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Bacillus thuringiensis pathogenicity islands encode regulatory circuits controlling insecticidal Cry toxin expression during vegetative growth.

PLOS Pathogens

Dear Dr. Peng,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Raffi V. Aroian

Academic Editor

PLOS Pathogens

Alice Prince

Section Editor

PLOS Pathogens

-->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

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Michael Malim

-->Editor-in-Chief

PLOS Pathogens

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Additional Editor Comments :

Two reviewers were enthusiastic about the manuscript with minor/no revisions. One reviewer has asked for some important control experiments that seem relevant. Please address all comments.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This is a solid and interesting study that, in my view, is suitable for publication after minor revision. The manuscript presents convincing evidence that VipR binds promoter regions and promotes vegetative-phase expression of several toxin genes in the strains examined. Furthermore, the distribution of VipR across Bt genomes reported in the manuscript supports the view that this may represent a broadly relevant regulatory mechanism within Bacillus thuringiensis. The work is mechanistically relevant and adds useful insight into phase-specific regulation of insecticidal gene expression in Bacillus thuringiensis.

Reviewer #2: Most cry genes depend on the sporulation-specific σE/σK regulatory cascade, leading to substantial Cry protein accumulation primarily during sporulation. In this work, the authors identify VipR, a BtPAI-1-encoded transcriptional regulator, as a key activator of cry gene expression during the vegetative phase. It uncovers a previously unrecognized regulatory mechanism that controls cry gene expression during the vegetative phase and revises the current model of Cry protein regulation. Moreover, it demonstrates that BtPAI-1 functions as a mobile genetic unit that encodes both insecticidal genes and the regulatory circuits that coordinate insecticidal protein production.

Reviewer #3: In summary, this paper reports evidence of a regulatory program, particularly turning on virulence factors against target insects, that are motile along pathegenicity islands. The evidence supports that these virulence factors are beyond those sigma factors that are essential to sporulation and that coincidentally also drive expression of those virulence factors during sporulation. The evidence supports the genetic mobility and its linkages to particular virulence factors. Importantly, this new regulatory protein drives vegetative expression, increasing the often crystalline accumulation of virulence factors within wild-type Bt. This genetically mobile transcriptional regulator is found in various types of wild-type Bt, including several that have been characterized (i.e. HD1). The strengths of the paper include it's clear writing, thoroughness of experimental exploration for the claims made, and generalizability of impact. While not entirely novel, as there are at least two papers out on VipR, the novelty of vipR shared among independent groups, is indicative of its early significance while I think will only expand over time. I find no conceptual weakness and few technical weaknesses, which I'll describe in detail in Part III.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: I did not identify any major issues that would preclude publication after minor revision.

Reviewer #2: 1. Line 176-178 “This pronounced reduction in insecticidal activity correlated with the diminished accumulation of Vip3Aa and Cry proteins in HD-1ΔvipR during the vegetative phase. ” But it is not clear the respective proportions of Vip3A and Cry in virulence. The expression of vip3A peaks in the vegetative phase, and Vip3A may exert mainly insecticidal activity in the fermentation broth of HD-1. To determine whether the reduced Cry accumulation in the vipR mutant affected insecticidal activity during the vegetative phase, the most suitable control strain should be ∆vip3A. This mutant strain knocks out vip3A while retaining vipR, eliminating interference from Vip3A.

2. In Fig 2, Panel I, J,K, and L, non-specific competitor (poly(dI-dC)) or unlabeled mutation probes (core binding sequence has been mutated) should be added to exclude non-specific binding. In addition, in panel L, why is there a lack of a 200-fold cold probe competition assay for cry2A?

Reviewer #3: I have no major issues, but I do think that the method of protein quantification is Necessary to add in the methods.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Major points within a minor revision

1. Some conclusions are overstated and should be restricted to the strains and targets directly examined.

The manuscript occasionally generalizes the proposed two-phase regulatory model too broadly. In my view, the data support such a model for the strains and promoters that were experimentally tested. However, extension of this framework to all strains carrying VipR-binding motifs or related BtPAI-1 architectures should be phrased more cautiously as a hypothesis or broader model, rather than as a demonstrated general principle.

2. The temporal annotation throughout the manuscript needs to be harmonized carefully.

There are several inconsistencies in reported sampling time points across the Results, figure panels, figure legends, and Materials and Methods. Because the manuscript’s central conclusions depend on phase-specific expression and protein accumulation, this is not a minor editorial detail. The authors should systematically reconcile all time-point information and ensure that each figure and corresponding description refer to the same experimental design and sampling scheme.

3. The manuscript would benefit from a clearer mechanistic presentation of VipR promoter interactions.

The authors convincingly show promoter binding and positive effects on transcript accumulation, but the mechanistic interpretation remains underdeveloped. In particular, it would be highly informative to include a simple schematic for one or a few representative promoters showing the approximate position of the VipR-binding motif relative to the -35/-10 elements, the transcription start site (if known), and the start codon. This would help the reader assess plausible regulatory mechanisms and seems a reasonable addition based on the promoter and motif analyses already presented.

Reviewer #2: 1. Line 306-308. “These results indicate that increased vipR expression promotes the transcription of multiple toxin genes across diverse Bt genetic backgrounds.” This conclusion may be premature here. Only two strains (HD-1 and CT-43) have been tested, and both harbor BtPAI-1. Thus, evidence for “diverse Bt genetic backgrounds” is insufficient. The authors may draw this conclusion in Line 450, after determining the transcription level of the cry9Aa gene in strain BGSC 4AE1 with introduced vipR.

2. Line 80 " Lepidoptera, Coleoptera" should be in regular font.

Reviewer #3: I have some minor issues, none of which are absolutely required to fix but would help clarify things for the reader, if addressed. One throughout is font size in some of the Figures. This can be troubling, I know. Perhaps the less relevant genes, the IS elements and functional genes other than ICPs and VIPs, don't have to be labeled within the Supplemental Figure 5, for example. They aren't pertinent to the findings or conclusions. Going through the manuscript, line 168 "equivalent cell numbers" is confusing. Cell numbers or counts of parasporal crystals are not counted. The line refers to Figure 1C, which is a Western blot which I find hard to believe that equivalent cell numbers were scored or loaded into these lanes. This brings me to my next small issue, which I honestly have with any manuscript that quantifies protein-spore mixes for biological assays: concentration and normalization of cells/proteins for input into assays and gels. LC50s are recorded as ug/g, but how protein concentration was determined is never mentioned. This part is important. If, for example, ∆vipR cells generate lower Cry concentrations, is this compensated for an increase in spores per culture volume? If BCA analysis was used, spores might confound protein measurements due to reducing sugars. Or perhaps other extracellular proteins are upregulated. When normalized for a protein level for an assay or a gel, are there more spores then? This is complicated as it's possible that the LC50 would appear to decrease if some other protein were made in compensation for the loss of vegetative synthesis of VIPs and ICPs. But given that ICPs are the major product in the broth that is lost in ∆vipR strains, there is an increase in its LC50, reflective of a decrease in the expression of known virulence factors. One could imagine a scenario in which total Crys were instead quantitated, and the LC50 (if cultures were normalized to ug/g Cry content) might not change, but in that case, one would have an increase in spores to get the same ug/g of Cry proteins. At the very least, I would need to know how protein concentrations were measured, how inputs were normalized, so we can reflect on what that means given the complexities of a spore crystal lysate. Finally, Figure 1A: at a glance, these images do not seem support the claim that there is a difference in parasporal association with crystals. Instead, it looks like the ∆vipR strain expresses Cry proteins earlier since the 30h mark is the most prevalently stained while HD-1 Crystals aren't obvious until 42h. I would zoom in further to show the that the ∆vipR strain has many spores without crystals, a qualitative zoom-in of WHAT exactly is quantified in Figure 1B. These are minor issues.

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Reviewer #1: Yes:  Dr. Heiko Liesegang

Reviewer #2: No

Reviewer #3: No

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Dear Dr. Peng,

We are pleased to inform you that your manuscript ' Bacillus thuringiensis pathogenicity islands encode regulatory circuits controlling insecticidal Cry toxin expression during vegetative growth.' has been provisionally accepted for publication in PLOS Pathogens.  You have addressed all significant comments and all reviewers, including myself, believe this manuscript provides important research ready to publish.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Raffi V. Aroian

Academic Editor

PLOS Pathogens

Alice Prince

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The study represents a solid study that strengthens the field.

Thanks the authors for carefully addressing all comments raised during the first review round. The revised manuscript has been substantially improved, and the conclusions are now appropriately aligned with the experimental evidence presented. I have no further concerns and support publication of the manuscript in its current form.

Reviewer #2: In this work, the authors identify VipR, a BtPAI-1-encoded transcriptional regulator, as a key activator of cry gene expression during the vegetative phase. It uncovers a previously unrecognized regulatory mechanism that controls cry gene expression during the vegetative phase and revises the current model of Cry protein regulation. Moreover, it demonstrates that BtPAI-1 functions as a mobile genetic unit that encodes both insecticidal genes and the regulatory circuits that coordinate insecticidal protein production.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: I had no major issues in my review of the original manuscript.

Reviewer #2: The authors have comprehensively revised the manuscript point by point in accordance with all the revision comments and requirements put forward in the previous review. All raised questions have been responded to clearly and reasonably, and the revisions have effectively addressed the deficiencies existing in the original manuscript.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Reviewer #1 considers all previously raised concerns to be adequately addressed. The authors have appropriately limited the generalization of VipR function to the genetic contexts directly investigated in this study, harmonized temporal annotations throughout the manuscript, and revised several interpretations to more closely align with the experimental evidence presented.

Reviewer #2: There are no additional revision comments or concerns for this manuscript.

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Reviewer #1: No

Reviewer #2: No