellular matrix (ECM) and the other does not (referred to as "cheaters"). The authors go on to demonstrate that the ECM producing cells utilize a bacillunoic acid toxin system to selectively kill cheaters, keeping the cheater population in check which maintains the stability of the community. Further, the authors demonstrate that coordination of ECM production and synthesis of bacillunoic acid/immunity via acetyl-CoA carboxylase is mediated in part by Spo0A. I find the work as a whole to be compelling and thorough, and I expect it to be of broad interest to various fields of research.

   Read the original source
7. 

   Version published to 10.1101/2022.05.14.491907v1 on bioRxiv

   May 14, 2022