Bam complex associated proteins in Escherichia coli are functionally linked to peptidoglycan biosynthesis, membrane fluidity and DNA replication

Reviewer #1 (Public Review):

Summary:

The overall goal of the manuscript is to delineate pathways that are conditionally essential with the Bam complex and associated chaperones. The Bam complex is made of several proteins, including BamA and BamD, which are essential. The protein complex works to insert proteins in the asymmetric outer membrane. Substrates are translated in the cytoplasm prior to transport across the cell envelope to the Bam complex. Transport includes non-essential periplasmic chaperones, SurA, Skp, and DegP. According to the authors, the pathways were assumed to be redundant. The Bam complex also includes non-essential components, BamBCE. These were thought to be accessory components that interact with BamA and BamD to coordinate optimal activity. While some roles have been assigned to BamE and BamB, a detailed understanding of the role of each accessory Bam protein is lacking. In this study, more specific roles for each non-essential Bam component are proposed.

Strengths:

The overall findings are intriguing and could advance our understanding as to how the Gram-negative cell envelope is assembled. These studies could provide new targets for antimicrobial treatment. In general, the manuscript was well-written.

Weaknesses:

While the overall findings are interesting, I had some concerns with the data analysis, presentation, and conclusions. Not all the conclusions are supported by data. The proposed revisions include experimental and editorial work. The manuscript is generally well-written and could provide impactful data to advance the field if the concerns are addressed.

Major concerns:

Overall Comments:

(1) The cutoffs the authors used to define "conditionally essential" mutants are not reported. The results also lack validation for lethality using a titratable system. It would be ideal to validate several genes in each dataset to determine cutoffs (i.e. 5-fold decrease in insertion mutants) for conditional lethality. It was not done (or described) here.

(2) Also, two mutations that both make the cells sick could provide an additive effect (i.e. dapF and BamB), which doesn't necessarily mean the pathways are linked. The authors should revise their wording. They have not shown genetic linkage in some cases.

(3) Mutations throughout the manuscript are not complemented. It would be ideal to add complementation data to show the gene-phenotype relationship is specific.

(4) Also, I would argue the term "conditionally essential genes" should be replaced with "synthetically lethal". Strains were compared in the same conditions but with different genetic backgrounds.

Reviewer #2 (Public Review):

Summary:
Bryant et al. apply phenotypic profiling and saturating transposon mutagenesis to investigate the role of the non-essential lipoproteins BamB, BamC, and BamE, along with chaperones DegP, Skp, and SurA, in the biogenesis of the bacterial outer membrane. This generated a set of genetic interactions that revealed that changes in LPS and outer membrane fluidity impact Bam activity, and that the cyclic form of enterobacterial common antigen becomes essential in the absence of the chaperone surA. The study also uncovers that peptidoglycan crosslinking and DNA replication control are conditionally essential with the absence of certain Bam components, suggesting a coordination between outer membrane protein (OMP) biogenesis and other cellular processes such as lipid and peptidoglycan synthesis, as well as DNA replication.

Strengths:

(1) This is probably the first comprehensive analysis of genetic interactions involving Bam-associated proteins and should provide rich insight to refine the mechanistic understanding of this complex machine and the process of OM biogenesis.

(2) Good quality data and analysis. Well-presented manuscript.

Weaknesses:

(1) An important control in any genetic interaction study is to do complementation tests to demonstrate that the phenotype observed is indeed due to the missing gene under analysis. Although the Keio library was designed to avoid polar effects, it is impossible to predict other undesirable effects of the deletions (hitting of a non-annotated sRNA or RNA stability effects, for example). Thus, before one can safely conclude that a proposed genetic interaction is real, complementation tests should be carried out. This seems particularly important in the case of a new and surprising interaction, such as that between bamB and DNA replication and repair genes.

(2) Why not include the suppressor interactions in the work? There are probably plenty, and in principle, they should be as informative as the conditional essential (or synthetic lethal) ones. The only one highlighted in the paper is that between bamB and diaA, since it nicely fits with the synthetic lethal effects with initiation inhibitors seqA and hda. Even if the authors cannot make sense of the suppressor interactions, their inclusion in the paper should make the dataset richer and more valuable to the community.

(3) The enrichment analysis in Figure 2B deserves some clarification. What is the meaning of gene ratio? How can single genes of a pathway yield an enrichment signal? Why weren´t seqA and hda included in the DNA replication class in 2B?

(4) The writing puts too much emphasis on demonstrating that bam lipoproteins and chaperones are specialized instead of fully redundant. However, I have the impression this is a long-settled conclusion in the field, as the manuscript itself describes at several points when reviewing the literature.

Reviewer #3 (Public Review):

In this work, Bryant, et al. investigate genetic interactions between non-essential members of the outer membrane protein biogenesis pathway and other genes in the genome using a transposon-directed insertion sequencing (TraDIS) approach in E. coli K-12. The authors identify interactions with other components of the envelope including LPS, peptidoglycan, and enterobacterial common antigen biogenesis, and they tie these interactions to specific members of the outer membrane biogenesis pathway. Although many of these interactions are known and have been previously investigated in the field, the study provides several synthetic phenotypes that could be useful for further investigations.

The strengths of the paper include their unbiased, TraDIS approach, and follow up on the interactions they observe. The interactions with genes of unknown function also are of interest as they may suggest experiments to find the functions of these genes. The largest weakness of this paper is the use of a gene deletion allele for bamB that is known to be polar leading to decreased expression of an essential gene. This largely invalidates all results related to DNA replication. In addition, it is a weakness that the paper does not adequately address its place in the field through discussion of existing results on the interactions they investigate.

Author response:

We would like to thank the reviewers for their time and for their kind comments about our work. We expect that their comments will help us to improve the manuscript and so will plan the following experiments/revisions to address some of their comments:

Reviewer 1 (Public Review):

(1) The cutoffs the authors used to define "conditionally essential" mutants are not reported. The results also lack validation for lethality using a titratable system. It would be ideal to validate several genes in each dataset to determine cutoffs (i.e. 5-fold decrease in insertion mutants) for conditional lethality. It was not done (or described) here.

We will report the cutoffs used when we generate the revised manuscript. Our experiments identified hundreds of lethal combinations and we have six datasets, validation of several genes from each would require generation of at least 20 depletion strains and subsequent testing of each. Validation using a depletion system would therefore be a significant undertaking and is typically not the standard when using these approaches. However, should time permit then we will attempt a subset of these experiments.

(2) Also, two mutations that both make the cells sick could provide an additive effect (i.e. dapF and BamB), which doesn't necessarily mean the pathways are linked. The authors should revise their wording. They have not shown genetic linkage in some cases.

We will revise the text to address this.

(3) Mutations throughout the manuscript are not complemented. It would be ideal to add complementation data to show the gene-phenotype relationship is specific.

We thank the reviewers for highlighting this and will complete the complementation experiments.

(4) Also, I would argue the term "conditionally essential genes" should be replaced with "synthetically lethal". Strains were compared in the same conditions but with different genetic backgrounds.

We take the reviewers point and will revise the text accordingly.

Reviewer 2 (Public Review):

Weaknesses:

(1) An important control in any genetic interaction study is to do complementation tests to demonstrate that the phenotype observed is indeed due to the missing gene under analysis. Although the Keio library was designed to avoid polar effects, it is impossible to predict other undesirable effects of the deletions (hitting of a non-annotated sRNA or RNA stability effects, for example). Thus, before one can safely conclude that a proposed genetic interaction is real, complementation tests should be carried out. This seems particularly important in the case of a new and surprising interaction, such as that between bamB and DNA replication and repair genes.

We thank the reviewers for highlighting this and will complete the complementation experiments.

(2) Why not include the suppressor interactions in the work? There are probably plenty, and in principle, they should be as informative as the conditional essential (or synthetic lethal) ones. The only one highlighted in the paper is that between bamB and diaA, since it nicely fits with the synthetic lethal effects with initiation inhibitors seqA and hda. Even if the authors cannot make sense of the suppressor interactions, their inclusion in the paper should make the dataset richer and more valuable to the community.

These data are available in supplementary table 1. However, we appreciate this is not obvious and so will make a new supplementary table and include a brief description of the data for the revised paper.

(3) The enrichment analysis in Figure 2B deserves some clarification. What is the meaning of gene ratio? How can single genes of a pathway yield an enrichment signal? Why weren´t seqA and hda included in the DNA replication class in 2B?

We apologise for the confusion caused and will include a description of the analysis in the methods section.

(4) The writing puts too much emphasis on demonstrating that bam lipoproteins and chaperones are specialized instead of fully redundant. However, I have the impression this is a long-settled conclusion in the field, as the manuscript itself describes at several points when reviewing the literature.

We will revise the text to reduce this emphasis.

Reviewer #3 (Public Review):

In this work, Bryant, et al. investigate genetic interactions between non-essential members of the outer membrane protein biogenesis pathway and other genes in the genome using a transposon-directed insertion sequencing (TraDIS) approach in E. coli K-12. The authors identify interactions with other components of the envelope including LPS, peptidoglycan, and enterobacterial common antigen biogenesis, and they tie these interactions to specific members of the outer membrane biogenesis pathway. Although many of these interactions are known and have been previously investigated in the field, the study provides several synthetic phenotypes that could be useful for further investigations.

The strengths of the paper include their unbiased, TraDIS approach, and follow up on the interactions they observe. The interactions with genes of unknown function also are of interest as they may suggest experiments to find the functions of these genes. The largest weakness of this paper is the use of a gene deletion allele for bamB that is known to be polar leading to decreased expression of an essential gene. This largely invalidates all results related to DNA replication. In addition, it is a weakness that the paper does not adequately address its place in the field through discussion of existing results on the interactions they investigate.

We appreciate the reviewers’ comments and concerns about the bamB allele, and we will address these concerns by completing complementation experiments for the CRISPRi depletion experiments and the run-out assays. However, despite the statement that it is known to be polar, several previous studies have also used the bamB Keio library strain. Many of these studies transfer the allele to a clean background and use the derivative in which the cassette has been removed as we have done here (Cox et al., 2017, Gunasinghe et al., 2018, Psonis et al., 2019, Storek et al., 2019, Ranava et al. 2021, Steenhuis et al., 2021, Thewasano et al., 2023). Therefore, we feel somewhat justified in our choice of strain.

We are unable to find a reference for the Keio bamB strain causing polar effects and would have appreciated the reviewers’ guidance here. However, we believe the concern about polar effects stems from the observations of Ruiz et al., (2005), in which it was observed that a yfgL::ISE1 allele causes polar effects. This was hypothesised to be due to the ORF contained within the IS being transcribed in the opposite orientation to yfgL and the downstream der gene. They subsequently observed that a strain carrying a Tn5KAN-I-SceI insertion in yfgL (yfgL::kan) did not cause polar effects and this was hypothesised to be due to the kan cassette being co-oriented with yfgL. In addition, Charlson et al., 2006 generated a yfgL deletion by replacing the majority of the gene with a kan cassette in a manner similar to that of the Keio library that was subsequently flipped out. This study also found no evidence of polar effects on der. In theory, the strain used here, and in previous studies by other groups, should provide minimal disruption to transcription through generation of a mini-gene from the original bamB sequence to maintain operon expression. This is in contrast to the disruption caused by the yfgL::ISE1 allele.

While we do appreciate the concern, several pieces of evidence lend themselves to counter the statement that our strain choice largely invalidates the results. The der GTPase is essential, hence the concern about polar effects leading to the bamB phenotypes we see. However, depletion of der leads to cold sensitivity, whereas we find that the bamB strain used here actually performs better in colder temperatures. In addition, the der depletion is sensitive to doxycycline, whereas the bamB mutant has increased fitness in this condition (Fig 1) (Bharat and Brown, 2015, Hwang and Inouye, 2008). Hence, should the mutation lead to decreased expression of der then we would expect the bamB strain to phenocopy the der depletion, which it does not. Regardless of this information, we will still address these concerns by completing complementation experiments.