Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads

ABSTRACT Pseudomonas protegens CHA0, a bacterial strain able to suppress plant pathogens as well as efficiently kill lepidopteran pest insects, has been studied as a biocontrol agent to prevent ensuing agricultural damage. However, the success of this method is dependent on efficient plant colonization by the bacterial inoculant, while it faces competition from the resident microbiota as well as predators such as bacteriophages. One of these naturally occurring phages, ΦGP100, was found to drastically reduce the abundance of CHA0 once inoculated into plant microcosms, resulting in the loss of plant protection effect against a phytopathogen. Here, we investigated the molecular determinants implicated in the interaction between CHA0 and the phage ΦGP100 using a high-density transposon-sequencing approach. We show that lipopolysaccharide cell surface decorations, specifically the longer OBC3-type O-antigenic polysaccharide (O-PS, O-antigen) of the two dominant O-PS of CHA0, are essential for the attachment and infection of ΦGP100. Moreover, when exploring the distribution of the OBC3 cluster in bacterial genomes, we identified several parts of this gene cluster that are conserved in phylogenetically distant bacteria. Through heterologous complementation, we integrated an OBC3-type gene copy from a phylogenetically distant bacterium and were able to restore the phage sensitivity of a CHA0 mutant which lacked the ability to form long O-PS. Finally, we evidence that the OBC3 gene cluster of CHA0 displays a high genomic plasticity and likely underwent several horizontal acquisitions and genomic rearrangements. Collectively, this study underlines the complexity of phage-bacteria interactions and the multifunctional aspect of bacterial cell surface decorations. IMPORTANCE The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.


Sincerely, Eric Eric Cascales
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): This study identified molecular determinants in the biocontrol agent Pseudomonas protegens CHA0 that confer sensitivity to phage ΦGP100 employing a transposon-sequencing (Tn-Seq) approach.The use of Tn-Seq to identify factors involved in phages sensitivity by identifying positively selected mutants in the presence of the phage is a refreshingly new experimental approach.While it would not change the main results of this study I wonder whether number of genes detected as potentially involved in the bacterial sensitivity is a bit high (about 300) and may reflect a relatively long exposure time (10 h, how many generations?)or that the threshold set is too low (I may have overlooked it, but I could not find the criteria used to judge whether a gene is considered important or not).If the threshold is increased the role of LPS biogenesis may become even more pronounced.Did the authors also observe negatively selected genes that would be worthwhile to mention?
The authors also performed a number of additional experiments to prove the results of the global Tn-Seq analysis and provide clear evidence that LPS biogenesis is key for ΦGP100.While this is not unexpected, I think this is a very complete and convincing study that is extremely well designed, executed and presented.The data and discussion on the evolutionary aspects of LPS and phage resistance were a pleasure to read.
Pseudomonas sp CHAO is a biocontrol agent in preventing damage by lepidopteran pests and to do it has to colonize the plant roots.Phages such as 0GP100 can prevent this colonization by lysing the pseudomonas species.Certain markers on the LPS namely OBC-3 type O antigenic polysaccharide allows attachment of the phage.The authors were interested in finding out the genes responsible for phage resistance by using a Tn library approach and identified genes responsible for attachment.Further studies were also carried out by deletion studies highlighting the importance of the OBC 3 Type O LPS.The role of the genes involved in infection was tested by plaque assays.The distribution of the obc3 gene cluster was checked and shown to be present in other bacterial species.Phylogenetic analysis showed that this gene cluster could have been obtained through horizontal gene transfer.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Title: Molecular and evolu�onary basis of O-an�genic polysaccharide 2 driven phage sensi�vity in environmental pseudomonads
Comments to the authors: Pseudomonas sp CHAO is a biocontrol agent in preven�ng damage by lepidopteran pests and to do it has to colonize the plant roots.Phages such as 0GP100 can prevent this coloniza�on by lysing the pseudomonas species.Certain markers on the LPS namely OBC-3 type O an�genic polysaccharide allow atachment of the phage.The authors were interested in finding out the genes responsible for phage resistance by using a Tn library approach and iden�fied genes responsible for atachment.Further studies were also carried out by dele�on studies highligh�ng the importance of the OBC 3 Type O LPS.The role of the genes involved in infec�on was tested by plaque assays.
The distribu�on of the obc3 gene cluster was checked and shown to be present in other bacterial species.Phylogene�c analysis showed that this gene cluster could have been obtained through horizontal gene transfer.
The paper is very comprehensive and detailed, and the authors have provided different lines of evidence showing the role of the long O-PS for the infec�on by 0GP100 phage.The role of the depolymerase gene of the phage was shown by the forma�on of turbid plaques in the plaque assay using CHAO as the host.The phage appears to belong to the Zobellviridae family based on genomic analysis of the phage.

Point-by-point response to the referees
Manuscript number: Spectrum02049-23 Title: "Molecular and evolutionary basis of O-antigenic polysaccharide driven phage sensitivity in environmental pseudomonads" The line numbers we refer to along this file correspond to the line numbers found in the Marked-Up Manuscript (PDF) file.

Reviewer #1 (Comments for the Author):
This study identified molecular determinants in the biocontrol agent Pseudomonas protegens CHA0 that confer sensitivity to phage ΦGP100 employing a transposon-sequencing (Tn-Seq) approach.The use of Tn-Seq to identify factors involved in phages sensitivity by identifying positively selected mutants in the presence of the phage is a refreshingly new experimental approach.While it would not change the main results of this study I wonder whether number of genes detected as potentially involved in the bacterial sensitivity is a bit high (about 300) and may reflect a relatively long exposure time (10 h, how many generations?)or that the threshold set is too low (I may have overlooked it, but I could not find the criteria used to judge whether a gene is considered important or not).If the threshold is increased the role of LPS biogenesis may become even more pronounced.
>> We appreciate the comments provided by reviewer #1.Reviewer #1 made a valid point about the lengthy exposure time (10 hours, which equals to approximately 3,000 bacterial generations -We have now added this information to line 395 of the revised manuscript).The exposure time also accounts for the similarities in Tn-seq results across different phage concentrations.This is now stated in lines 141-142.
To determine the importance of a gene, we applied a threshold based on log2 fold change and the P value resulting from comparing gene count in the presence and absence of phages.While this information is available in the related-figure captions, we appreciate the reviewer bringing it to our attention and have included it in the Materials and Methods section on line 408-410.
Did the authors also observe negatively selected genes that would be worthwhile to mention? >> We appreciate that the reviewer #1 raised this question.Certainly, the insertion of the transposon could result in bacterial mutants that are highly sensitive to the phage, more than the wild-type strain.
In this scenario, genes that were disturbed by the transposon insertion are subjected to negative selection throughout the experiment.We identified 3,476, 4,029, and 4,074 genes that were significantly less represented in the Tn-mutant library after the phage exposure, based on the applied threshold (Fig. 1 and Fig. S1).We manually screened for genes related to membrane biosynthesis or DNA processing but found that this set of genes consisted of essential ones required for the development and growth of the bacterial host.If certain bacterial genes that hinder the phage infection process are selectively disadvantaged, they are most probably integrated within all these essential genes identified regarding this experimental setup.That is why we chose not to elaborate further on this, and to focus on the cell surface decorations.
The authors also performed a number of additional experiments to prove the results of the global Tn-Seq analysis and provide clear evidence that LPS biogenesis is key for ΦGP100.While this is not unexpected, I think this is a very complete and convincing study that is extremely well designed, executed and presented.The data and discussion on the evolutionary aspects of LPS and phage resistance were a pleasure to read.
>> We thank reviewer #1 for all the comments and questions which greatly contributed to improving our manuscript.

Reviewer #2 (Comments for the Author):
Pseudomonas sp CHAO is a biocontrol agent in preventing damage by lepidopteran pests and to do it has to colonize the plant roots.Phages such as 0GP100 can prevent this colonization by lysing the pseudomonas species.Certain markers on the LPS namely OBC-3 type O antigenic polysaccharide allows attachment of the phage.The authors were interested in finding out the genes responsible for phage resistance by using a Tn library approach and identified genes responsible for attachment.Further studies were also carried out by deletion studies highlighting the importance of the OBC 3 Type O LPS.The role of the genes involved in infection was tested by plaque assays.
The distribution of the obc3 gene cluster was checked and shown to be present in other bacterial species.Phylogenetic analysis showed that this gene cluster could have been obtained through horizontal gene transfer.
>> We thank reviewer #2 for this accurate summary appreciating our manuscript.Re: Spectrum02049-23R1 (Molecular and evolutionary basis of O-antigenic polysaccharide driven phage sensitivity in environmental pseudomonads) Dear Jordan, dear Christoph, Thank you for submitting your revised manuscript.I am pleased to accept it for publication in Microbiology Spectrum.You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.

Publication Fees:
We have partnered with Copyright Clearance Center to collect author charges.You will soon receive a message from no-reply@copyright.com with further instructions.For questions related to paying charges through RightsLink, please contact Copyright Clearance Center by email at ASM_Support@copyright.com or toll free at +1.877.622.5543.Hours of operation: 24 hours per day, 7 days per week.Copyright Clearance Center makes every attempt to respond to all emails within 24 hours.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Spectrum.

Sincerely, Eric
Eric Cascales Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred