Dear Dr. Canals:

Thank you very much for submitting your manuscript "The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid" (PPATHOGENS-D-19-01144) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Andreas J Baumler

Associate Editor

PLOS Pathogens

Renée Tsolis

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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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 molecular basis of pathogen adaptation to different environments is still not completely understood. The Hinton lab has made major contributions to this field and established powerful web based platforms to make this data easily accessible to the field. In the current paper, they have focused on a strain of ST313, which currently dominates the worldwide Typhimurium infections. Specifically, they performed a comparative high saturation INSEQ screen to identify genes required for growth of D23580 in LB, in a SPI2 inducing minimal medium and in macrophages. The data iscarefully evaluated and the pros and cons of the approach are well discussed.

The data provide an exciting complementary dataset not only for comparison to similar data on the model strains commonly used in the field, but also for enriching the Hinton lab's spectacular online resources on Salmonella gene function. This will be of exceptionally high value. On top of this general importance, the screen identified an intriguing case of functional replacement of an otherwise essential chromosomal gene by a plasmid-encoded aatRNA synthetase, cysSpBT1. The biochemical characteristics of the chromosomal and the plasmid encoded enzyme were compared and my point towards a role in the stringent response. This would be an intriguing starting point for future studies.

The paper is very well written, and will serve as a valuable resource for functional information for the field. I only have one minor comment.

Reviewer #2: In this study, Canals et al perform a genetic screen to determine unique fitness determinants of an iNTS Salmonella isolate during growth in cultured macrophages and in laboratory conditions. A few “hits” were validated in separate assays, for example arginine biosynthesis is required for optimal replication in macrophages in iNTS and gastroenteritis-associated Salmonella Typhimurium. Overall, the authors found that the genetic requirements for growth under these conditions are similar to that to other, gastroenteritis-associated and typhoidal Salmonella strains. The authors found that many plasmid-encoded genes appeared to be essential for efficient replication of iNTS inside cultured macrophages. A plasmid-encoded aminoacyl tRNA synthetase, CysRS, had increased activity compared to the chromosomally-encoded paralogue but was less stable in vitro. Through bioinformatics, other plasmid encoded aminoacyl tRNA synthetases are identified.

iNTS is a significant public health problem in developing countries, yet the molecular mechanisms of pathogenesis are incompletely understood. This study touches on an important topic as compares the genetic requirements for growth in iNTS, gastroenteritis-associated strains, and typhoidal strain (done previously by the Hinton group). The data are solid; the effort by the authors in generating and analyzing the data, especially cross-referencing other studies, are very much appreciated. The authors deserve a lot of credit for carefully discussing the limitations of their study. I have two comments:

1. The study revealed little differences between iNTS and gastroenteritis-associated strains. Since only a small number of conditions were tested, only limited conclusions can be drawn from this comparison. If the conclusion of the study was that there are little differences in the genetic requirements for iNTS and gastroenteritis-associated strains (favored by the authors), then this analysis is suffers from not being comprehensive as only a few conditions were tested; conversely, if the main conclusion of the study was that there are indeed differences with relevance to iNTS pathogenesis, then this study did not find provide experimental evidence to support this conclusion.

2. Very little new biology, beyond what one would expect already (the authors do a great job at incorporating current knowledge into their analysis), is revealed. The authors argue that differences in the activity and thermal stability of CysRS may allow the organism to cope better with stress, for example by inducing the stringent response. This is plausible, but not shown experimentally and it is not clear whether this phenomenon is relevant for iNTS pathogenesis.

Reviewer #3: Canals et al present a transposon-insertion sequencing study of the S. Typhimurium ST313 strain D23580. The study examines the fitness of D23580 transposon insertion mutants during in vitro growth and during infection of a murine macrophage cell line, and identifies a range of required gene products and intergenic regions needed for growth in a given context. Altogether, this is a well-written manuscript that provides substantial insight into an important pathogen's biology, highlights the importance of a very interesting class of plasmid-encoded required genes, and in general should serve as a great resource for the community to leverage and perform followup studies.

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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: no additional experiments required.

Reviewer #2: (No Response)

Reviewer #3: 1. Considering the employed transposon's potential for driving the overexpression of genes downstream of the aph gene's promoter, the potential for interrupting downstream gene transcription in operons when the transposon is inserted in the 'antisense'-orientation (relative to the aph promoter), and the high level of genome saturation achieved in the study (an insertion every 10nt on average), the authors should (and should be able to) include an analysis of transposon insertion orientation bias within genes and intergenic regions, and correlate each entity's bias in the input and output pools (e.g., "gene X: Input='no bias'; Output='biased towards antisense insertions'"). One can envision a number of scenarios where polarity stemming from one/both/neither insertion orientations could improve or diminish the organism's fitness in the employed assays. Although the authors touch on aspects of polarity in the text, for example when rationalizing why some genes exhibit low insertion density, a comprehensive accounting of insertion orientation bias would provide further insight into (and confidence in) the TIS results, as well as potentially help to further rationalize discrepancies concerning necessity/dispensability of genes.

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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: Line 176: the authors should comment whether this observation is truly novel. Have other publications previously overlooked that essential genes are often highly expressed?

Reviewer #2: (No Response)

Reviewer #3: 1. On Line 277, please change "reveals" to "suggests" (akin to what is written in Lines 37 and 496). Bacterial mutants which are defective in the production of a given host-disarming gene product are, conceivably, subject to rescue by other co-infecting mutants in the pool (i.e., the rest of the pool is still competent for the host-disarming function). Individually screening the mutants, albeit beyond the scope of this manuscript, would have greater resolution in identifying novel virulence factors related to intra-macrophage survival, and thus such novel virulence factors could still exist in D23580.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Dear Dr. Canals,

We are pleased to inform that your manuscript, "The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Andreas J Baumler

Associate Editor

PLOS Pathogens

Renée Tsolis

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer Comments (if any, and for reference):